ORCID Profile
0000-0002-7030-2288
Current Organisations
University of Queensland
,
RMIT University
,
University of Wollongong
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Membrane Biology | Biological And Medical Chemistry | Basic Pharmacology | Receptors and Membrane Biology | Structural Biology (incl. Macromolecular Modelling) | Medicinal and Biomolecular Chemistry | Cell Physiology | Biological Physics | Protein Targeting And Signal Transduction | Biologically Active Molecules | Structural Chemistry | Reproduction | Basic Pharmacology | Biophysics | Cellular Nervous System | Biological Sciences Not Elsewhere Classified | Peripheral Nervous System | Cell Metabolism | Cell Neurochemistry | Paediatrics and Reproductive Medicine | Characterisation of Biological Macromolecules | Biomolecular Modelling and Design | Cheminformatics and Quantitative Structure-Activity Relationships | Chemical Engineering Not Elsewhere Classified | Proteins and Peptides | Cell Physiology | Other Physical Sciences | Other Biological Sciences | Neurosciences | Animal Physiology—Systems | Bioinformatics
ANZSRC Socio-Economic Objective (SEO)
Biological sciences | Nervous system and disorders | Treatments (e.g. chemicals, antibiotics) | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in the Biological Sciences | Chemical sciences | Expanding Knowledge in the Chemical Sciences | Human Diagnostics | Other | Diagnostics | Reproductive system and disorders | Immune system and allergy | Infectious diseases | Cardiovascular system and diseases | Cancer and related disorders | Processed food products and beverages not elsewhere classified | Expanding Knowledge in Technology | Human Pharmaceutical Treatments (e.g. Antibiotics) | Public health not elsewhere classified |
Related Links
Publications
Voltage sensitivity of inhibitory postsynaptic currents in Aplysia buccal ganglia
Publisher: Elsevier BV
Date: 10-1976
Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1
Publisher: Wiley
Date: 21-07-2018
DOI: 10.1111/BPH.13910
Cyclic-RGDfK peptide conjugated succinoyl-TPGS nanomicelles for targeted delivery of docetaxel to integrin receptor over-expressing angiogenic tumours
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.NANO.2015.04.007
Abstract: Docetaxel (DTX) is an anticancer drug that is used alone and in combination with other drugs to treat tumours. However, it suffers from the drawback of non-specific cytotoxicity. To improve the therapeutic potential of DTX, we report the synthesis of cRGDfK peptide-conjugated succinoyl-TPGS (tocopheryl polyethylene glycol succinate) nanomicelles for targeted delivery of DTX. Among RGD (Arg-Gly-Asp) peptides, cRGDfK peptide shows specificity towards αvβ3 integrin receptors that are most commonly over-expressed in tumour cells. To cRGDfK peptide, succinoylated TPGS was synthesised and conjugated to cRGDfK peptide using a carbodiimide reaction. Peptide-conjugated DTX loaded nanomicelles (PDNM) displayed small particle size with a narrow distribution, controlled drug release and high physicochemical stability. Cytotoxicity, cellular uptake, apoptosis and anti-angiogenic comparisons of unconjugated nanomicelles to PDNM in DU145 human prostate cancer cells and HUVECs (Human Umblical Vein Endothelial Cells) clearly revealed the importance of the cRGDfK peptide in enhancing the drug delivery performance of nanomicelles. Common to many chemotherapeutic agents for cancer, systemic toxicity remains a big concern. In this article, the authors attempted to address this issue by conjugating RGD based peptides to Docetaxel, which would target integrins expressed on tumor cell surface. The experimental data revealed enhanced drug delivery.
Purinergic receptor activation inhibits mitogen-stimulated proliferation in primary neurospheres from the adult mouse subventricular zone
Publisher: Elsevier BV
Date: 08-2007
DOI: 10.1016/J.MCN.2007.04.013
Abstract: The expression pattern of purinergic receptors was examined in subventricular zone-derived primary neurospheres. Primary neurospheres expressed mRNA for P2X4 and P2X7 receptors, all P2Y receptors, with the exception of P2Y4, and the A1, A2a and A2b adenosine receptors. ATPgammaS, ADPbetaS and UTP evoked transient increases in cytoplasmic Ca(2+) concentration in dissociated primary neurospheres, demonstrating the functional expression of P2Y1 and P2Y2 receptors. Ca(2+) transients were not attenuated by the removal of extracellular Ca(2+) and were reversibly inhibited by the P2Y1 selective antagonist, MRS 2179. P2Y and adenosine receptor agonists reduced the size and frequency of primary neurospheres. The effects of ADPbetaS and adenosine were reversed by subtype-selective receptor antagonists, demonstrating that P2Y1 and A2a receptors mediate inhibitory effects on primary neurosphere proliferation. The modulation of neural precursor cell proliferation by P2Y and adenosine receptors therefore represents a potential regulatory mechanism within the neurogenic microenvironment.
Novel ω-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes
Publisher: Elsevier BV
Date: 11-2000
Coordinate regulation of stretch-activated channels and myogenic tone by polycystins 1 and 2
Publisher: Informa UK Limited
Date: 2010
An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0GC01366H
Abstract: An environmentally sustainable production platform for a variety of correctly folded cyclic disulfide-rich peptides with enhanced yields.
Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 09-06-2005
Abstract: Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K(+) (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1+GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca(2+)-activated large conductance K(+) channel (BK or MaxiK alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1+GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K(+) channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-cl ed oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K(+) channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1+GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular alent cations (Ca(2+), Mg(2+), Zn(2+), and Ba(2+)) in defolliculated oocytes. In voltage-cl ed DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K(+) currents, but Cs(+)-blocked hyperpolarization-activated inward currents including I(H) were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-cl conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca(2+)-activated K(+) channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.
Stabilization of α-Conotoxin AuIB: Influences of Disulfide Connectivity and Backbone Cyclization
Publisher: Mary Ann Liebert Inc
Date: 2011
Abstract: α-Conotoxins are peptides isolated from the venom ducts of cone snails that target nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential applications for treating a range of conditions in humans, including pain. However, like all peptides, conotoxins are susceptible to degradation, and to enhance their therapeutic potential it is important to elucidate the factors contributing to instability and to develop approaches for improving stability. AuIB is a unique member of the α-conotoxin family because the nonnative "ribbon" disulfide isomer exhibits enhanced activity at the nAChR in rat parasympathetic neurons compared with the native "globular" isomer. Here we show that the ribbon isomer of AuIB is also more resistant to disulfide scrambling, despite having a nonnative connectivity and flexible structure. This resistance to disulfide scrambling does not correlate with overall stability in serum because the ribbon isomer is degraded in human serum more rapidly than the globular isomer. Cyclization via the joining of the N- and C-termini with peptide linkers of four to seven amino acids prevented degradation of the ribbon isomer in serum and stabilized the globular isomers to disulfide scrambling. The linker length used for cyclization strongly affected the relative proportions of the disulfide isomers produced by oxidative folding. Overall, the results of this study provide important insights into factors influencing the stability and oxidative folding of α-conotoxin AuIB and might be valuable in the design of more stable antagonists of nAChRs.
Molecular dynamics simulations of dihydro‐β‐erythroidine bound to the human α4β2 nicotinic acetylcholine receptor
Publisher: Wiley
Date: 07-06-2019
DOI: 10.1111/BPH.14698
Medicinal chemistry, pharmacology, and therapeutic potential of α-conotoxins antagonizing the α9α10 nicotinic acetylcholine receptor.
Publisher: Elsevier BV
Date: 06-2020
DOI: 10.1016/J.PHARMTHERA.2020.107792
Abstract: α-Conotoxins are disulfide-rich and well-structured peptides, most of which can block nicotinic acetylcholine receptors (nAChRs) with exquisite selectivity and potency. There are various nAChR subtypes, of which the α9α10 nAChR functions as a heteromeric ionotropic receptor in the mammalian cochlea and mediates postsynaptic transmission from the medial olivocochlear. The α9α10 nAChR subtype has also been proposed as a target for the treatment of neuropathic pain and the suppression of breast cancer cell proliferation. Therefore, α-conotoxins targeting the α9α10 nAChR are potentially useful in the development of specific therapeutic drugs and pharmacological tools. Despite dissimilarities in their amino acid sequence and structures, these conopeptides are potent antagonists of the α9α10 nAChR subtype. Consequently, the activity and stability of these peptides have been subjected to chemical modifications. The resulting synthetic analogues have not only functioned as molecular probes to explore ligand binding sites of the α9α10 nAChR, but also have the potential to become candidates for drug development. From the perspectives of medicinal chemistry and pharmacology, we highlight the structure and function of the α9α10 nAChR and review studies of α-conotoxins targeting it, including their three-dimensional structures, structure optimization strategies, and binding modes at the α9α10 nAChR, as well as their therapeutic potential.
ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation
Publisher: Wiley
Date: 15-11-2022
DOI: 10.1111/JNC.15535
Abstract: αO‐Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis . The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage‐activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein‐coupled GABA B receptors (GABA B R) inhibiting HVA N‐type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba 2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABA B R antagonist CGP 55845. In transfected HEK293T cells co‐expressing human GABA B R1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization‐activated Ba 2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABA B R‐coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly‐rectifying K + currents mediated by human GIRK1/2 channels co‐expressed with GABA B R in HEK293T cells. This study highlights GABA B R as a potentially important receptor target for the activity of αO‐conotoxin GeXIVA to mediate analgesia. image
Analgesic conopeptides targeting G protein-coupled receptors reduce excitability of sensory neurons
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.NEUROPHARM.2017.05.020
Abstract: Conotoxins (conopeptides) are a erse group of peptides isolated from the venom of marine cone snails. Conus peptides modulate pain by interacting with voltage-gated ion channels and G protein-coupled receptors (GPCRs). Opiate drugs targeting GPCRs have long been used, nonetheless, many undesirable side effects associated with opiates have been observed including addiction. Consequently, alternative avenues to pain management are a largely unmet need. It has been shown that various voltage-gated calcium channels (VGCCs) respond to GPCR modulation. Thus, regulation of VGCCs by GPCRs has become a valuable alternative in the management of pain. In this review, we focus on analgesic conotoxins that exert their effects via GPCR-mediated inhibition of ion channels involved in nociception and pain transmission. Specifically, α-conotoxin Vc1.1 activation of GABA
Molecular Determinants Conferring the Stoichiometric-Dependent Activity of α-Conotoxins at the Human α9α10 Nicotinic Acetylcholine Receptor Subtype
Publisher: American Chemical Society (ACS)
Date: 07-05-2018
DOI: 10.1021/ACS.JMEDCHEM.8B00115
Abstract: α9α10 nicotinic acetylcholine receptors (nAChRs) putatively exist at different stoichiometries. We systematically investigated the molecular determinants of α-conotoxins Vc1.1, RgIA#, and PeIA inhibition at hypothetical stoichiometries of the human α9α10 nAChR. Our results suggest that only Vc1.1 exhibits stoichiometric-dependent inhibition at the α9α10 nAChR. The hydrogen bond between N154 of α9 and D11 of Vc1.1 at the α9(+)-α9(-) interface is responsible for the stoichiometric-dependent potency of Vc1.1.
Dicarba α-Conotoxin Vc1.1 Analogues with Differential Selectivity for Nicotinic Acetylcholine and GABAB Receptors
Publisher: American Chemical Society (ACS)
Date: 17-06-2013
DOI: 10.1021/CB4002393
Abstract: Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3β4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptide's innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptide's potent analgesic activity.
A novel mechanism of inhibition of high-voltage activated calcium channels by α-conotoxins contributes to relief of nerve injury-induced neuropathic pain
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2011
DOI: 10.1016/J.PAIN.2010.09.007
Abstract: α-Conotoxins that are thought to act as antagonists of nicotinic acetylcholine receptors (nAChRs) containing α3-subunits are efficacious in several preclinical models of chronic pain. Potent interactions of Vc1.1 with other targets have suggested that the pain-relieving actions of α-conotoxins might be mediated by either α9α10 nAChRs or a novel GABA(B) receptor-mediated inhibition of N-type calcium channels. Here we establish that three α-conotoxins, Vc1.1, AuIB and MII have distinct selectivity profiles for these three potential targets. Their potencies after intramuscular administration were then determined for reversal of allodynia produced by partial nerve ligation in rats. Vc1.1, which potently inhibits α9α10 nAChRs and GABA(B)/Ca(2+) channels but weakly blocks α3β2 and α3β4 nAChRs, produced potent, long-lasting reversal of allodynia that were prevented by pre-treatment with the GABA(B) receptor antagonist, SCH50911. α-Conotoxin AuIB, a weak α3β4 nAChR antagonist, inhibited GABA(B)/Ca(2+) channels but did not act on α9α10 nAChRs. AuIB also produced reversal of allodynia. These findings suggest that GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels can mediate the sustained anti-allodynic actions of some α-conotoxins. However, MII, a potent α3β2 nAChR antagonist but inactive on α9α10 and α3β4 nAChRs and GABA(B)/Ca(2+) channels, was demonstrated to have short-acting anti-allodynic action. This suggests that α3β2 nAChRs may also contribute to reversal of allodynia. Together, these findings suggest that inhibition of α9α10 nAChR is neither necessary nor sufficient for relief of allodynia and establish that α-conotoxins selective for GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels relieve allodynia, and could therefore be developed to manage chronic pain.
Structural basis of the potency and selectivity of Urotoxin, a potent Kv1 blocker from scorpion venom
Publisher: Elsevier BV
Date: 04-2020
DOI: 10.1016/J.BCP.2019.113782
Abstract: Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC
Structure and Activity of α-Conotoxin PeIA at Nicotinic Acetylcholine Receptor Subtypes and GABAB Receptor-coupled N-type Calcium Channels
Publisher: Elsevier BV
Date: 03-2011
Drysdalin, an antagonist of nicotinic acetylcholine receptors highlights the importance of functional rather than structural conservation of amino acid residues
Publisher: Wiley
Date: 10-01-2019
DOI: 10.1096/FBA.1027
Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae: A Structural Analogue of α-Conotoxins
Publisher: MDPI AG
Date: 25-01-2023
DOI: 10.3390/MD21020081
Abstract: Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11–18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1β1εδ, α3β2, α3β4, α4β2, α7, and α9α10) at 1 µM concentrations.
The Engineering of an Orally Active Conotoxin for the Treatment of Neuropathic Pain
Publisher: Wiley
Date: 08-06-2010
Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons
Publisher: Springer Science and Business Media LLC
Date: 28-11-2000
Abstract: The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-cl recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the litude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP litude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.
Ionic selectivity of native ATP-activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia
Publisher: Wiley
Date: 07-2001
DOI: 10.1111/J.1469-7793.2001.00423.X
Abstract: 1. The relative permeability of the native P2X receptor channel to monovalent and alent inorganic and organic cations was determined from reversal potential measurements of ATP-evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole-cell patch cl technique. 2. The P2X receptor-channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na(+) > Li(+) > Cs(+) > Rb(+) > K(+), and permeability ratios relative to Cs(+) (P(X)/P(Cs)) ranging from 1.11 to 0.86. 3. The selectivity for the alent alkaline earth cations was also weak with the sequence Ca(2+) > Sr(2+) > Ba(2+) > Mn(2+) > Mg(2+). ATP-evoked currents were strongly inhibited when the extracellular alent cation concentration was increased. 4. The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size. 5. Acidification to pH 6.2 increased the ATP-induced current litude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current litude. Cell dialysis with either anti-P2X(2) and/or anti-P2X(4) but not anti-P2X(1) antibodies attenuated the ATP-evoked current litude. Taken together, these data are consistent with homomeric and/or heteromeric P2X(2) and P2X(4) receptor subtypes expressed in rat submandibular neurones. 6. The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X(2) receptors expressed in mammalian cells.
Effects of linker sequence modifications on the structure, stability, and biological activity of a cyclic α‐conotoxin
Publisher: Wiley
Date: 11-2016
DOI: 10.1002/BIP.22848
Abstract: The cyclic conotoxin analogue cVc1.1 is a promising lead molecule for the development of new treatments for neuropathic and chronic pain. The design of this peptide includes a linker sequence that joins the N and C termini together, improving peptide stability while maintaining the structure and activity of the original linear Vc1.1. The effect of linker length on the structure, activity and stability of cyclised conotoxins has been studied previously but the effect of altering the composition of the linker sequence has not been investigated. In this study, we designed three analogues of cVc1.1 with linker sequences that varied in charge, hydrophobicity and hydrogen bonding capacity and examined the effect on structure, stability, membrane permeability and biological activity. The three designed peptides were successfully synthesized using solid phase peptide synthesis approaches and had similar structures and stability compared with cVc1.1. Despite modifications in charge, hydrophobicity and hydrogen bonding potential, which are all factors that can affect membrane permeability, no changes in the ability of the peptides to pass through membranes in either PAMPA or Caco-2 cell assay were observed. Surprisingly, modification of the linker sequence was deleterious to biological activity. These results suggest the linker sequence might be a useful part of the molecule for optimization of bioactivity and not just the physiochemical properties of cVc1.1. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 864-875, 2016.
A Novel Lid-Covering Peptide Inhibitor of Nicotinic Acetylcholine Receptors Derived from αD-Conotoxin GeXXA
Publisher: MDPI AG
Date: 05-06-2017
DOI: 10.3390/MD15060164
An ATP-sensitive K+ conductance in dissociated neurones from adult rat intracardiac ganglia
Publisher: Wiley
Date: 08-2001
DOI: 10.1111/J.1469-7793.2001.00713.X
Abstract: 1. An ATP-sensitive K(+) (K(ATP)) conductance has been identified using the perforated patch recording configuration in a population (52%) of dissociated neurones from adult rat intracardiac ganglia. The presence of the sulphonylurea receptor in approximately half of the intracardiac neurones was confirmed by labelling with fluorescent glibenclamide-BODIPY. 2. Under current cl conditions in physiological solutions, levcromakalim (10 microM) evoked a hyperpolarization, which was inhibited by the sulphonylurea drugs glibenclamide and tolbutamide. 3. Under voltage cl conditions in symmetrical (140 mM) K(+) solutions, bath application of levcromakalim evoked an inward current with a density of 8 pA pF(-1) at -50 mV and a slope conductance of approximately 9 nS, which reversed close to the potassium equilibrium potential (E(K)). Cell dialysis with an ATP-free intracellular solution also evoked an inward current, which was inhibited by tolbutamide. 4. Bath application of either glibenclamide (10 microM) or tolbutamide (100 microM) depolarized adult intracardiac neurones by 3-5 mV, suggesting that a K(ATP) conductance is activated under resting conditions and contributes to the resting membrane potential. 5. Activation of a membrane current by levcromakalim was concentration dependent with an EC(50) of 1.6 microM. Inhibition of the levcromakalim-activated current by glibenclamide was also concentration dependent with an IC(50) of 55 nM. 6. Metabolic inhibition with 2,4-dinitrophenol and iodoacetic acid or superfusion with hypoxic solution (P(O2) approximately 16 mmHg) also activated a membrane current. These currents exhibited similar I-V characteristics to the levcromakalim-induced current and were inhibited by glibenclamide. 7. Activation of K(ATP) channels in mammalian intracardiac neurones may contribute to changes in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion.
Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues
Publisher: Elsevier BV
Date: 02-2009
Cyclic RGDfK Peptide Functionalized Polymeric Nanocarriers for Targeting Gemcitabine to Ovarian Cancer Cells
Publisher: American Chemical Society (ACS)
Date: 28-03-2016
DOI: 10.1021/ACS.MOLPHARMACEUT.5B00935
Abstract: Current cancer chemotherapies commonly suffer from nonspecificity, drug resistance, poor bioavailability, and narrow therapeutic indices. To achieve the optimum drug efficacy, we designed a polymeric drug delivery system for targeted intracellular delivery of a clinically approved, water-soluble anticancer drug, gemcitabine hydrochloride (GEM). We utilized the unique ability of a cyclic pentapeptide cRGDfK to specifically target αvβ3 integrin receptors that are overexpressed on SKOV-3 human ovarian cancer cells. This significantly increased the effective intracellular drug concentration even at low doses, thereby remarkably improving the chemotherapeutic potential of GEM. cRGDfK-conjugated, GEM-loaded nanoparticles reduced the nonspecific hemolytic cytotoxicity of the drug, simultaneously influencing intracellular processes such as mitochondrial membrane potential (DΨm), reactive oxygen species (ROS) levels, and apoptosis, thereby favorably influencing drug antiproliferative efficacy.
Hydrophobic residues at position 10 of a-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.BCP.2014.07.025
Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are a erse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki=44nM) though selectivity reverted to α3β2 (IC50=0.7nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants.
Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII
Publisher: Proceedings of the National Academy of Sciences
Date: 14-09-2005
Abstract: Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo . By cyclizing the α-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.
TEA inhibits ACh-induced EDRF release: Endothelial Ca2+-dependent K+ channels contribute to vascular tone
Publisher: American Physiological Society
Date: 09-1994
DOI: 10.1152/AJPHEART.1994.267.3.H1135
Abstract: The effects of K(+)-channel blockers on the acetylcholine (ACh)-induced relaxation of vascular smooth muscle, intracellular free Ca2+ concentration ([Ca2+]i) elevation, and ACh-evoked outward K+ current of endothelial cells of rabbit aorta were studied using bioassay, spectrofluorimetry, and patch-cl techniques, respectively. In bioassay experiments, ACh caused relaxation of endothelium-denuded aortic rings in a concentration-dependent manner when perfused through an endothelium-intact donor segment of aorta but not when perfused directly onto the recipient aortic ring. ACh-induced relaxation was inhibited by perfusion of tetraethylammonium ions (TEA 5 mM) through the donor but not by perfusion directly onto the recipient segment. Glibenclamide had no effect on ACh-induced relaxation of the bioassay ring in either situation. ACh increased [Ca2+]i at the endothelial surface of aortic strips but not at the adventitial surface. TEA inhibited ACh-induced [Ca2+]i elevation, whereas glibenclamide had no effect. In patch-cl experiments with freshly isolated endothelial cells, ACh evoked a biphasic outward current which was completely abolished by TEA (3 mM). It is concluded that Ca(2+)-dependent K+ channels are important for increasing [Ca2+]i during agonist stimulation and consequently for the synthesis/release of endothelium-derived relaxing factors (EDRFs). Furthermore, endothelial ATP-sensitive K+ channels do not contribute to ACh-induced relaxation or evoke an increase in endothelial [Ca2+]i of rabbit thoracic aorta.
ZNF265 - A novel spliceosomal protein able to induce alternative splicing
Publisher: Rockefeller University Press
Date: 09-07-2001
Abstract: The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-β1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF35. Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265–EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain–containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.
NMR structure of µ-conotoxin GIIIC: Leucine 18 induces local repacking of the N-terminus resulting in reduced Nav channel potency
Publisher: MDPI AG
Date: 22-10-2018
DOI: 10.3390/MOLECULES23102715
Abstract: μ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of μ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of μ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical μ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (NaV), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.
Contribution of membrane receptor signalling to chronic visceral pain
Publisher: Elsevier BV
Date: 05-2018
DOI: 10.1016/J.BIOCEL.2018.02.017
Abstract: Irritable bowel syndrome and inflammatory bowel disease are major forms of chronic visceral pain, which affect over 15% of the global population. In order to identify new therapies, it is important to understand the underlying causes of chronic visceral pain. This review provides recent evidence demonstrating that inflammation or infection of the gastrointestinal tract triggers specific changes in the neuronal excitability of sensory pathways responsible for the transmission of nociceptive information from the periphery to the central nervous system. Specific changes in the expression and function of a variety of ion channels and receptors have been documented in inflammatory and chronic visceral pain conditions relevant to irritable bowel syndrome and inflammatory bowel disease. An increase in pro-nociceptive mechanisms enhances peripheral drive from the viscera and provides an underlying basis for enhanced nociceptive signalling during chronic visceral pain states. Recent evidence also highlights increases in anti-nociceptive mechanisms in models of chronic visceral pain, which present novel targets for pharmacological treatment of this condition.
Cyclic analogues of α-conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain
Publisher: Wiley
Date: 13-02-2018
DOI: 10.1111/BPH.14115
Dimerization of α-Conotoxins as a Strategy to Enhance the Inhibition of the Human α7 and α9α10 Nicotinic Acetylcholine Receptors
Publisher: American Chemical Society (ACS)
Date: 26-02-2020
Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation
Publisher: Rockefeller University Press
Date: 31-03-2014
Abstract: Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein–coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5′-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.
Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type CaV channels
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1016/J.BCP.2014.02.008
Abstract: Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba(2+) currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels.
Trends in peptide drug discovery
Publisher: Springer Science and Business Media LLC
Date: 03-02-2021
DOI: 10.1038/S41573-020-00135-8
Abstract: Since the introduction of insulin almost a century ago, more than 80 peptide drugs have reached the market for a wide range of diseases, including diabetes, cancer, osteoporosis, multiple sclerosis, HIV infection and chronic pain. In this Perspective, we summarize key trends in peptide drug discovery and development, covering the early efforts focused on human hormones, elegant medicinal chemistry and rational design strategies, peptide drugs derived from nature, and major breakthroughs in molecular biology and peptide chemistry that continue to advance the field. We emphasize lessons from earlier approaches that are still relevant today as well as emerging strategies such as integrated venomics and peptide-display libraries that create new avenues for peptide drug discovery. We also discuss the pharmaceutical landscape in which peptide drugs could be particularly valuable and analyse the challenges that need to be addressed for them to reach their full potential.
Mechanisms of conotoxin inhibition of N-type (Cav 2.2) calcium channels
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.BBAMEM.2013.01.019
Abstract: N-type (Ca(v)2.2) voltage-gated calcium channels (VGCC) transduce electrical activity into other cellular functions, regulate calcium homeostasis and play a major role in processing pain information. Although the distribution and function of these channels vary widely among different classes of neurons, they are predominantly expressed in nerve terminals, where they control neurotransmitter release. To date, genetic and pharmacological studies have identified that high-threshold, N-type VGCCs are important for pain sensation in disease models. This suggests that N-type VGCC inhibitors or modulators could be developed into useful drugs to treat neuropathic pain. This review discusses the role of N-type (Ca(v)2.2) VGCCs in nociception and pain transmission through primary sensory dorsal root ganglion (DRG) neurons (nociceptors). It also outlines the potent and selective inhibition of N-type VGCCs by conotoxins, small disulfide-rich peptides isolated from the venom of marine cone snails. Of these conotoxins, ω-conotoxins are selective N-type VGCC antagonists that preferentially block nociception in inflammatory pain models, and allodynia and/or hyperalgesia in neuropathic pain models. Another conotoxin family, α-conotoxins, were initially proposed as competitive antagonists of muscle and neuronal nicotinic acetylcholine receptors (nAChR). Surprisingly, however, α-conotoxins Vc1.1 and RgIA, also potently inhibit N-type VGCC currents in the sensory DRG neurons of rodents and α9 nAChR knockout mice, via intracellular signaling mediated by G protein-coupled GABAB receptors. Understanding how conotoxins inhibit VGCCs is critical for developing these peptides into analgesics and may result in better pain management. This article is part of a Special Issue entitled: Calcium channels.
α‐Conotoxin Bt1.8 from Conus betulinus selectively inhibits α6/α3β2β3 and α3β2 nicotinic acetylcholine receptor subtypes
Publisher: Wiley
Date: 22-06-2021
DOI: 10.1111/JNC.15434
Abstract: α‐Conotoxins are small disulfide‐rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α‐conotoxin Bt1.8, which was cloned from Conus betulinus . Bt1.8 selectively inhibited ACh‐evoked currents in Xenopus oocytes expressing rat(r) α6/α3β2β3 and rα3β2 nAChRs with an IC 50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3β2β3 and hα3β2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3β2β3 subtype compared to rα3β2. The amino acid sequence of Bt1.8 is significantly different from other reported α‐conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3β2β3 and α3β2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7‐conotoxins which may explain its potent inhibition at α6/α3β2β3 nAChRs. image
Ionic currents in response to membrane depolarization in an Aplysia neurone.
Publisher: Wiley
Date: 04-1979
DOI: 10.1113/JPHYSIOL.1979.SP012728
Abstract: 1. Action potentials recorded in the soma of R15 neurones in the abdominal ganglia of Aplysia juliana were not suppressed by selective inhibition of either Na or Ca conductance alone. It was necessary to block both conductances to suppress action potentials. 2. Membrane currents generated by step depolarizations of the soma consisted of early transient and delayed steady-state currents. The early transient current could have one or two components depending on the activating depolarization. 3. The early more rapid component had a reversal potential at +54 mV and the reversal potential changed with extracellular Na concentration in accord with the Nernst equation. It was blocked by substitution of impermeant cations for Na, by TTX and by internal injections of Zn. It was concluded that this component was normally a Na current. 4. The later slower component of the transient current had a reversal potential at about +65 mV and the reversal potential changed with extracellular Ca concentration is accord with the Nernst equation. It was blocked by substitution of Mg for Ca or addition of Mn, Co, Ni or verapamil to the extracellular solution. It was concluded that this component was normally a Ca current. 5. Na and Ca currents were generated at different threshold potentials, Na currents first appearing at about -20 mV and Ca currents at -5 to 0 mV. 6. The time-to-peak of both Na and Ca currents was affected by the holding potential, by the litude of the activating depolarization, by temperature and by alent ion concentration. 7. The peak Na and Ca conductances both increased sigmoidally with increasing depolarization, the maximum Na conductance of 10--15 microS being approximately twice the maximum Ca conductance. Peak conductances for Na and Ca reached half-maximum at -8 and +3 mV, respectively. 8. The litude of the delayed steady-state current could be varied by changing the extracellular K+ ion concentration or by adding tetraethylammonium to the extracellular solution. The reversal potential for 'tail currents' was -67 mV and shifted 18 mV when the extracellular K concentration was doubled. It was concluded that the delayed steady-state current was K current. 9. With prolonged depolarizations, K current decayed with a time constant of the order of 1 sec. Peak K conductance increased with increasing depolarization with the half-maximum occurring at a potential more positive than +20 mV. The maximum rate of fractional activation of K conductance was independent of the litude of the cl step.
Conotoxin αD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors
Publisher: Springer Science and Business Media LLC
Date: 23-09-2015
DOI: 10.1038/SREP14261
Abstract: Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis , we identified a novel dimeric nAChR-inhibiting αD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of αD-GeXXA, GeXXA-CTD, retains inhibitory activity against the α9α10 nAChR subtype. Furthermore, we identified that His7 of the rat α10 nAChR subunit determines the species preference of αD-GeXXA and is probably part of the binding site of this toxin. These results together suggest that αD-GeXXA cooperatively binds to two inter-subunit interfaces on the top surface of nAChR, thus allosterically disturbing the opening of the receptor. The novel antagonistic mechanism of αD-GeXXA via a new binding site on nAChRs provides a valuable basis for the rational design of new nAChR-targeting compounds.
ω-Conotoxin inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.NEUROPHARM.2008.06.049
Abstract: A number of omega-conotoxins are potent and selective antagonists of N-type voltage-gated calcium channels (VGCCs) and are potentially effective as analgesic agents. omega-Conotoxins CVID and CVIB, venom peptides from Conus catus, inhibit N-type and N/P/Q-type VGCCs, respectively, in rat dorsal root ganglion sensory neurons. In the present study, we tested the effects of five different omega-conotoxins, CVID, CVIB, MVIIA, MVIIC and GVIA, on excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurons of rat spinal cord. The N-type VGCC antagonists CVID (200nM) and MVIIA (500nM) completely and irreversibly inhibited excitatory postsynaptic currents (EPSCs) in the dorsal horn superficial lamina. The N- and P/Q-type VGCC antagonist CVIB (200nM) reversibly reduced evoked EPSC litude an average of 34+/-8%, whereas MVIIC (200nM) had no effect on excitatory synaptic transmission. In neurons receiving polysynaptic input, CVIB reduced both the EPSC litude and the "success rate" calculated as the relative number of primary afferent stimulations that resulted in postsynaptic responses. These results indicate that (i) the analgesic action of omega-conotoxins that antagonise N-type VGCCs may be attributed to inhibition of neurotransmission between primary afferents and superficial dorsal horn neurons, (ii) nociceptive synaptic transmission between primary afferents and superficial lamina neurons is mediated predominantly by N-type VGCCs, and (iii) in contrast to the irreversible inhibition by CVID, MVIIA and GVIA, the inhibition of excitatory monosynaptic transmission by CVIB is reversible.
Determination of the α-Conotoxin Vc1.1 Binding Site on the α9α10 Nicotinic Acetylcholine Receptor
Publisher: American Chemical Society (ACS)
Date: 29-04-2013
DOI: 10.1021/JM400041H
Abstract: α-Conotoxin Vc1.1 specifically and potently inhibits the nicotinic acetylcholine receptor subtype α9α10 (α9α10 nAChR) and is a potential novel treatment for neuropathic pain. Here, we used a combination of computational modeling and electrophysiology experiments to determine the Vc1.1 binding site on the α9α10 nAChR. Interactions of Vc1.1 with two probable binding sites, α9α10 and α10α9, were modeled. Mutational energies calculated by assuming specific interactions in the α10α9 binding site correlated better with electrophysiological recordings than those assuming interactions with the α9α10 binding site. Two novel Vc1.1 analogues, [N9F]Vc1.1 and [N9W]Vc1.1, were predicted to have large differences in affinity between the two binding sites. Data from functional studies were consistent with computational predictions that assumed preferred binding of Vc1.1 to the α10α9 pocket. Moreover, our modeling study suggested that a single hydrogen bond formed between Vc1.1 and position 59 of the α10α9 pocket confers specificity to rat versus human α9α10 nAChRs.
Characteristics of sodium and calcium conductance changes produced by membrane depolarization in an Aplysia neurone.
Publisher: Wiley
Date: 04-1979
DOI: 10.1113/JPHYSIOL.1979.SP012729
Abstract: 1. The time course and voltage dependence of Na and Ca conductance changes produced by depolarization of the soma of the neurone R15 in the abdominal ganglion of Aplysia juliana were examined at temperatures of 10--14 degrees C. 2. During a maintained depolarization, Na currents turned on then decayed (inactivated). Inactivation was exponential with time constant tauh. Activation (after correction for inactivation) was reasonably well described by the expression G'Na(t) = G'Na (infinity) (1 - exp [-t/taum])3 over a wide range of potentials. 3. taum and tauh were both voltage dependent. In the range -20 to +40 mV, taum varied from 5 to 0.5 msec and tauh from 25 to 8 msec (13.5 degrees C). Steady-state Na conductance (corrected for inactivation) was voltage dependent also, increasing sigmoidally with depolarization to a maximum of 25--30 muS at +10 to +20 mV. Half-maximal Na conductance occurred at a membrane potential of -8 mV and from -15 to -5 mV, a 5 mV change in membrane potential produced an e-fold change in steady-state Na conductance. 4. Steady-state inactivation of Na conductance (hNa(infinity)) was voltage dependent with half-inactivation occurring at a membrane potential of -32 mV. Recovery from Na inactivation followed an exponential time course with a voltage-dependent time constant. 5. During a maintained depolarization Ca currents activated then decayed (inactivated) more slowly than Na currents. The decay was exponential with time constant tauH. The decay of Ca current was not an artifact porduced by an outward current. The litude of calcium tail currents, produced by voltage steps back to epsilonK at different times during the decay of ICa, decayed also with a time constant close to tauH. 6. Ca conductance (after correction for inactivation) could be described approximately by the expression G'Ca(t) = G'Ca(infinity) (1 - exp [-t/tauM])p but it was necessary to vary p from 1 to 2 at different potentials. No value of p gave as good a fit to this model as that obtained for Na currents. 7. taum and tauH were voltage dependent. In the range of potentials from 0 to +60 mV, tauM varied from 9 to 5 msec and tauH from 300 to 50 msec (13.5 degrees C). Steady-state Ca conductance (corrected for inactivation) was voltage dependent also, increasing sigmoidally with depolarization to a maximum of 10--15 muS at +30 to +40 mV. Half-maximal Ca conductance occurred at a membrane potential of +12 mV, and from +10 to +20 mV a 6 mV change in membrane potential produced an e-fold change in Ca conductance. 8. Steady-state inactivation of Ca conductance (hCa(infinity)) varied with holding potential (VH). Half-inactivation occurred with depolarization to -20 mV. At potentials more negative than -40 mV, hCa(infinity) was less than at -40 mV, i.e. hyperpolarization produced Ca 'inactivation'. 9...
Formulation and dosage of therapeutic nanosuspension for active targeting of docetaxel (WO 2014210485A1).
Publisher: Informa UK Limited
Date: 18-06-2016
DOI: 10.1080/13543776.2016.1180365
Abstract: Non-specificity and drug resistance are two major limitations of all chemotherapeutic agents. Ligand-conjugated nanomedicine is the most versatile approach for targeted cancer therapy. Attaching a targeting ligand to the nanoparticle surface increases drug concentration at the desired sites, decreases the dose needed and lessens side effects. The subject of this patent evaluation describes the preparation of a therapeutic nanosuspension of an anticancer drug, docetaxel (DTX). The nanoparticle matrix comprised a polylactic acid-polyethylene glycol block copolymer (PLA-PEG). The nanoparticles were actively directed towards prostate-specific membrane antigen (PSMA) over-expressing cancer cells using a targeting ligand S,S-2-{3-[1-carboxy-5-amino-pentyl-]ureido}-pantanedioic acid (GL2). The dose-limiting toxicity and maximum tolerated dose were determined for GL2-conjugated and DTX-loaded polymeric nanosuspensions. The efficacy of nanosuspensions was evaluated in people with various cancer types. The investigators claim the method of preparation of therapeutic nanosuspension, optimized composition of the formulation and dosage regimen for the clinical studies to effectively treat gastroesophageal and breast cancers.
Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by Go -protein subunits
Publisher: Society for Neuroscience
Date: 06-04-2005
DOI: 10.1523/JNEUROSCI.4971-04.2005
Abstract: G-protein modulation of neuronal nicotinic acetylcholine receptor (nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTPγS increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current litudes were increased approximately twofold in the presence of GTPγS. In inside-out membrane patches, the open probability (NP o ) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 m m GTPγS relative to control but was unchanged in the presence of GDPβS. The modulation of nAChR-mediated whole-cell currents was agonist specific currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTPγS. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G o α or Gβγ subunits to inside-out membrane patches and in glutathione S -transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 n m Gβγ increased the open probability of ACh-activated single-channel currents fivefold, whereas G o α (50 n m ) produced no significant increase in NP o . Neuronal nAChR subunits α3-α5 and β2 exhibited a positive interaction with G o α and Gβγ, whereas β4 and α7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.
Chemical modification of conotoxins to improve stability and activity
Publisher: American Chemical Society (ACS)
Date: 07-2007
DOI: 10.1021/CB700091J
Abstract: Conotoxins are small disulfide-rich peptides from the venom of cone snails. Along with other conopeptides, they target a wide range of membrane receptors, ion channels, and transporters, and because of their high potency and selectivity for defined subtypes of these receptors, they have attracted a great deal of attention recently as leads in drug development. However, like most peptides, conopeptides potentially suffer from the disadvantages of poor absorption, poor stability, or short biological half-lives. Recently, various chemical approaches, including residue substitutions, backbone cyclization, and disulfide-bridge modification, have been reported to increase the stability of conopeptides. These manufactured interventions add to the array of post-translational modifications that occur naturally in conopeptides. They enhance the versatility of these peptides as tools in neuroscience and as drug leads.
In vivo and in vitro testing of native α-conotoxins from the injected venom of Conus purpurascens
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.NEUROPHARM.2017.09.020
Abstract: α-Conotoxins inhibit nicotinic acetylcholine receptors (nAChRs) and are used as probes to study cholinergic pathways in vertebrates. Model organisms, such as Drosophila melanogaster, express nAChRs in their CNS that are suitable to investigate the neuropharmacology of α-conotoxins in vivo. Here we report the paired nanoinjection of native α-conotoxin PIA and two novel α-conotoxins, PIC and PIC[O7], from the injected venom of Conus purpurascens and electrophysiological recordings of their effects on the giant fiber system (GFS) of D. melanogaster and heterologously expressed nAChRs in Xenopus oocytes. α-PIA caused disruption of the function of giant fiber dorsal longitudinal muscle (GF-DLM) pathway by inhibiting the Dα7 nAChR a homolog to the vertebrate α7 nAChR, whereas PIC and PIC[O7] did not. PIC and PIC[O7] reversibly inhibited ACh-evoked currents mediated by vertebrate rodent (r)α1β1δγ, rα1β1δε and human (h)α3β2, but not hα7 nAChR subtypes expressed in Xenopus oocytes with the following selectivity: rα1β1δε > rα1β1δγ ≈ hα3β2 >> hα7. Our study emphasizes the importance of loop size and α-conotoxin sequence specificity for receptor binding. These studies can be used for the evaluation of the neuropharmacology of novel α-conotoxins that can be utilized as molecular probes for diseases such as, Alzheimer's, Parkinson's, and cancer. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'
Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine
Publisher: Wiley
Date: 09-2006
α-conotoxins PnIA and [A10L]PnIA stabilize different states of the α7-L247T nicotinic acetylcholine receptor
Publisher: Elsevier BV
Date: 07-2003
Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine
Publisher: Wiley
Date: 02-2005
(E)-3-Furan-2-yl-N-p-tolyl-acrylamide and its Derivative DM489 Decrease Neuropathic Pain in Mice Predominantly by α7 Nicotinic Acetylcholine Receptor Potentiation
Publisher: American Chemical Society (ACS)
Date: 19-10-2020
Identification of a Novel O-Conotoxin Reveals an Unusual and Potent Inhibitor of the Human α9α10 Nicotinic Acetylcholine Receptor
Publisher: MDPI AG
Date: 09-06-2017
DOI: 10.3390/MD15060170
Conotoxins and their potential pharmaceutical applications
Publisher: Wiley
Date: 03-1999
DOI: 10.1002/(SICI)1098-2299(199903/04)46:3/4<219::AID-DDR7>3.0.CO;2-S
Analgesic ω-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels (Molecular Pharmacology (2010) 77, (139-148))
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 14-07-2011
DOI: 10.1124/MOL.80.2.356
VIP and PACAP potentiation of nicotinic ACh-evoked currents in rat parasympathetic neurons is mediated by G-protein activation
Publisher: Wiley
Date: 07-2000
DOI: 10.1046/J.1460-9568.2000.00116.X
Abstract: The effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on isolated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined under voltage cl using whole-cell patch-cl recording techniques. VIP and PACAP (</= 10 nM) selectively and reversibly increased the affinity of nicotinic acetylcholine receptor channels (nAChRs) for their agonists resulting in a potentiation of acetylcholine (ACh)-evoked whole-cell currents at low agonist concentrations. VIP-induced potentiation was observed with either ACh or nicotine as the cholinergic agonist. The VIP- but not the PACAP-induced potentiation of ACh-evoked currents was inhibited by [Ac-Tyr1, D-Phe2]-GRF 1-29, amide (100 nM), a selective antagonist of VPAC1 and VPAC2 receptors whereas the PACAP38- but not the VIP-induced potentiation was inhibited by 100 nM PACAP6-38, a PAC1 and VPAC2 receptor antagonist. The signal transduction pathway mediating VIP- and PACAP-induced potentiation of nicotinic ACh-evoked currents involves a pertussis toxin (PTX)-sensitive G-protein. Intracellular application of 200 microM GTPgammaS or GDPbetaS inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. GTPgammaS alone potentiated ACh- and nicotine-evoked currents and the magnitude of these currents was not further increased by VIP or PACAP. The G-protein subtype modulating the neuronal nAChRs was examined by intracellular dialysis with antibodies directed against alphao, alphai-1,2, alphai-3 or beta G-protein subunits. Only the anti-Galphao and anti-Gbeta antibodies significantly inhibited the effect of VIP and PACAP on ACh-evoked currents. The potentiation of ACh-evoked currents by VIP and PACAP may be mediated by a membrane-delimited signal transduction cascade involving the PTX-sensitive Go protein.
Neural control of the heart: Developmental changes in ionic conductances in mammalian intrinsic cardiac neurons
Publisher: Elsevier BV
Date: 06-2002
DOI: 10.1016/S1566-0702(02)00036-X
Abstract: The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, Ih, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, IK(IR), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+ Using the perforated-patch recording configuration, an ATP-sensitive K+ (KATP) conductance was identified in > or = 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion.
Funnel web spider venom produces spontaneous action potentials in nerve
Publisher: Elsevier BV
Date: 1977
Identification of a Novel Class of Nicotinic Receptor Antagonists
Publisher: Elsevier BV
Date: 08-2006
Chemical and Functional Identification and Characterization of Novel Sulfated α-Conotoxins from the Cone Snail Conus anemone
Publisher: American Chemical Society (ACS)
Date: 29-01-2004
DOI: 10.1021/JM031010O
Abstract: An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.
Alanine Scan of α-Conotoxin RegIIA Reveals a Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist
Publisher: Elsevier BV
Date: 2015
Nicotinic acetylcholine receptors: Key targets for attenuating neurodegenerative diseases
Publisher: Elsevier BV
Date: 04-2023
Single-Disulfide Conopeptide Czon1107, an Allosteric Antagonist of the Human α3β4 Nicotinic Acetylcholine Receptor
Publisher: MDPI AG
Date: 31-07-2022
DOI: 10.3390/MD20080497
Abstract: Conopeptides are peptides in the venom of marine cone snails that are used for capturing prey or as a defense against predators. A new cysteine-poor conopeptide, Czon1107, has exhibited non-competitive inhibition with an undefined allosteric mechanism in the human (h) α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, the binding mode of Czon1107 to hα3β4 nAChR was investigated using molecular dynamics simulations coupled with mutagenesis studies of the peptide and electrophysiology studies on heterologous hα3β4 nAChRs. Overall, this study clarifies the structure–activity relationship of Czon1107 and hα3β4 nAChR and provides an important experimental and theoretical basis for the development of new peptide drugs.
Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 05-10-2015
Abstract: α-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The α3β2 and α3β4 nAChR subtypes are expressed in the central and peripheral nervous systems and play a critical role in various pathophysiological conditions ranging from nicotine addiction to the development and progression of lung cancer. Here we used the α4/7-conotoxin RegIIA, a disulfide-bonded peptide from the venom of Conus regius, and its analog [N11A,N12A]RegIIA to probe the specific pharmacological properties of rat and human nAChR subtypes. nAChR subtypes were heterologously expressed in Xenopus oocytes and two-electrode voltage cl recordings used to investigate the effects of the peptides on nAChR activity. RegIIA potently inhibited currents evoked by acetylcholine (ACh) at rat α3β2 (IC50 = 10.7 nM), whereas a 70-fold lower potency was observed at human α3β2 nAChR (IC50 = 704.1 nM). Conversely, there were no species-specific differences in sensitivity to RegIIA at the α3β4 nAChR. Receptor mutagenesis and molecular dynamics studies revealed that this difference can be attributed primarily to a single amino acid change: Glu198 on the rat α3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species- and subunit-specific receptor-antagonist interaction.
muO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits.
Publisher: Proceedings of the National Academy of Sciences
Date: 07-11-2006
Abstract: The tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Na v 1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that μO-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Na v 1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetrodotoxin-resistant current characteristic of Na v 1.8 but not Na v 1.9 or tetrodotoxin-sensitive VGSC currents. MrVIB blocked human Na v 1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03–3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Na v 1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.
Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider
Publisher: Proceedings of the National Academy of Sciences
Date: 24-01-2022
Abstract: Pain development and discomfort are universal features of spider envenomation, yet severe pain arising from bites by Old World spiders is poorly understood. Molecular analyses of the venom of the King Baboon spider revealed abundant expression of the inhibitory cystine knot peptide Pm1a. Synthetic Pm1a induces pain in mice while simultaneously enhancing proexcitatory sodium currents and decreasing inhibitory potassium currents. These concomitant effects promote hyperexcitability in pain-sensing neurons that can be reversed by pharmacological inhibition of voltage-gated sodium channels. The coordinated modulation of excitatory and inhibitory ion channels involved in pain propagation may represent an economical and effective defense strategy in pain-inducing defensive venoms.
Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors
Publisher: Springer Science and Business Media LLC
Date: 30-11-2017
DOI: 10.1038/S41598-017-16809-0
Abstract: Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [ 125 I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3β4, α4β4, α3β2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.
ω-Conotoxin CVID Inhibits a Pharmacologically Distinct Voltage-sensitive Calcium Channel Associated with Transmitter Release from Preganglionic Nerve Terminals
Publisher: Elsevier BV
Date: 02-2003
γ-Aminobutyric acid type B (GABAB ) receptor expression is needed for inhibition of N-type (Cav 2.2) calcium channels by analgesic α-conotoxins
Publisher: Elsevier BV
Date: 07-2012
The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells
Publisher: Society for Neuroscience
Date: 04-04-2007
DOI: 10.1523/JNEUROSCI.5060-06.2007
Abstract: Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX + ) cells more stringently, these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic, early postnatal, and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX high ) in the embryonic brain coexpressed the neuronal marker βIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro . However, the DCX + population from the early postnatal brain and the adult subventricular zone and hippoc us, which expressed low levels of DCX (DCX low ), was enriched for neurosphere-forming cells, with only a small subpopulation of these cells coexpressing the neuronal markers βIII-tubulin or microtubule-associated protein 2. Similarly, the DCX low population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX high cells, which were shown to be similar to the E14 DCX high cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX + cells, with some cells displaying characteristics of immature or mature neurons, whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX high cells, regardless of location, are restricted to the neuronal lineage or are bone fide neurons, whereas some DCX low cells retain their multipotentiality.
Analgesic ω-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 05-11-2010
Abstract: Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular ersity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.
Physiological roles of ion channels in adult neural stem cells and their progeny
Publisher: Wiley
Date: 16-06-2010
DOI: 10.1111/J.1471-4159.2010.06822.X
Abstract: Elucidation of the machinery of adult neurogenesis is indispensable for the treatment of neurodegenerative diseases by therapeutic drugs and/or by neural stem cell (NSC) transplantation. It is well known that membrane ion channels play a critical role in cell function, including proliferation, apoptosis and migration in a wide range of cells. In NSC research, interdisciplinary collaboration between cell biologists and membrane physiologists has been pursued principally to monitor ion channel and synaptic currents as a hallmark of neuronal differentiation and maturation of NSC progeny. Nevertheless, less attention had been paid to a functional role of ion channels in NSCs or their immature progeny. Recently, however, evidence regarding their functional relevance has started to accumulate. In focusing on the early stages of the neurogenic process during which NSCs give rise to neuroblasts, this review highlights the latent ability of ion channels to act as functional regulators of adult neurogenesis.
The relationship of brevetoxin 'length' and A-ring functionality to binding and activity in neuronal sodium channels
Publisher: Elsevier BV
Date: 08-1995
DOI: 10.1016/1074-5521(95)90187-6
Abstract: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential (ii) occurrence of subconductance states (iii) induction of longer mean open times of the channel and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.
Allosteric α1-Adrenoreceptor Antagonism by the Conopeptide ρ-TIA
Publisher: Elsevier BV
Date: 09-2003
Structure of α-conotoxin BuIA: influences of disulfide connectivity on structural dynamics
Publisher: Springer Science and Business Media LLC
Date: 20-04-2007
Abstract: α-Conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in α-conotoxins is variable, leading to the classification of sub-families. BuIA is the only α-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin. In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer. These findings are opposite to the general trends observed for α-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.
Automated patch clamp screening of amiloride and 5-N,N-hexamethyleneamiloride (HMA) analogs identifies 6-iodoamiloride as a potent acid-sensing ion channel inhibitor
Publisher: Cold Spring Harbor Laboratory
Date: 14-03-2022
DOI: 10.1101/2022.03.12.484055
Abstract: Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K + -sparing diuretic amiloride is a moderate non-specific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch-cl protocol and identified 6-iodoamiloride as a more potent ASIC1 inhibitor. Follow-up IC 50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 97 (hASIC1 97 IC 50 88 nM cf. amiloride 11 IC 50 1.7 μM). A similar improvement in activity was observed in ASIC3-mediated currents from rat small diameter dorsal root ganglion neurons (rDRG single-concentration 97 IC 50 230 nM cf. 11 IC 50 2.7 μM). 6-iodoamiloride represents the amiloride analogue of choice for studying the effects of ASIC inhibition on cell physiology.
Identification of a sensory neuron Cav2.3 inhibitor within a new superfamily of macro-conotoxins
Publisher: Cold Spring Harbor Laboratory
Date: 04-07-2022
DOI: 10.1101/2022.07.04.498665
Abstract: Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, that defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and two additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of voltage-gated ion channels, Mu8.1 preferentially inhibited the R-type (Cav2.3) calcium channel. Ca 2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K + (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study demonstrates the feasibility of large, disulfide-rich venom-component investigation, an endeavor that will lead to the discovery of novel structures and functions in the previously underexplored group of macro-conotoxins.
Analgesic α-conotoxins modulate GIRK1/2 channels via GABAB receptor activation and reduce neuroexcitability
Publisher: Cold Spring Harbor Laboratory
Date: 04-12-2020
DOI: 10.1101/2020.12.02.407627
Abstract: Activation of G protein-coupled inwardly rectifying potassium (GIRK or Kir3) channels leads to membrane hyperpolarization and d ening of neuronal excitability. Here we show that the analgesic α-conotoxin Vc1.1 potentiates inwardly rectifying K + currents (I Kir ) mediated through native and recombinant GIRK1/2 channels by activation of the G protein-coupled GABA B receptor (GABA B R) via a Pertussis toxin (PTX)-sensitive G protein. Recombinant co-expression of human GIRK1/2 subunits and GABA B R in HEK293T cells resulted in a Ba 2+ -sensitive I Kir potentiated by baclofen and Vc1.1 which was inhibited by PTX, intracellular GDP-β-S, or the GABA B R-selective antagonist CGP 55845. In adult mouse DRG neurons, GABA B R-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell resting membrane potential with concomitant reduction of excitability consistent with Vc1.1 and baclofen analgesic effects in vivo . This study provides new insight into Vc1.1 as an allosteric agonist for GABA B R-mediated potentiation of GIRK channels and may aid in the development of novel non-opioid treatments for chronic pain.
Voltage-Gated R-Type Calcium Channel Inhibition via Human μ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gbg Protein Subunits
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 21-10-2016
Abstract: Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of μ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa litude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the ersity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner.
Localization of Nav1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction, pheromone sensing and immune function
Publisher: Informa UK Limited
Date: 03-2012
DOI: 10.4161/CHAN.19484
Abstract: Loss-of-function mutations in the pore-forming α subunit of the voltage-gated sodium channel 1.7 (Nav 1.7) cause congenital indifference to pain and anosmia. We used immunohistochemical techniques to study Nav 1.7 localization in the rat olfactory system in order to better understand its role in olfaction. We confirm that Nav 1.7 is expressed on olfactory sensory axons and report its presence on vomeronasal axons, indicating an important role for Nav 1.7 in transmission of pheromonal cues. Following neuroepithelial injury, Nav 1.7 was transiently expressed by cells of monocytic lineage. These findings support an emerging role for Nav 1.7 in immune function. This sodium channel may provide an important pharmacological target for treatment of inflammatory injury and inflammatory pain syndromes.
Analgesic α‐conotoxins modulate native and recombinant GIRK1/2 channels via activation of GABAB receptors and reduce neuroexcitability
Publisher: Wiley
Date: 25-11-2022
DOI: 10.1111/BPH.15690
Abstract: Activation of GIRK channels via G protein‐coupled GABA B receptors has been shown to attenuate nociceptive transmission. The analgesic α‐conotoxin Vc1.1 activates GABA B receptors resulting in inhibition of Ca v 2.2 and Ca v 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α‐conotoxins on recombinant and native GIRK‐mediated K + currents and on neuronal excitability. The effects of analgesic α‐conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly‐rectifying K + currents in HEK293T cells recombinantly co‐expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABA B receptors. The effects of α‐conotoxin Vc1.1 and baclofen were studied on GIRK‐mediated K + currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons. Analgesic α‐conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly‐rectifying K + currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABA B receptors. GABA B receptor‐dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin‐sensitive G protein and is inhibited by the selective GABA B receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABA B receptor‐dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability. This is the first report of GIRK channel potentiation via allosteric α‐conotoxin Vc1.1‐GABA B receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo.
Divalent ion currents and the delayed potassium conductance in an Aplysia neurone.
Publisher: Wiley
Date: 07-1980
DOI: 10.1113/JPHYSIOL.1980.SP013325
Abstract: 1. In Na- and Ca-free external solutions, Sr or Ba (but not Mg) could act as carriers of inward current during action potentials in the neurone, R15 of the Aplysia abdominal ganglion. These action potentials exhibited a prolonged plateau phase, the duration of which was dependent on the concentration and species of alent cation and activity of the neurone. 2. Depolarization of the soma membrane in Na-free Ba solution generated a prolonged, 'late' inward current the litude of which was dependent on the external Ba concentration. The Ba current was insensitive to tetrodotoxin but could be blocked by Mn2+ and Co2+ ions. 3. The peak current-voltage relation and threshold for activation of the late inward current was shifted to more negative potentials on replacement of Ca with Ba. The zero-current (reversal) potentials for both Sr and Ba were more negative than for Ca, indicating that the 'Ca' channel is less permeable to Sr2+ or Ba2+ ions than to Ca2+ ions. 4. Inactivation of the 'Ca' channel is slower in Ba than in Ca solution. The time course of Ba currents during a maintained depolarization of 2 sec could be reasonably described by the expression, I'Ba(t) = I'Ba (infinity) [1-exp(-t/tau M)]2exp(-t/tau H). 5. Time constants for activation (tau M) and inactivation (tau H) were voltage-dependent. In the range -10 to +30 mV, tau M varied from 15 to 5 msec and tau H from 2.0 to 0.5 sec (12 degrees C). Steady-state Ba conductance (corrected for inactivation) was voltage-dependent, increasing sigmoidally with depolarization to a maximum of approximately 12 microS at potentials beyond +15 mV. 6. Steady-state inactivation of Ba conductance (hBa(infinity)) varied with holding potential (VH). Conditioning holding potentials more negative than the resting potential (-40 to -50 mV) produced depression of Ba currents. Complete inactivation of Ba currents occurred at holding potentials more positive than 0 mV or with repetitive activation at frequencies greater than 1 Hz. 7. The alent ions, Ba2+ and Sr2+, reversible depressed the total delayed K+ current at a rate dependent on the frequency of activation. Ba and Sr shifted the delayed K+ current-voltage curve to more positive voltages and depressed the delayed outward current at all membrane potentials. 8. Comparison of the effect of Ba on delayed K+ currents with those obtained in the presence of Mn2+ ions indicated that Ba2+ ions depress both the voltage-dependent and Ca-dependent components of the delayed K+ current. However, the mechanism by which Ba acts to inhibit the two components of the delayed K+ current appears to be different.
Isolation, characterization and total regioselective synthesis of the novel μo-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.BCP.2012.05.008
Abstract: The μO-conotoxins are notable for their unique selectivity for Na(v)1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na(v)1.8 (IC₅₀ 95.9±74.3 nM) and Na(v)1.4 (IC₅₀ 81±16 nM), with significantly lower affinity for other Na(v) subtypes (IC₅₀ 431-6203 nM Na(v)1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management.
The tarantula toxin β/δ-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity
Publisher: Springer Science and Business Media LLC
Date: 20-04-2017
DOI: 10.1038/S41598-017-01129-0
Abstract: Voltage-gated sodium (Na V ) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between Na V channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a’s potent and selective inhibitory effect on the fast inactivation process of Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the erse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.
Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1
Publisher: Springer Science and Business Media LLC
Date: 20-08-2015
DOI: 10.1038/SREP13264
Abstract: Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC 50 of 13 μM and rat N-type (Ca v 2.2) and recombinant human Ca v 2.3 calcium channels via GABA B receptor activation, with an IC 50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.
Single amino acid substitutions in α-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor
Publisher: Elsevier BV
Date: 12-1999
α-Conotoxin Dendrimers Have Enhanced Potency and Selectivity for Homomeric Nicotinic Acetylcholine Receptors
Publisher: American Chemical Society (ACS)
Date: 03-03-2015
DOI: 10.1021/JACS.5B00244
Abstract: Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.
Intravenous anaesthetics inhibit nicotinic acetylcholine receptor‐mediated currents and Ca2+ transients in rat intracardiac ganglion neurons
Publisher: Wiley
Date: 2005
P2Y purinoceptor activation mobilizes intracellular Ca2+ and induces a membrane current in rat intracardiac neurones
Publisher: Wiley
Date: 07-2000
DOI: 10.1111/J.1469-7793.2000.00287.X
Abstract: 1. The mobilization of Ca2+ by purinoceptor activation and the relative contributions of intra- and extracellular sources of Ca2+ were investigated using microfluorimetric measurements of fura-2 loaded in cultured neurones from rat intracardiac ganglia. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of mRNA for the G protein-coupled P2Y2 and P2Y4 receptors. 3. Brief application of either 300 microM ATP or 300 microM UTP caused transient increases in [Ca2+]i of 277 +/- 22 nM and 267 +/- 39 nM, respectively. Removal of external Ca2+ did not significantly reduce these [Ca2+]i responses. 4. The order of purinoceptor agonist potency for [Ca2+]i increases was ATP = UTP > 2-MeSATP > ADP >> adenosine, consistent with the profile for P2Y2 purinoceptors. ATP- and UTP-induced rises in [Ca2+]i were completely and reversibly blocked by 10 microM PPADS (a P2 purinoceptor antagonist) and partially inhibited by 100 microM suramin (a relatively non-specific purinoceptor antagonist). 5. In the presence of the endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (10 microM) in Ca2+-free media, the [Ca2+]i responses evoked by ATP were progressively decreased and abolished. 6. ATP- and UTP-induced [Ca2+]i rises were insensitive to pertussis toxin, caffeine (5 mM) and ryanodine (10 microM) but were significantly reduced by U-73122, a phospholipase C (PLC) inhibitor. 7. In fura-2-loaded cells, perforated patch whole-cell recordings show that ATP and UTP evoked slow outward currents at -60 mV, concomitant with the rise in [Ca2+]i, in approximately 30 % of rat intracardiac neurones. 8. In conclusion, these results suggest that in r intracardiac neurones, ATP binds to P2Y2 purinoceptors to transiently raise [Ca2+]i and activate an outward current. The signalling pathway appears to involve a PTX-insensitive G protein coupled to PLC generation of IP3 which triggers the release of Ca2+ from a ryanodine-insensitive Ca2+ store(s).
Structure–Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1
Publisher: American Chemical Society (ACS)
Date: 10-05-2018
DOI: 10.1021/ACSCHEMBIO.8B00190
Abstract: α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABA
Cardiac vagal ganglia
Publisher: Elsevier
Date: 2022
Effects of Cyclization on Stability, Structure, and Activity of α-Conotoxin RgIA at the α9α10 Nicotinic Acetylcholine Receptor and GABAB Receptor
Publisher: American Chemical Society (ACS)
Date: 15-09-2011
DOI: 10.1021/JM201060R
Abstract: α-Conotoxin RgIA is of interest as a lead in the development of drugs for neuropathic pain. It modulates the α9α10 nicotinic acetylcholine receptor (nAChR) and the GABA(B) receptor, both of which are implicated in antinociception. However, because of its peptidic nature, RgIA is potentially susceptible to generic problems encountered by peptide-based drugs of poor oral bioavailability, short biological half-life, and low stability. Here, we improved the biopharmaceutical properties of RgIA by backbone cyclization using 3-7 residue peptidic linkers. Cyclization with a six-residue linker does not perturb the overall structure of RgIA, improves selectivity for the GABA(B) receptor over the α9α10 nAChR, and improves stability in human serum. The results provide insights to further improve the therapeutic properties of RgIA and other conotoxins being considered as drug leads and confirm that cyclization is a readily applicable strategy to improve the stability of peptides with proximate N- and C-termini.
Functional maturation of isolated neural progenitor cells from the adult rat hippocampus
Publisher: Wiley
Date: 29-04-2004
Distinct activities of novel neurotoxins from Australian venomous snakes for nicotinic acetylcholine receptors
Publisher: Springer Science and Business Media LLC
Date: 10-2007
DOI: 10.1007/S00018-007-7352-Z
Abstract: Envenomation from Australian elapid snakes results in a myriad of neurological effects due to post-synaptic neurotoxins that bind and inhibit nicotinic acetylcholine receptors (nAChRs) of neurons and muscle fibres. However, despite the significant physiological effects of these toxins, they have remained largely undercharacterised at the molecular level. This study describes the identification and comparative analysis of multiple neurotoxin isoforms from ten Australian snakes, including functional characterisation of two of these isoforms, Os SNTX-1 from Oxyuranus scutellatus and the more potent Pt LNTX-1 from Pseudonaja textilis. Electrophysiological recordings from adrenal chromaffin cells demonstrate that both neurotoxins act as competitive antagonists of nAChRs in a concentration-dependent manner. Their effects upon spontaneous and nerve-evoked membrane responses at the hibian neuromuscular junction provide further evidence that both toxins bind muscle nAChRs in an irreversible manner. This study represents one of the most comprehensive descriptions to date of the sequences and activity of in idual Australian elapid neurotoxins.
Neuronal voltage-gated sodium channel subtypes: key roles in inflammatory and neuropathic pain.
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.BIOCEL.2006.06.008
Abstract: Voltage-gated sodium channels (VGSCs) play an important role in neuronal excitability. Regulation of VGSC activity is a complex phenomenon that occurs at multiple levels in the cell, including transcriptional regulation, post-translational modification and membrane insertion and retrieval. Multiple VGSC subtypes exist that vary in their biophysical and pharmacological properties and tissue distribution. Any alteration of the VGSC subtype profile of a neuron or the mechanisms that regulate VGSC activity can cause significant changes in neuronal excitability. Inflammatory and neuropathic pain states are characterised by alterations in VGSC subtype composition and activity in sensory neurons. This review focuses on the VGSC subtypes involved in such pain states.
Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2.
Publisher: Elsevier BV
Date: 07-2004
Conotoxin modulation of voltage-gated sodium channels.
Publisher: Elsevier BV
Date: 2007
DOI: 10.1016/J.BIOCEL.2007.08.017
Abstract: The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the microO-conotoxins which inhibit VGSC Na+ conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the microO-conotoxins, which may develop into potential drug leads for the treatment of pain states.
α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors.
Publisher: BMJ
Date: 17-02-2016
N-acetyl-d-glucosamine-conjugated PAMAM dendrimers as dual receptor-targeting nanocarriers for anticancer drug delivery.
Publisher: Elsevier BV
Date: 09-2020
Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor
Publisher: Elsevier BV
Date: 07-2009
Kv 3.1 channels stimulate adult neural precursor cell proliferation and neuronal differentiation
Publisher: Wiley
Date: 12-04-2013
Chemical synthesis and folding of APETx2, a potent and selective inhibitor of acid sensing ion channel 3
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.TOXICON.2009.03.014
Abstract: Acid sensing ion channels (ASICs) are pH-sensitive channels that are distributed in the central and peripheral nervous system and which are believed to play a key role in pain perception. APETx2, a 42-residue peptide toxin isolated from the sea anemone Anthopleura elegantissima, is the only known selective inhibitor of ASIC3 channels. Here we describe the total chemical synthesis of APETx2 by solid-phase peptide synthesis and native chemical ligation. The folded synthetic toxin had an IC(50) of 57 nM for inhibition of rat ASIC3 channels expressed in Xenopus oocytes, in agreement with the IC(50) reported for the native toxin (63 nM). The native chemical ligation approach should provide an efficient route for synthesis of other pharmacologically useful disulfide-rich toxins from venomous animals.
Improving efficacy, oral bioavailability, and delivery of paclitaxel using protein-grafted solid lipid nanoparticles
Publisher: American Chemical Society (ACS)
Date: 13-10-2016
DOI: 10.1021/ACS.MOLPHARMACEUT.6B00691
Abstract: Oral delivery of anticancer drugs remains challenging despite the most convenient route of drug administration. Hydrophobicity and nonspecific toxicities of anticancer agents are major impediments in the development of oral formulation. In this study, we developed wheat germ agglutinin (WGA)-conjugated, solid lipid nanoparticles to improve the oral delivery of the hydrophobic anticancer drug, paclitaxel (PTX). This study was focused to improve the PTX loading in biocompatible lipid matrix with high bioconjugation efficiency. WGA-conjugated, PTX-loaded solid lipid nanoparticles (LPSN) exhibited enhanced anticancer activity against A549 lung cancer cells after internalization through lectin receptors than free PTX. Biodistribution studies in rats revealed that LPSN significantly improved the oral bioavailability and lung targetability of PTX, which could be due to cumulative bioadhesive property of the nanocarrier system and the targeting ligand WGA.
Erratum: Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca 2+ transients in rat intracardiac ganglion neurons (British Journal of Pharmacology (2005) 144 (98-107) doi:10.1038/sj.bjp.0705942)
Publisher: Wiley
Date: 2005
Reactive oxygen species modulate neuronal excitability in rat intrinsic cardiac ganglia
Publisher: Elsevier BV
Date: 10-2009
Trastuzumab-grafted PAMAM dendrimers for the selective delivery of anticancer drugs to HER2-positive breast cancer
Publisher: Springer Science and Business Media LLC
Date: 07-04-2016
DOI: 10.1038/SREP23179
Abstract: Approximately 20% of breast cancer cases are human epidermal growth factor receptor 2 (HER2)-positive. This type of breast cancer is more aggressive and tends to reoccur more often than HER2-negative breast cancer. In this study, we synthesized trastuzumab (TZ)-grafted dendrimers to improve delivery of docetaxel (DTX) to HER2-positive breast cancer cells. Bioconjugation of TZ on the surface of dendrimers was performed using a heterocrosslinker, MAL-PEG-NHS. For imaging of cancer cells, dendrimers were also conjugated to fluorescein isothiocyanate. Comparative in vitro studies revealed that these targeted dendrimers were more selective, and had higher antiproliferation activity, towards HER2-positive MDA-MB-453 human breast cancer cells than HER2-negative MDA-MB-231 human breast cancer cells. When compared with unconjugated dendrimers, TZ-conjugated dendrimers also displayed higher cellular internalization and induction of apoptosis against MDA-MB-453 cells. Binding of TZ to the dendrimer surface could help site-specific delivery of DTX and reduce systemic toxicity resulting from its lack of specificity. In addition, in vivo studies revealed that the pharmacokinetic profile of DTX was significantly improved by the conjugated nanosystem.
α‐Conotoxins active at α3‐containing nicotinic acetylcholine receptors and their molecular determinants for selective inhibition
Publisher: Wiley
Date: 11-06-2017
DOI: 10.1111/BPH.13852
Analgesic conotoxins: Block and G protein-coupled receptor modulation of N-type (Ca V 2.2) calcium channels
Publisher: Wiley
Date: 13-04-2012
Analgesic alpha-conotoxins Vc1.1 and RgIA inhibit N-type calcium channels in sensory neurons of alpha9 nicotinic receptor knockout mice.
Publisher: Informa UK Limited
Date: 2010
Abstract: Alpha-conotoxins Vc1.1 and RgIA are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. We have reported previously that the alpha9alpha10 nicotinic acetylcholine receptor (nAChR) selective-conotoxins Vc1.1 and RgIA potently and selectively inhibit high voltage-activated (HVA) N-type calcium channel currents in dissociated neurons from rat dorsal root ganglia (DRG). Our data indicated that Vc1.1 does not interact directly with N-type Ca(2+) channels but inhibits them via GABA(B) receptor activation. The present study investigated Vc1.1 and RgIA inhibition of N-type Ca(2+) channels currents in DRG neurons of wild-type and alpha9 knockout (KO) mice to determine if the alpha9 nAChR was necessary for inhibition of the Ca(2+) channel current. Application of Vc1.1 (100 nM) inhibited N-type Ca(2+) channel currents to 69.2 +/- 3.5% of control in DRG neurons isolated from wild-type mice. In >70% of DRG neurons isolated from the alpha9 KO mice, both Vc1.1 and RgIA selectively inhibited N-type Ca(2+) channel currents with an IC(50) of 24.6 nM and 22.4 nM, respectively. The GABA(B) receptor antagonist CGP55845 (1 microM) antagonized the effect of Vc1.1 and RgIA on the N-type calcium channels in alpha9 KO mice. RT-PCR and western blot analysis confirmed the absence of the alpha9 nAChR in mice carrying a null mutation for the nAChR alpha9 gene. These results demonstrate that the inhibition of N-type Ca(2+) channel channels by Vc1.1 and RgIA is not mediated by the expression of alpha9alpha10 nAChRs in DRG neurons.
Neuropharmacology of venom peptides
Publisher: Elsevier BV
Date: 12-2017
A Distinct Functional Site in Ω-Neurotoxins: Novel Antagonists of Nicotinic Acetylcholine Receptors from Snake Venom
Publisher: American Chemical Society (ACS)
Date: 15-10-2015
DOI: 10.1021/ACSCHEMBIO.5B00492
Abstract: Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: Ω-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of α-neurotoxins are not conserved. We evaluated the subtype specificity and structure-function relationships of Oh9-1, an Ω-neurotoxin from Ophiophagus hannah venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in Xenopus oocytes indicated Oh9-1 is selective for rat muscle type α1β1εδ (adult) and α1β1γδ (fetal) and rat neuronal α3β2 subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve in idual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to α1β1εδ and α3β2 subtypes. Oh9-1's loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at α1β1εδ receptors but had no effect on the interaction with the α3β2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike α-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the β-strand of Oh9-1's loop-II interact with α1β1εδ, but only one side interacts with α3β2. Phylogenetic analysis revealed functional organization of the Ω-neurotoxins independent of α-neurotoxins. Thus, Ω-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like α-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh binding pocket via a different set of functional residues.
Monoclonal antibody-conjugated dendritic nanostructures for siRNA delivery
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-9220-1_14
Abstract: Small interfering RNA (siRNA) is a promising tool for gene therapy-based disease treatments. However, delivery of siRNA to the target cells requires a specific and reliable carrier system. Herein we describe a targeted carrier system that can deliver siRNA to cancer cells overexpressing the human epidermal growth factor 2 (HER2) receptor. Trastuzumab-conjugated poly(amido)amine dendrimers can be synthesized using the protocols described here.
ACh- and caffeine-induced Ca2+ mobilization and current activation in rabbit arterial endothelial cells
Publisher: American Physiological Society
Date: 11-1998
DOI: 10.1152/AJPHEART.1998.275.5.H1748
Abstract: Fura 2 microfluorometry and perforated-patch whole cell recording were carried out simultaneously to investigate the relationship between intracellular free Ca 2+ concentration ([Ca 2+ ] i ) and membrane current activation in response to ACh and caffeine in freshly dissociated arterial endothelial cells. ACh and caffeine evoked transient increases in [Ca 2+ ] i . The initial increase in [Ca 2+ ] i was accompanied by a transient outward current, which caused membrane hyperpolarization. The litudes of the [Ca 2+ ] i transient and outward current were dependent on caffeine concentration (EC 50 ∼ 1 mM). Cyclopiazonic acid raised resting [Ca 2+ ] i levels by ≥50 nM and failed to completely block caffeine- or ACh-induced [Ca 2+ ] i transients but slowed [Ca 2+ ] i recovery fourfold. The reversal potential of caffeine-induced currents was dependent on external K + and Cl − concentrations. Caffeine-induced current litudes, but not [Ca 2+ ] i responses, were attenuated by external tetraethylammonium, Zn 2+ , and La 3+ . A consistent temporal relationship between agonist-activated membrane current and [Ca 2+ ] i increases was not observed, and, in some cases, time differences were greater than expected for simple diffusion of Ca 2+ throughout the cell. These results suggest that Ca 2+ -dependent current activation monitors local [Ca 2+ ] i changes adjacent to the plasmalemma, whereas single-cell photometry provides a measure of global changes in [Ca 2+ ] i .
Calcium‐activated potassium channels in native endothelial cells from rabbit aorta: conductance, Ca2+ sensitivity and block.
Publisher: Wiley
Date: 09-1992
DOI: 10.1113/JPHYSIOL.1992.SP019318
Abstract: 1. Isolated native endothelial cells, obtained by treatment of rabbit aortic endothelium with papain and dithiothreitol, were voltage cl ed, and single channel (unitary) and spontaneous transient outward currents (STOCs) were recorded from both whole cells and excised membrane patches. 2. In inside-out patches, the reversal potential of unitary currents was dependent on the extracellular K+ concentration and had a single-channel slope conductance of 220 pS in symmetrical 140 mM-K+ solutions. The open-state probability (Po) of the unitary K+ currents was sensitive to the intracellular Ca2+ concentration with half-maximal activation at approximately 1 microM at +20 mV. The ionic selectivity and Ca2+ sensitivity indicate that a large conductance, Ca(2+)-activated K+ channel is present in freshly dissociated rabbit aortic endothelial cells. 3. The frequency and litude of whole-cell unitary currents and litude of spontaneous transient outward currents were voltage-dependent. Whole-cell outward K+ currents evoked by depolarizing voltage r s had litudes often corresponding to the simultaneous opening of more than five single Ca(2+)-activated K+ channels. Lowering the intracellular EGTA concentration tenfold, and hence the Ca2+ buffering capacity of the cell, increased unitary K+ current activity and shifted the relationship between Po and membrane potential by approximately -20 mV. 4. Bradykinin (1 microM), adenosine 5'-triphosphate (3 microM) and acetylcholine (3 microM) applied extracellularly evoked a biphasic increase in N Po (where N is number of channels activated) of the Ca(2+)-activated K+ channel studied in the whole-cell recording configuration. The development of a biphasic response to agonist stimulation requires a source of extracellular Ca2+. The sustained increase in N Po of the Ca(2+)-activated K+ channel was attenuated upon the removal of external Ca2+ (Mg2+ replacement) or in the presence of the Ca2+ entry blocker, Ni2+, and the potassium channel blockers tetrabutylammonium (TBA) or tetraethylammonium (TEA). 5. Unitary and spontaneous transient outward currents were inhibited by extracellularly applied TEA (0.5 mM), TBA (0.5-5 mM) and charybdotoxin (100 nM). Ca(2+)-activated K+ currents were blocked completely by 5 mM-TEA, whereas 3,4-diaminopyridine (1 mM), Ba2+ (10 mM) and apamin (0.1-1 microM) did not abolish these K+ currents. 6. The K+ channel opener cromakalim (10 microM) evoked a sustained increase in N Po of the Ca(2+)-activated K+ channels which was not potentiated by the addition of bradykinin. Glibenclamide (10 microM) alone increased N Po and partially inhibited the cromakalim-induced increase in N Po with respect to control.(ABSTRACT TRUNCATED AT 400 WORDS)
Kir and Kv channels regulate electrical properties and proliferation of adult neural precursor cells
Publisher: Elsevier BV
Date: 02-2008
DOI: 10.1016/J.MCN.2007.10.003
Abstract: The functional significance of the electrophysiological properties of neural precursor cells (NPCs) was investigated using dissociated neurosphere-derived NPCs from the forebrain subventricular zone (SVZ) of adult mice. NPCs exhibited hyperpolarized resting membrane potentials, which were depolarized by the K(+) channel inhibitor, Ba(2+). Pharmacological analysis revealed two distinct K(+) channel families: Ba(2+)-sensitive K(ir) channels and tetraethylammonium (TEA)-sensitive K(v) (primarily K(DR)) channels. Ba(2+) promoted mitogen-stimulated NPC proliferation, which was mimicked by high extracellular K(+), whereas TEA inhibited proliferation. Based on gene and protein levels in vitro, we identified K(ir)4.1, K(ir)5.1 and K(v)3.1 channels as the functional K(+) channel candidates. Expression of these K(+) channels was immunohistochemically found in NPCs of the adult mouse SVZ, but was negligible in neuroblasts. It therefore appears that expression of K(ir) and K(v) (K(DR)) channels in NPCs and related changes in the resting membrane potential could contribute to NPC proliferation and neuronal lineage commitment in the neurogenic microenvironment.
μ-Theraphotoxin-Pn3a inhibition of CaV3.3 channels reveals a novel isoform-selective drug binding site
Publisher: Cold Spring Harbor Laboratory
Date: 04-10-2021
DOI: 10.1101/2021.10.04.463006
Abstract: Low voltage-activated calcium currents are mediated by T-type calcium channels Ca V 3.1, Ca V 3.2, and Ca V 3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. Ca V 3 isoforms’ biophysical properties, overlapping expression and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. Notably, Ca V 3.3’s contribution to normal and pathophysiological function has remained largely unexplored. We have identified Pn3a as the first subtype-selective spider venom peptide inhibitor of Ca V 3.3, with -fold lower potency against the other T-type isoforms. Pn3a modifies Ca V 3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing Ca V 3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of K V 1.7 channels bearing voltage sensor sequences from all four Ca V 3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (Ca V 3.3 DII ). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a into all T-type channel isoforms highlighting it as subtype-specific pharmacophore with therapeutic potential. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of Ca V 3.3 channels.
α-Conotoxin Vc1.1 Structure–Activity Relationship at the Human α9α10 Nicotinic Acetylcholine Receptor Investigated by Minimal Side Chain Replacement
Publisher: American Chemical Society (ACS)
Date: 14-08-2019
DOI: 10.1021/ACSCHEMNEURO.9B00389
Abstract: α-Conotoxin Vc1.1 inhibits the nicotinic acetylcholine receptor (nAChR) α9α10 subtype and has the potential to treat neuropathic chronic pain. To date, the crystal structure of Vc1.1-bound α9α10 nAChR remains unavailable thus, understanding the structure-activity relationship of Vc1.1 with the α9α10 nAChR remains challenging. In this study, the Vc1.1 side chains were minimally modified to avoid introducing large local conformation perturbation to the interactions between Vc1.1 and α9α10 nAChR. The results suggest that the hydroxyl group of Vc1.1, Y10, forms a hydrogen bond with the carbonyl group of α9 N107 and a hydrogen bond donor is required. However, Vc1.1 S4 is adjacent to the α9 D166 and D169, and a positive charge residue at this position increases the binding affinity of Vc1.1. Furthermore, the carboxyl group of Vc1.1, D11, forms two hydrogen bonds with α9 N154 and R81, respectively, whereas introducing an extra carboxyl group at this position significantly decreases the potency of Vc1.1. Second-generation mutants of Vc1.1 [S4 Dab, N9A] and [S4 Dab, N9W] increased potency at the α9α10 nAChR by 20-fold compared with that of Vc1.1. The [S4 Dab, N9W] mutational effects at positions 4 and 9 of Vc1.1 are not cumulative but are coupled with each other. Overall, our findings provide valuable insights into the structure-activity relationship of Vc1.1 with the α9α10 nAChR and will contribute to further development of more potent and specific Vc1.1 analogues.
The three‐dimensional structure of the analgesic α‐conotoxin, RgIA
Publisher: Wiley
Date: 31-01-2008
DOI: 10.1016/J.FEBSLET.2008.01.027
Abstract: The alpha-conotoxin RgIA is a selective antagonist of the alpha9alpha10 nicotinic acetylcholine receptor and has been shown to be a potent analgesic and reduces nerve injury associated inflammation. RgIA was chemically synthesized and found to fold into two disulfide isomers, globular and ribbon. The native globular isomer inhibited ACh-evoked currents reversibly in oocytes expressing rat alpha9alpha10 nAChRs but the ribbon isomer was inactive. We determined the three-dimensional structure of RgIA using NMR methods to assist in elucidating the molecular role of RgIA in analgesia and inflammation.
ω‐Conotoxin CVIB differentially inhibits native and recombinant N‐ and P/Q‐type calcium channels
Publisher: Wiley
Date: 2007
DOI: 10.1111/J.1460-9568.2006.05299.X
Abstract: Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.
Liposome reconstitution and modulation of recombinant N-methyl-D-aspartate receptor channels by membrane stretch
Publisher: Proceedings of the National Academy of Sciences
Date: 30-01-2007
Abstract: In this study, the heteromeric N -methyl- d -aspartate (NMDA) receptor channels composed of NR1a and NR2A subunits were expressed, purified, reconstituted into liposomes, and characterized by using the patch cl technique. The protein exhibited the expected electrophysiological profile of activation by glutamate and glycine and internal Mg 2+ blockade. We demonstrated that the mechanical energy transmitted to membrane-bound NMDA receptor channels can be exerted directly by tension developed in the lipid bilayer. Membrane stretch and application of arachidonic acid potentiated currents through NMDA receptor channels in the presence of intracellular Mg 2+ . The correlation of membrane tension induced by either mechanical or chemical stimuli with the physiological Mg 2+ block of the channel suggests that the synaptic transmission can be altered if NMDA receptor complexes experience local changes in bilayer thickness caused by dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the cell membranes. The ability to study gating properties of NMDA receptor channels in artificial bilayers should prove useful in further study of structure–function relationships and facilitate discoveries of new therapeutic agents for treatment of glutamate-mediated excitotoxicity or analgesic therapies.
α-conotoxin EpI, a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors
Publisher: Elsevier BV
Date: 06-1998
Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain
Publisher: Elsevier BV
Date: 06-2012
DOI: 10.1016/J.NEUROPHARM.2012.01.016
Abstract: The large ersity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the α-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of α-conotoxins. Intrathecal administration of α-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 h without significant side effects. In vitro patch-cl electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA(B)-dependent mechanism, and is more likely related to their action at nAChRs containing combinations of α3, α7 or other subunits. Intrathecal nAChR subunit-selective conotoxins are therefore promising tools for the effective treatment of neuropathic pain.
Fulditoxin, representing a new class of dimeric snake toxins, defines novel pharmacology at nicotinic ACh receptors.
Publisher: Wiley
Date: 09-02-2020
DOI: 10.1111/BPH.14954
Ionic currents in molluscan soma
Publisher: Annual Reviews
Date: 03-1980
DOI: 10.1146/ANNUREV.NE.03.030180.001041
Abstract: The Niemann-Pick type C1 (NPC1) protein is a large transmembrane protein located in lysosomes/endosomes. NPC1 binds cholesterol (CLR) and transports it to cellular membrane and endoplasmic reticulum. Mutations in NPC1 cause Niemann-Pick type C (NPC) disease, a rare autosomal disorder characterized by intracellular accumulations of CLR and subsequent neurodegeneration leading to premature death. Among known disease-causing mutations in NPC1, Q92R is the one that is located in the N-terminal cholesterol-binding domain [NTD]. Here we study the effect of the mutation on the ability of NPC1 (NTD) to bind and retain CLR in the binding pocket using structural analysis. We compare characteristics of the Q92R and Q92S mutant type (MT) protein, which is predicted to be benign. We provide detailed investigation of the CLR-NPC1 (NTD) binding process and propose the mechanism, by which Q92R mutation causes NPC disease. We show that although Q92 residue neither directly participates in catalytic activity of the NPC1 (NTD), nor defines its CLR-binding specificity - it is important for the overall protein structure as well as for providing favorable electrostatic environment for CLR transfer. Our results suggest that a negative electrostatic potential of the CLR binding site (the S-opening) might promote NPC2 interaction with NPC1 (NTD) and/or proper CLR orientation and its enforced transfer. We show that in contrast to the benign Q92S mutation, Q92R significantly reduces electrostatic potential around S-opening, and thus likely affects NPC1 (NTD)-NPC2 interaction and/or CLR transfer from NPC2 to NPC1.
On-resin strategy to label α-Conotoxins: Cy5-RgIA, a Potent α9α10 nicotinic acetylcholine receptor imaging probe
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/CH19456
Abstract: In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with tert-butyloxycarbonyl solid phase peptide synthesis (Boc-SPPS), and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5, resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine (100µM)-evoked current reversibly with an IC50 of 5.0nM (Hill coefficient=1.7) for Cy5-RgIA and an IC50 of 1.6 (Hill coefficient=1.2) for α-RgIA at the α9α10 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus oocytes. Cy5-RgIA was then used to successfully visualise nAChRs in the RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for invivo experiments. The strategy is compatible with Boc- and 9-fluorenylmethoxycarbonyl (Fmoc)-chemistry, allows site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 fluorescence resonance energy transfer (FRET) pairs.
Rational Design of α-Conotoxin RegIIA Analogues Selectively Inhibiting the Human α3β2 Nicotinic Acetylcholine Receptor through Computational Scanning
Publisher: American Chemical Society (ACS)
Date: 10-07-2020
Mechanism of Action and Structure–Activity Relationship of α-Conotoxin Mr1.1 at the Human α9α10 Nicotinic Acetylcholine Receptor
Publisher: American Chemical Society (ACS)
Date: 22-09-2022
DOI: 10.1021/ACS.JMEDCHEM.2C00494
Abstract: α-Conotoxins (α-CTxs) can selectively target nicotinic acetylcholine receptors (nAChRs) and are important drug leads for the treatment of cancer, chronic pain, and neuralgia. Here, we chemically synthesized a formerly defined rat α7 nAChR targeting α-CTx Mr1.1 and evaluated its activity at human nAChRs. Mr1.1 was most potent at the human (h) α9α10 nAChR with a half-maximal inhibitory concentration (IC
Chemical engineering and structural and pharmacological characterization of the α-scorpion toxin OD1
Publisher: American Chemical Society (ACS)
Date: 03-04-2013
DOI: 10.1021/CB400012K
Abstract: Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.
NEDD4-2 as a potential candidate susceptibility gene for epileptic photosensitivity.
Publisher: Wiley
Date: 09-01-2007
Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia
Publisher: Elsevier BV
Date: 11-2001
DOI: 10.1016/S0306-4522(01)00347-5
Abstract: The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurones of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), or the desensitising agonist, alpha,beta-methylene ATP. In contrast, EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 microM) had no effect on membrane potential of the postsynaptic neurone or on the litude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 microM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gamma S, reversibly depressed EPSPs litude. The inhibitory effects of ATP and ATP gamma S on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurones from rat submandibular ganglia, focal application of ATP (100 microM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 microM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurones to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability.
Basal nonselective cation permeability in rat cardiac microvascular endothelial cells
Publisher: Elsevier BV
Date: 09-2002
Abstract: The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-cl technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca(2+)-activated Cl(-) conductance was present. When the intracellular Ca(2+) was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na(+) by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K(+) > Cs(+) > Na(+) > Li(+) and was inhibited by Ca(2+), La(3+), Gd(3+), and amiloride. The K(+)/Na(+) permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K(+) was replaced by NMDG(+), but was not sensitive to substitution by Cs(+). Finally, microfluorimetric experiments indicated the existence of a basal Ca(2+) entry pathway, inhibited by La(3+) and Gd(3+). The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca(2+)-dependent processes.
An ATP‐sensitive potassium conductance in rabbit arterial endothelial cells.
Publisher: Wiley
Date: 15-06-1995
DOI: 10.1113/JPHYSIOL.1995.SP020755
Abstract: 1. Whole-cell patch cl recording was used to study an ATP-sensitive, sulphonylurea-inhibitable potassium (K+) conductance in freshly dissociated endothelial cells from rabbit arteries. 2. The ATP-sensitive K+ conductance was activated by micromolar concentrations of the K+ channel opener, levcromakalim, and by metabolic inhibition of endothelial cells using dinitrophenol and iodoacetic acid. The current-voltage (I-V) relationship obtained in isotonic K+ solutions was linear between -150 and -50 mV and had a slope conductance of approximately 1 nS. 3. The permeability of the ATP-sensitive K+ conductance determined from reversal potential measurements exhibited the following ionic selectivity sequence: Rb+ > K+ > Cs+ >> Na+ > NH4+ > Li+. 4. Membrane currents activated by either levcromakalim or metabolic inhibition were inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide, with half-maximal inhibitory concentrations of 43 nM and 224 microM and Hill coefficients of 1.1 and 1.2, respectively. Levcromakalim-induced currents were also inhibited by millimolar concentrations of Ba2+ or tetraethylammonium ions in the external solution. 5. Levcromakalim (3 microM) and metabolic inhibition hyperpolarized endothelial cells by approximately 10-15 mV in normal physiological salt solutions. The hyperpolarization induced by levcromakalim or metabolic inhibition was inhibited by bath application of 10 microM glibenclamide. 6. Internal perfusion of the cytosol of whole-cell voltage-cl ed endothelial cells with an ATP-free pipette solution activated a membrane current which was reversibly inhibited by internal perfusion with a 3 mM MgATP pipette solution. This current was insensitive to other adenine and guanine nucleotides in the pipette solution. The inward current evoked in a nominally ATP-free internal solution was further increased by bath application of levcromakalim. 7. Levcromakalim (25 microM) did not induce a change in the intracellular Ca2+ concentration of fura-2-loaded endothelial cells, whereas metabolic inhibition caused a slow and sustained increase in intracellular Ca2+ concentration, which was attenuated by 10 microM glibenclamide applied externally.(ABSTRACT TRUNCATED AT 400 WORDS)
α-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors
Publisher: Elsevier BV
Date: 07-2010
Transforming conotoxins into cyclotides: Backbone cyclization of P‐superfamily conotoxins
Publisher: Wiley
Date: 11-2015
DOI: 10.1002/BIP.22699
Abstract: Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides but until recently it had not been applied to peptides with multiple disulfide bonds. Conotoxins are disulfide-rich conopeptides derived from the venoms of cone snails that have applications in drug design and development. However, because of their peptidic nature, they can suffer from poor bioavailability and poor stability in vivo. In this study two P-superfamily conotoxins, gm9a and bru9a, were backbone cyclized by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclized derivatives had conformations similar to the native peptides showing that backbone cyclization can be applied to three disulfide-bonded peptides with cystine knot motifs. Cyclic gm9a was more potent at high voltage-activated (HVA) calcium channels than its acyclic counterpart, highlighting the value of this approach in developing active and stable conotoxins containing cyclic cystine knot motifs.
The α2δ Auxiliary Subunit Reduces Affinity of ω-Conotoxins for Recombinant N-type (Cav2.2) Calcium Channels
Publisher: Elsevier BV
Date: 08-2004
Author response: µ-Theraphotoxin Pn3a inhibition of CaV3.3 channels reveals a novel isoform-selective drug binding site
Publisher: eLife Sciences Publications, Ltd
Date: 19-04-2022
Asymmetrical displacement currents
Publisher: Springer Science and Business Media LLC
Date: 02-1978
DOI: 10.1038/271586C0
Conotoxins targeting neuronal voltage-gated sodium channel subtypes: Potential analgesics?
Publisher: MDPI AG
Date: 08-11-2012
Total Synthesis of the Analgesic Conotoxin MrVIB through Selenocysteine‐Assisted Folding
Publisher: Wiley
Date: 31-05-2011
Ketamine inhibits synaptic transmission and nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglia in situ
Publisher: Elsevier BV
Date: 03-2020
DOI: 10.1016/J.NEUROPHARM.2019.107932
Abstract: The intravenous anaesthetic ketamine, has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR)-mediated currents in dissociated rat intracardiac ganglion (ICG) neurons (Weber et al., 2005). This effect would be predicted to depress synaptic transmission in the ICG and would account for the inhibitory action of ketamine on vagal transmission to the heart (Inoue and König, 1988). This investigation was designed to examine the activity of ketamine on (i) postsynaptic responses to vagal nerve stimulation, (ii) the membrane potential, and (iii) membrane current responses evoked by exogenous application of ACh and nicotine in ICG neurons in situ. Intracellular recordings were made using sharp intracellular microelectrodes in a whole mount ICG preparation. Preganglionic nerve stimulation and recordings in current- and voltage-cl modes were used to assess the action of ketamine on ganglionic transmission and nAChR-mediated responses. Ketamine attenuated the postsynaptic responses evoked by nerve stimulation. This reduction was significant at clinically relevant concentrations at high frequencies. The excitatory membrane potential and current responses to focal application of ACh and nicotine were inhibited in a concentration-dependent manner by ketamine. In contrast, ketamine had no effect on either the directly-evoked action potential or excitatory responses evoked by focal application of γ-aminobutyric acid (GABA). Taken together, ketamine inhibits synaptic transmission and nicotine- and ACh-evoked currents in adult rat ICG. Ketamine inhibition of synaptic transmission and nAChR-mediated responses in the ICG contributes significantly to its attenuation of the bradycardia observed in response to vagal stimulation in the mammalian heart.
Advances in Xanthan Gum-Based Systems for the Delivery of Therapeutic Agents
Publisher: MDPI AG
Date: 25-01-2023
DOI: 10.3390/PHARMACEUTICS15020402
Abstract: In the last three decades, polymers have contributed significantly to the improvement of drug delivery technologies by enabling the controlled and sustained release of therapeutic agents, versatility in designing different delivery systems, and feasibility of encapsulation of both hydrophobic and hydrophilic molecules. Both natural and synthetic polymers have been explored for the delivery of various therapeutic agents. However, due to the disadvantages of synthetic polymers, such as lack of intrinsic biocompatibility and bioactivity, hydrophobicity, and expensive and complex procedure of synthesis, there is a move toward the use of naturally occurring polymers. The biopolymers are generally derived from either plants or microorganisms and have shown a wide range of applications in drug administration due to their hydrophilic nature, biodegradability, biocompatibility, no or low toxicity, abundance, and readily available, ease of chemical modification, etc. This review describes the applications of a biopolymer, xanthan gum (XG), in the delivery of various therapeutic agents such as drugs, genetic materials, proteins, and peptides. XG is a high molecular weight, microbial heteropolysaccharide and is produced as a fermented product of Gram-negative bacteria, Xanthomonas c estris. Traditionally, it has been used as a thickener in liquid formulations and an emulsion stabiliser. XG has several favourable properties for designing various forms of drug delivery systems. Furthermore, the structure of XG can be easily modified using different temperature and pH conditions. Therefore, XG and its derivatives have been explored for various applications in the food, pharmaceutical, and cosmetic industries.
Backbone cyclization of analgesic conotoxin GeXIVA facilitates direct folding of the ribbon isomer.
Publisher: Elsevier BV
Date: 10-2017
Biophysical properties of Nav 1.8/Nav 1.2 chimeras and inhibition by μo-conotoxin MrVIB
Publisher: Wiley
Date: 09-07-2012
Analgesic α-Conotoxins Vc1.1 and Rg1A Inhibit N-Type Calcium Channels in Rat Sensory Neurons via GABABReceptor Activation
Publisher: Society for Neuroscience
Date: 22-10-2008
DOI: 10.1523/JNEUROSCI.3594-08.2008
Abstract: α-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the α9α10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other α-conotoxins tested. Vc1.1 inhibited the ω-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca V 2.2, Ca V 2.1, Ca V 2.3, or Ca V 1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPβS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA B receptor antagonists, [ S -( R *, R *)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2 S )-3[[(1 S )-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca 2+ channel currents. Together, the results identify Ca V 2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which α-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca V 2.2) Ca 2+ channel currents, namely acting as agonists via G-protein-coupled GABA B receptors.
DM506 (3-Methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole fumarate), a Novel Derivative of Ibogamine, Inhibits α7 and α9α10 Nicotinic Acetylcholine Receptors by Different Allosteric Mechanisms
Publisher: American Chemical Society (ACS)
Date: 30-06-2023
Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells
Publisher: Wiley
Date: 30-04-2004
Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones
Publisher: Elsevier BV
Date: 04-1999
DOI: 10.1016/S0304-3940(99)00180-9
Abstract: The effects of Met-enkephalin on Ca2+-dependent K+ channel activity were investigated using the cell-attached patch recording technique on isolated parasympathetic neurones of rat intracardiac ganglia. Large-conductance, Ca2+-dependent K+ channels (BK(Ca)) were examined as an assay of agonist-induced changes in the intracellular free calcium ion concentration ([Ca2+]i). These BK(Ca) channels had a conductance of approximately 200 pS and were charybdotoxin- and voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BK(Ca) channel activity, which was inhibited by 10 microM ryanodine. Met-enkephalin (10 microM) evoked a similar increase in BK(Ca) channel activity, which was dependent on the presence of extracellular Ca2+ and inhibited by either ryanodine (10 microM) or naloxone (1 microM). In Fura-2-loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca2+]i. Met-enkephalin-induced mobilization of intracellular Ca+ may play a role in neuronal excitability and firing behaviour in mammalian intracardiac ganglia.
Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 20-05-2004
Resting membrane potential and potassium currents in cultured parasympathetic neurones from rat intracardiac ganglia.
Publisher: Wiley
Date: 10-1992
DOI: 10.1113/JPHYSIOL.1992.SP019343
Abstract: 1. Whole-cell K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-cl ed parasympathetic neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. Rat intracardiac neurones had a mean resting membrane potential of -52 mV and mean input resistance of 850 M omega. The current-voltage relationship recorded during slow voltage r s indicated the presence of both leakage and voltage-dependent currents. The contribution of Na+, K+ and Cl- to the resting membrane potential was examined and relative ionic permeabilities PNa/PK = 0.12 and PCl/PK 5 s. The instantaneous current-voltage relationship, examined under varying external K+ concentrations, was linear, and the reversal (zero current) potential shifted in accordance with that predicted by the Nernst equation for a K(+)-selective electrode. The shift in reversal potential of the tail currents as a function of the extracellular K+ concentration gave a relative permeability, PNa/PK = 0.02 for the delayed outward K+ channel(s).(ABSTRACT TRUNCATED AT 400 WORDS)
Voltage-dependent sodium and calcium currents in cultured parasympathetic neurones from rat intracardiac ganglia.
Publisher: Wiley
Date: 10-1992
DOI: 10.1113/JPHYSIOL.1992.SP019344
Abstract: 1. Depolarization-activated Na+ and Ca2+ currents underlying the rising phase of the action potential in mammalian parasympathetic ganglion cells were investigated in voltage-cl ed neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. A current component isolated by replacing intracellular K+ with Cs+ or arginine and adding 0.1 mM Cd2+ to the external solution was dependent on extracellular [Na+] and reversibly blocked in the presence of 300 nM tetrodotoxin (TTX). Peak litudes of Na+ currents elicited by step depolarization from a holding potential of -100 mV were 351 +/- 18 pA F (140 mM extracellular Na+). 3. The sodium current-voltage (I-V) curve exhibited a threshold for activation at -40 mV and reached a maximum at -10 mV. The Na+ conductance increased sigmoidally with increasing depolarization reaching half-maximal activation at -25 mV, with a maximum slope corresponding to 7.5 mV per e-fold change in conductance. 4. During a maintained depolarization, Na+ currents turned on and then decayed (inactivated) with an exponential time course. The time constant of inactivation was voltage dependent decreasing from 0.85 ms at -20 mV to 0.3 ms at +60 mV (23 degrees C). The steady-state inactivation of the Na+ conductance was voltage-dependent with half-inactivation occurring at -61 mV and near-complete inactivation at -20 mV. Recovery from inactivation also followed an exponential time course with a time constant that increased at depolarized membrane potentials. 5. A voltage- and Ca(2+)-dependent current was isolated by replacement of intracellular K+ with either Cs+ or arginine and of extracellular Na+ with tetraethylammonium and the addition of TTX. Extracellular Ba2+ or Na+ (in the absence of external alent cation) could substitute for Ca2+. Peak Ca2+ current increased with increasing extracellular [Ca2+] and above 10 mM (Kd approximately 4 mM) approached saturation. The peak Ca2+ current density was 45 +/- 4 pA F (2.5 mM-extracellular Ca2+). 6. The Ca2+ I-V relation exhibited a high threshold for activation (-20 mV) and reached a maximum at +20 mV. Changing the holding potential from -100 to -40 mV did not alter the I-V relationship. Peak Ca2+ conductance increased sigmoidally with increasing depolarization reaching half-maximal activation at -4 mV, with a maximal slope of 4 mV per e-fold change in Ca2+ conductance. 7. The kinetics of activation and inactivation of the Ca2+ current were voltage dependent and the time course of inactivation was fitted by the sum of two exponentials.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells
Publisher: Elsevier BV
Date: 1987
DOI: 10.1016/0040-8166(87)90015-2
Abstract: The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch cl and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.
Calcium Imaging of Non-adherent Cells
Publisher: Springer US
Date: 2023
Glycinergic Modulation of Pain in Behavioral Animal Models
Publisher: Frontiers Media SA
Date: 25-05-2022
DOI: 10.3389/FPHAR.2022.860903
Abstract: Animal models of human pain conditions allow for detailed interrogation of known and hypothesized mechanisms of pain physiology in awake, behaving organisms. The importance of the glycinergic system for pain modulation is well known however, manipulation of this system to treat and alleviate pain has not yet reached the sophistication required for the clinic. Here, we review the current literature on what animal behavioral studies have allowed us to elucidate about glycinergic pain modulation, and the progress toward clinical treatments so far. First, we outline the animal pain models that have been used, such as nerve injury models for neuropathic pain, chemogenic pain models for acute and inflammatory pain, and other models that mimic painful human pathologies such as diabetic neuropathy. We then discuss the genetic approaches to animal models that have identified the crucial glycinergic machinery involved in neuropathic and inflammatory pain. Specifically, two glycine receptor (GlyR) subtypes, GlyRα1(β) and GlyRα3(β), and the two glycine transporters (GlyT), GlyT1 and GlyT2. Finally, we review the different pharmacological approaches to manipulating the glycinergic system for pain management in animal models, such as partial vs . full agonism, reversibility, and multi-target approaches. We discuss the benefits and pitfalls of using animal models in drug development broadly, as well as the progress of glycinergic treatments from preclinical to clinical trials.
Local anaesthetic blockade of neuronal nicotinic ACh receptor‐channels in rat parasympathetic ganglion cells
Publisher: Wiley
Date: 03-1994
DOI: 10.1111/J.1476-5381.1994.TB14789.X
Abstract: 1 The effects of the local anaesthetics QX-222 and procaine on nicotinic acetylcholine (ACh)-evoked currents in cultured parasympathetic cardiac neurones of the rat were investigated by use of the whole-cell, perforated-patch, and outside-out recording configurations of the patch cl method. 2 QX-222 and procaine, applied to the extracellular surface, reversibly inhibited the peak litude of the whole-cell nicotinic ACh-evoked current in a concentration-dependent manner, with half-maximal inhibitory concentrations (IC50) of 28 microM and 2.8 microM, respectively, at -80 mV. In these neurones, the sustained inward current mediated by M1 muscarinic receptor activation was unaltered by QX-222, and neither local anaesthetic affected the adenosine 5'-triphosphate (ATP)-evoked current. 3 QX-222 and procaine block of nicotinic ACh-evoked inward current was voltage-dependent and enhanced by hyperpolarization. An e-fold change in their dissociation equilibrium constants (Kd) resulted from a 62 mV and a 122 mV change in membrane potential, respectively. 4 Both local anaesthetics produce a concentration-dependent increase in the half-time of decay of the nicotinic ACh-evoked inward current. 5 Measurements of unitary currents in outside-out patches showed that QX-222 reversibly increased the mean burst duration and closed time and reduced the mean channel open time and open-state probability of the nicotinic ACh receptor-channel (AChR) in a concentration-dependent manner. 6 The Kd and voltage sensitivity of local anaesthetic block of the nicotinic AChR in rat intracardiac neurones suggests that the pore-forming region of this channel differs from that of the AChR in frog and rat skeletal muscle and from the neuronal alpha 4 beta 2 ACh receptor-channel.
Mechanism of direct Cav 2.2 channel block by the κ-opioid receptor agonist U50488H
Publisher: Elsevier BV
Date: 10-2016
DOI: 10.1016/J.NEUROPHARM.2016.05.019
Abstract: U50488H is a benzeneacetamide κ-opioid receptor (κ-OR) agonist analgesic, widely used for investigating the pharmacology of G protein-coupled κ-ORs. However, U50488H is also known to directly block various voltage-gated ion channels in a G protein-independent manner. We investigated the direct actions of U50488H on various high voltage-activated (HVA) and low voltage-activated (LVA) neuronal Ca(2+) channels heterologously expressed in human embryonic kidney (HEK293) cells. U50488H inhibited HVA rat Cav1.3 (rCav1.3), human Cav2.1 (hCav2.1), hCav2.2, hCav2.3, and LVA hCav3.1 and hCav3.2 channels in a concentration-dependent manner, with similar potencies characterised with half-maximal inhibitory concentration (IC50) values of ∼30 μM. U50488H concentrations causing direct Cav inhibition are typically >100 times higher than those producing κ-OR activation. Investigation of the mechanism of U50488H block of the Cav2.2 channel revealed that U50488H interacted with all major kinetic states of the channel - resting, open, and inactivated. U50488H did not affect the voltage dependence of activation but shifted the steady-state inactivation curve by ∼11 mV to more hyperpolarized potentials. U50488H also increased the rate of Ba(2+) current inactivation during a step depolarization and significantly delayed recovery from slow inactivation, compared with control. Cav2.2 current inhibition was frequency dependent during repetitive step depolarization at 1 Hz and 3 Hz, consistent with use-dependent block. In summary, our results suggest that preferential interaction of U50488H with inactivated Cav2.2 channels significantly contributes to reduced Cav2.2 channel availability and slow recovery form inactivation. We conclude that U50488H non-selectively blocks heterologously expressed neuronal HVA and LVA Cav channels in the absence of κ-ORs. This cross-reactivity also suggests potentially common U50488H binding motifs across Cav channel targets.
Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia
Publisher: Rockefeller University Press
Date: 06-1995
Abstract: Acetylcholine-evoked currents mediated by activation of nicotinic receptors in rat parasympathetic neurons were examined using whole-cell voltage cl . The relative permeability of the neuronal nicotinic acetylcholine (nACh) receptor channel to monovalent and alent inorganic and organic cations was determined from reversal potential measurements. The channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Cs+ & K+ & Rb+ & Na+ & Li+, and permeability ratios relative to Na+ (Px/PNa) ranging from 1.27 to 0.75. The selectivity of the alkaline earths was also weak, with the sequence of Mg2+ & Sr2+ & Ba2+ & Ca2+, and relative permeabilities of 1.10 to 0.65. The relative Ca2+ permeability (PCa/PNa) of the neuronal nACh receptor channel is approximately fivefold higher than that of the motor endplate channel (Adams, D. J., T. M. Dwyer, and B. Hille. 1980. Journal of General Physiology. 75:493-510). The transition metal cation, Mn2+ was permeant (Px/PNa = 0.67), whereas Ni2+, Zn2+, and Cd2+ blocked ACh-evoked currents with half-maximal inhibition (IC50) occurring at approximately 500 microM, 5 microM and 1 mM, respectively. In contrast to the muscle endplate AChR channel, that at least 56 organic cations which are permeable to (Dwyer et al., 1980), the majority of organic cations tested were found to completely inhibit ACh-evoked currents in rat parasympathetic neurons. Concentration-response curves for guanidinium, ethylammonium, diethanolammonium and arginine inhibition of ACh-evoked currents yielded IC50's of approximately 2.5-6.0 mM. The organic cations, hydrazinium, methylammonium, ethanolammonium and Tris, were measureably permeant, and permeability ratios varied inversely with the molecular size of the cation. Modeling suggests that the pore has a minimum diameter of 7.6 A. Thus, there are substantial differences in ion permeation and block between the nACh receptor channels of mammalian parasympathetic neurons and hibian skeletal muscle which represent functional consequences of differences in the primary structure of the subunits of the ACh receptor channel.
Isolation and structure-activity of mu-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 12-2006
Abstract: Mu-conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other mu-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.
Spider Venom Peptide Pn3a Inhibition of Primary Afferent High Voltage-Activated Calcium Channels.
Publisher: Frontiers Media SA
Date: 28-01-2020
DOI: 10.3389/FPHAR.2020.633679
Abstract: Despite potently inhibiting the nociceptive voltage-gated sodium (Na v ) channel, Na v 1.7, µ -theraphotoxin Pn3a is antinociceptive only upon co-administration with sub-therapeutic opioid agonists, or by itself at doses & ,000-fold greater than its Na v 1.7 IC 50 by a yet undefined mechanism. Na v channels are structurally related to voltage-gated calcium (Ca v ) channels, Ca v 1 and Ca v 2. These channels mediate the high voltage-activated (HVA) calcium currents ( I Ca ) that orchestrate synaptic transmission in nociceptive dorsal root ganglion (DRG) neurons and are fine-tuned by opioid receptor (OR) activity. Using whole-cell patch cl recording, we found that Pn3a (10 µM) inhibits ∼55% of rat DRG neuron HVA- I Ca and 60–80% of Ca v 1.2, Ca v 1.3, Ca v 2.1, and Ca v 2.2 mediated currents in HEK293 cells, with no inhibition of Ca v 2.3. As a major DRG I Ca component, Ca v 2.2 inhibition by Pn3a ( IC 50 = 3.71 ± 0.21 µM) arises from an 18 mV hyperpolarizing shift in the voltage dependence of inactivation. We observed that co-application of Pn3a and µ-OR agonist DAMGO results in enhanced HVA- I Ca inhibition in DRG neurons whereas co-application of Pn3a with the OR antagonist naloxone does not, underscoring HVA channels as shared targets of Pn3a and opioids. We provide evidence that Pn3a inhibits native and recombinant HVA Ca v s at previously reportedly antinociceptive concentrations in animal pain models. We show additive modulation of DRG HVA- I Ca by sequential application of low Pn3a doses and sub-therapeutic opioids ligands. We propose Pn3a's antinociceptive effects result, at least in part, from direct inhibition of HVA- I Ca at high Pn3a doses, or through additive inhibition by low Pn3a and mild OR activation.
Regulation of voltage-gated ion channels in excitable cells by the ubiquitin ligases Nedd4 and Nedd4-2
Publisher: Informa UK Limited
Date: 2011
Abstract: The electrical excitability of neurons is mediated primarily by voltage-gated ion channels, particularly voltage-gated Na(+) (Na(v)), K(+) (K(v)) and Cl(-) (ClC) channels. Cells regulate their electrical excitability by controlling not only the activity, but also the number of in idual ion channels in the plasma membrane. There exist several mechanisms for regulating levels of voltage-gated ion channels: transcription and translation, retention and export from the endoplasmic reticulum as well as insertion and retrieval from the plasma membrane. Alterations in voltage-gated ion channel activity, composition and distribution can contribute to the pathophysiology of epilepsy, hypertension, neuropathic and inflammatory pain. One mechanism for retrieval is ubiquitination. Here specific ubiquitin ligases bind to membrane proteins to modulate and regulate their cellular fate. In this review, we focus on Nedd4 and Nedd4-2 ubiquitin ligases and the mechanisms by which they regulate voltage-gated ion channels and describe a novel paradigm on the mechanisms that underpin aberrant ion channel function in neurological disorders.
Bombesin receptors as potential targets for anticancer drug delivery and imaging
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.BIOCEL.2019.105567
Abstract: The biggest challenge in delivering anticancer agents is the ability to direct these molecules specifically to cancer cells. With this in mind, modern research is focussing on improving the precision of cancer drug delivery by incorporating a ligand that has the ability to specifically recognize cancer cells. Peptides are emerging as a new tool in drug and gene delivery. Peptide-drug conjugates, peptide-modified drug delivery systems, and peptide-coupled imaging agents have been shown to increase on-site delivery. This has allowed better tumor mass contouring in imaging and increased therapeutic efficacy of chemotherapies, reducing adverse effects. Benefits of peptide ligands include their small size, easy and affordable production, high specificity and remarkable flexibility regarding their sequence and conjugation possibilities. Bombesin (Bn) receptors have shown great promise for tumor targeting due to their increased expression in a variety of human cancers, including prostate, breast, small cell lung, and pancreatic cells. This review discusses the overexpression of Bn receptors in different cancers and various approaches to target these receptors for therapeutic and diagnostic interventions in human malignancies.
Voltage-Gated Calcium Channels in Nociception
Publisher: Springer Berlin Heidelberg
Date: 2008
Solution structure of μ-conotoxin PIIIA, a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels
Publisher: Elsevier BV
Date: 07-2002
Regulation of the voltage-gated K+ channels KCNQ2/3 and KCNQ3/5 by serum- and glucocorticoid-regulated kinase-1
Publisher: American Physiological Society
Date: 07-2008
DOI: 10.1152/AJPCELL.00146.2008
Abstract: The voltage-gated KCNQ2/3 and KCNQ3/5 K + channels regulate neuronal excitability. We recently showed that KCNQ2/3 and KCNQ3/5 channels are regulated by the ubiquitin ligase Nedd4-2. Serum- and glucocorticoid-regulated kinase-1 (SGK-1) plays an important role in regulation of epithelial ion transport. SGK-1 phosphorylation of Nedd4-2 decreases the ability of Nedd4-2 to ubiquitinate the epithelial Na + channel, which increases the abundance of channel protein in the cell membrane. In this study, we investigated the mechanism(s) of SGK-1 regulation of M-type KCNQ channels expressed in Xenopus oocytes. SGK-1 significantly upregulated the K + current litudes of KCNQ2/3 and KCNQ3/5 channels ∼1.4- and ∼1.7-fold, respectively, whereas the kinase-inactive SGK-1 mutant had no effect. The cell surface levels of KCNQ2-hemagglutinin/3 were also increased by SGK-1. Deletion of the KCNQ3 channel COOH terminus in the presence of SGK-1 did not affect the K + current litude of KCNQ2/3/5-mediated currents. Coexpression of Nedd4-2 and SGK-1 with KCNQ2/3 or KCNQ3/5 channels did not significantly alter K + current litudes. Only the Nedd4-2 mutant S448A Nedd4-2 exhibited a significant downregulation of the KCNQ2/3/5 K + current litudes. Taken together, these results demonstrate a potential mechanism for regulation of KCNQ2/3 and KCNQ3/5 channels by SGK-1 regulation of the activity of the ubiquitin ligase Nedd4-2.
Structure–Activity Studies of Cysteine‐Rich α‐Conotoxins that Inhibit High‐Voltage‐Activated Calcium Channels via GABAB Receptor Activation Reveal a Minimal Functional Motif
Publisher: Wiley
Date: 07-03-2016
Abstract: α-Conotoxins are disulfide-rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α-conotoxins that also modulate voltage-gated calcium channels by acting as G protein-coupled GABA(B) receptor (GABA(B)R) agonists. These α-conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the ersity of α-conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α-conotoxins known to inhibit high voltage-activated calcium channels via GABA(B)R activation. Remarkably, all disulfide isomers of the active α-conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.
Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor
Publisher: American Chemical Society (ACS)
Date: 06-2023
α-Selenoconotoxins, a New Class of Potent α7 Neuronal Nicotinic Receptor Antagonists
Publisher: Elsevier BV
Date: 05-2006
Neuronally micro-conotoxins from Conus striatus utilize an alpha-helical motif to target mammalian sodium channels.
Publisher: Elsevier BV
Date: 08-0005
The Synthesis, Structural Characterization, and Receptor Specificity of the α-Conotoxin Vc1.1
Publisher: Elsevier BV
Date: 08-2006
Modulation of human NAv 1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs
Publisher: Informa UK Limited
Date: 26-01-2016
Bombesin-conjugated nanoparticles improve the cytotoxic efficacy of docetaxel against gastrin-releasing but androgen-independent prostate cancer
Publisher: Future Medicine Ltd
Date: 09-2015
DOI: 10.2217/NNM.15.107
Abstract: Aim: Bombesin (BBN)-conjugated polymeric nanoparticles to target docetaxel (DTX) to prostate cancer cells that overexpress gastrin-releasing peptides receptors. Materials & methods: In vitro cytotoxicity, uptake of nanoparticles and inhibition of cell migration were assessed against human prostate cancer cells. Preclinical pharmacokinetic and tissue-distribution studies of nanoparticles were performed in Balb/c mice and results compared with the marketed formulation Taxotere ® . Results: BBN-conjugated DTX-loaded nanoparticles exhibited higher cytotoxicity, inhibition of cell migration and colony formation than non-targeted nanoparticles or DTX alone. More BBN-conjugated nanoparticles were taken up at a faster rate than unconjugated nanoparticles. In vivo, this drug delivery improved pharmacokinetics of DTX by increasing mean residence time and decreasing clearance. Conclusion: This study provides an alternate approach for polysorbate-free delivery of DTX, with improved in vivo performance.
Two new classes of conopeptides inhibit the α1-adrenoceptor and noradrenaline transporter
Publisher: Springer Science and Business Media LLC
Date: 06-08-2001
DOI: 10.1038/NN0901-902
Globular and ribbon isomers of Conus geographus α-conotoxins antagonize human nicotinic acetylcholine receptors
Publisher: Elsevier BV
Date: 08-2021
M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons
Publisher: American Physiological Society
Date: 10-1997
DOI: 10.1152/JN.1997.78.4.1903
Abstract: Cuevas, J. and Adams, D. J. M 4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons. J. Neurophysiol. 78: 1903–1912, 1997. Modulation of high-voltage–activated Ca 2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the hotericin B perforated-patch whole cell recording configuration of the patch-cl technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-cl ed soma membrane reversibly depressed peak Ca 2+ channel current litude. The dose-reponse relationship obtained for ACh-induced inhibition of Ba 2+ current ( I Ba ) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of I Ba litude obtained with 100 μM ACh was ∼75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca 2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (≤300 nM) and m4-toxin (≤100 nM), but not by AF-DX 116 (300 nM) or m1-toxin (60 nM). The dose-response relationship obtained for antagonism of muscarine-induced inhibition of I Ba by m4-toxin gave an IC 50 of 11 nM. These results suggest that muscarinic agonist-induced inhibition of high-voltage–activated Ca 2+ channels in rat intracardiac neurons is mediated by the M 4 muscarinic receptor. M 4 receptor activation shifted the voltage dependence and depressed maximal activation of Ca 2+ channels but had no effect on the steady-state inactivation of Ca 2+ channels. Peak Ca 2+ channel tail current litude was reduced ≥30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinicreceptor-mediated inhibition. Both dihydropyridine- and ω-conotoxin GVIA–sensitive and -insensitive Ca 2+ channels were inhibited by ACh, suggesting that the M 4 muscarinic receptor is coupled to multiple Ca 2+ channel subtypes in these neurons. Inhibition of I Ba litude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 μM guanosine 5′-O-(2-thiodiphosphate) trilithium salt (GDP-β-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of I Ba by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.
Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav 2.2 channels
Publisher: Wiley
Date: 10-06-2014
DOI: 10.1111/BPH.12686
CHAPTER 4. Venoms-Based Drug Discovery: Bioassays, Electrophysiology, High-Throughput Screens and Target Identification
Publisher: Royal Society of Chemistry
Date: 2015
µ-Theraphotoxin Pn3a inhibition of CaV3.3 channels reveals a novel isoform-selective drug binding site
Publisher: eLife Sciences Publications, Ltd
Date: 20-07-2022
DOI: 10.7554/ELIFE.74040
Abstract: Low voltage-activated calcium currents are mediated by T-type calcium channels Ca V 3.1, Ca V 3.2, and Ca V 3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. Ca V 3 isoforms’ biophysical properties, overlapping expression, and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. We have identified μ-theraphotoxin Pn3a as the first subtype-selective spider venom peptide inhibitor of Ca V 3.3, with -fold lower potency against the other T-type isoforms. Pn3a modifies Ca V 3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing Ca V 3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of K V 1.7 channels bearing voltage sensor sequences from all four Ca V 3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (Ca V 3.3 DII ). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a, site-directed mutagenesis, and full domain II swaps between Ca V 3 channels highlighting it as a subtype-specific pharmacophore. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of Ca V 3.3 channels.
Structures of muO-conotoxins from Conus marmoreus. I nhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons.
Publisher: Elsevier BV
Date: 06-2004
Cysteine-Rich α-Conotoxin SII Displays Novel Interactions at the Muscle Nicotinic Acetylcholine Receptor
Publisher: American Chemical Society (ACS)
Date: 31-03-2022
DOI: 10.1021/ACSCHEMNEURO.1C00857
Abstract: α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 3
Interactions of the α3β2 Nicotinic Acetylcholine Receptor Interfaces with α-Conotoxin LsIA and its Carboxylated C-terminus Analogue: Molecular Dynamics Simulations
Publisher: MDPI AG
Date: 03-07-2020
DOI: 10.3390/MD18070349
Abstract: Notably, α-conotoxins with carboxy-terminal (C-terminal) amidation are inhibitors of the pentameric nicotinic acetylcholine receptors (nAChRs), which are therapeutic targets for neurological diseases and disorders. The (α3)2(β2)3 nAChR subunit arrangement comprises a pair of α3(+)β2(−) and β2(+)α3(−) interfaces, and a β2(+)β2(−) interface. The β2(+)β2(−) interface has been suggested to have higher agonist affinity relative to the α3(+)β2(−) and β2(+)α3(−) interfaces. Nevertheless, the interactions formed by these subunit interfaces with α-conotoxins are not well understood. Therefore, in order to address this, we modelled the interactions between α-conotoxin LsIA and the α3β2 subtype. The results suggest that the C-terminal carboxylation of LsIA predominantly influenced the enhanced contacts of the conotoxin via residues P7, P14 and C17 on LsIA at the α3(+)β2(−) and β2(+)α3(−) interfaces. However, this enhancement is subtle at the β2(+)β2(−) site, which can compensate the augmented interactions by LsIA at α3(+)β2(−) and β2(+)α3(−) binding sites. Therefore, the ergent interactions at the in idual binding interface may account for the minor changes in binding affinity to α3β2 subtype by C-terminal carboxylation of LsIA versus its wild type, as shown in previous experimental results. Overall, these findings may facilitate the development of new drug leads or subtype-selective probes.
Venom-Derived Peptides Inhibiting Voltage-Gated Sodium and Calcium Channels in Mammalian Sensory Neurons.
Publisher: Springer Nature Singapore
Date: 2021
DOI: 10.1007/978-981-16-4254-8_1
Abstract: Pain management is a serious worldwide problem that affects the physical and mental health of all affected humans. As an alternative to opioids, pharmaceutical companies are seeking other sources of potential analgesics that have fewer adverse side effects. Animal venoms are a natural cocktail of a complex mixture of salts, peptides, and proteins. Most animals that produce venoms release them for the purpose of prey capture and/or defense against other vertebrates. Over the last 30 years, many venom-derived peptides have been shown to be active against numerous voltage-gated ion channels in the mammalian somatosensory nervous system. Voltage-gated ion channels and in particular sodium, potassium, and calcium channels are fundamental to the transmission of all somatosensory information from the periphery to the central nervous system. This information can be chemical, mechanical, or thermal sensation that can result from touch to a more painful sensation of tissue injury. These voltage-gated ion channels open or close in response to changes in membrane potential to permit ion movement across the cell membrane. In this chapter, we screened the scientific literature characterizing venom-derived peptides that target voltage-gated sodium and calcium channels and exhibit analgesic properties. Depending on peptide activity, these can either inhibit voltage-gated sodium or calcium channels completely by binding to the pore of the channel or modulate the activity by binding to other regions such as the voltage sensor of the channel.
Peptide grafted and self-assembled poly(γ-glutamic acid)-phenylalanine nanoparticles targeting camptothecin to glioma
Publisher: Future Medicine Ltd
Date: 07-2017
Abstract: Aim: To synthesize cRGDfK peptide conjugated poly(γ-glutamic acid)-phenylalanine nanoparticles to improve the therapeutic efficacy of c tothecin (CPT) against glioblastoma multiforme. Methods: Peptide-conjugated, drug-loaded nanoparticles (cRGDfK-conjugated c tothecin-loaded PGA–PA nanoparticles [RCPN]) were prepared and physico-chemically characterized using different techniques. Nanoparticles were evaluated for in vitro anticancer activity, cellular uptake, induction of apoptosis and wound healing cell migration against U87MG human glioblastoma cells. Results: RCPN, with a particle size of nm and 65% CPT encapsulation efficiency, exhibited a dose- and time-dependent cytotoxicity to glioblastoma cells. Compared with native CPT or unconjugated nanoparticles, RCPN induced apoptosis, increased reactive oxygen species generation and inhibited U87MG cell migration. Conclusion: cRGDfK-mediated and hiphilic copolymer-based nanomedicines represent a new approach for improved delivery of anticancer drugs to and treatment of glioblastoma multiforme.
Embryonic toxin expression in the cone snail Conus victoriae
Publisher: Elsevier BV
Date: 06-2011
Electrical properties and synaptic transmission in mouse intracardiac ganglion neurons in situ
Publisher: Wiley
Date: 09-2021
DOI: 10.14814/PHY2.15056
Muscarinic and Nicotinic ACh Receptor Activation Differentially Mobilize Ca2+in Rat Intracardiac Ganglion Neurons
Publisher: American Physiological Society
Date: 09-2201
Abstract: The origin of intracellular Ca 2 + concentration ([Ca 2 + ] i ) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca 2 + ] i increases that were reduced to ∼60% of control in the presence of either atropine (1 μM) or mecamylamine (3 μM) and to % in the presence of both antagonists. Removal of external Ca 2 + reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca 2 + ] i response was reduced to 50% by 10 μM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca 2 + release from ryanodine-sensitive Ca 2 + stores may only contribute to the nAChR-induced [Ca 2 + ] i responses. Perforated-patch whole cell recording at –60 mV shows that the rise in [Ca 2 + ] i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca 2 + ] i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.
Editorial: Sensory Abnormalities and Primary Sensory Neurons
Publisher: Frontiers Media SA
Date: 04-02-2022
Ethanol reduces excitatory postsynaptic current duration at a crustacean neuromuscular junction [32]
Publisher: Springer Science and Business Media LLC
Date: 04-1977
DOI: 10.1038/266739A0
Abstract: The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (Mr, 84,500) with a subunit with an Mr of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.
Novel mechanism of voltage-gated N-type (Cav 2.2) calcium channel inhibition revealed through α-conotoxin Vc1.1 activation of the GABAB receptor
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 25-11-2015
Abstract: Neuronal voltage-gated N-type (Cav2.2) calcium channels are expressed throughout the nervous system and regulate neurotransmitter release and hence synaptic transmission. They are predominantly modulated via G protein-coupled receptor activated pathways, and the well characterized Gβγ subunits inhibit Cav2.2 currents. Analgesic α-conotoxin Vc1.1, a peptide from predatory marine cone snail venom, inhibits Cav2.2 channels by activating pertussis toxin-sensitive Gi/o proteins via the GABAB receptor (GABA(B)R) and potently suppresses pain in rat models. Using a heterologous GABA(B)R expression system, electrophysiology, and mutagenesis, we showed α-conotoxin Vc1.1 modulates Cav2.2 via a different pathway from that of the GABA(B)R agonists GABA and baclofen. In contrast to GABA and baclofen, Vc1.1 changes Cav2.2 channel kinetics by increasing the rate of activation and shifting its half-maximum inactivation to a more hyperpolarized potential. We then systematically truncated the GABA(B)(1a) C terminus and discovered that removing the proximal carboxyl terminus of the GABA(B)(1a) subunit significantly reduced Vc1.1 inhibition of Cav2.2 currents. We propose a novel mechanism by which Vc1.1 activates GABA(B)R and requires the GABA(B)(1a) proximal carboxyl terminus domain to inhibit Cav2.2 channels. These findings provide important insights into how GABA(B)Rs mediate Cav2.2 channel inhibition and alter nociceptive transmission.
Biomedical Applications of Trastuzumab: As a Therapeutic Agent and a Targeting Ligand
Publisher: Wiley
Date: 04-2015
DOI: 10.1002/MED.21345
Abstract: Trastuzumab (TZ) is a humanized monoclonal antibody targeted to the extracellular domain of human epidermal growth factor receptor 2 (HER2), a tyrosine kinase receptor. TZ is approved by the Food and Drug Administration (FDA) for the treatment of HER2-overexpressing early stage and metastatic breast cancer and HER2-overexpressing metastatic gastric cancer. For breast cancer, it is recommended as both a single agent and in combination with standard chemotherapy. In the last few years, TZ has also been used as a targeting ligand. Overexpression of HER2 in breast cancer and the presence of free surface functional groups on TZ provide an opportunity to use it as a targeting ligand. TZ can be conjugated to various nanoparticulate systems such as dendrimers, polymeric, and protein nanoparticles to target drug delivery. TZ-conjugated inorganic nanoparticles have been reported for imaging and diagnostic purposes. This review summarizes the applications of TZ both as a therapeutic agent and as a targeting ligand.
Stoichiometry dependent inhibition of rat α3β4 nicotinic acetylcholine receptor by the ribbon isomer of α-conotoxin AuIB
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.BCP.2018.07.007
Abstract: The ribbon isomer of α-conotoxin AuIB has 10-fold greater potency than the wild-type globular isomer at inhibiting nicotinic acetylcholine receptors (nAChRs) in rat parasympathetic neurons, and unlike its globular isoform, ribbon AuIB only targets a specific stoichiometry of the α3β4 nAChR subtype. Previous electrophysiological recordings of AuIB indicated that ribbon AuIB binds to the α3(+)α3(-) interface within the nAChR extracellular domain, which is displayed by the (α3)
A method for high-content functional imaging of intracellular calcium responses in gelatin-immobilized non-adherent cells
Publisher: Elsevier BV
Date: 10-2020
Cytosolic [Ca2+] measurements in endothelium of rabbit cardiac valves using imaging fluorescence microscopy
Publisher: American Physiological Society
Date: 05-1994
DOI: 10.1152/AJPHEART.1994.266.5.H2130
Abstract: Cytosolic Ca2+ plays a critical role in the secretion of endothelium-derived factors. A new preparation that allows fluorescence imaging of intracellular free Ca2+ concentration ([Ca2+]i) in endothelial cells of rabbit cardiac valves is described. Electron micrographs of the valves revealed no underlying smooth muscle cells that might influence endothelial cell responses or contribute to [Ca2+]i signaling. The valve leaflets, which were 100 microns in diameter, were visualized using a specially designed chamber and a long working distance fluorescence objective. The semilunar valves (pulmonary and aortic) responded to endothelium-dependent vasodilators, including acetylcholine, with an increase in [Ca2+]i. Synchronized [Ca2+]i transients were observed in the endothelial monolayer in response to agonist stimulation in K(+)-free solutions. The ability to monitor changes in [Ca2+]i in a native endothelial monolayer provides a more realistic assessment of stimulus-response coupling within in idual cells and communication between cells of native endothelium. In addition, this preparation affords an opportunity for comparative studies of endothelium-related pathophysiologies, which can be induced experimentally in animal models.
Sodium and calcium gating currents in an Aplysia neurone.
Publisher: Wiley
Date: 06-1979
DOI: 10.1113/JPHYSIOL.1979.SP012826
Abstract: 1. Currents generated by depolarizing the hyperpolarizing voltage pulses were recorded at temperatures of 4--12 degrees C in the voltage-cl ed soma of R15 in aplysia abdominal ganglia exposed to solutions which suppressed ionic currents. 2. Subtraction of linear capacitive and leakage currents from current generated by voltage pulses to levels more positive than -20mV revealed non-linear transient outward displacement currents at the onset of the cl step (on-current) and transient inward displacement currents after the membrane potential returned to the holding potential (off-current). Only on-currents were studied. 3. Pulses to membrane potentials of -20 to 0 mV generated a displacement current with rapid onset and exponential decay. At membrane potentials more positive than o mV a second displacement current with a much slower onset and slower exponential decay was seen. Because the different threshold potentials for the two displacement currents were close to the different threshold potentials for Na and Ca ion currents, the two displacement currents were called Na and Ca 'gating' currents. 4. The amount of charge transfer during Ca gating currents increased sigmoidally with increasing depolarization, reaching a maximum at +30 to +40 mV. Half-maximum charge transfer occurred at +15 mV. 5. Total charge movement during Ca gating currents was maximal with holding potentials of -30 to -40 mV. More positive or more negative holding potentials produced a decrease in charge movement. 6. The time course of the gating currents, but not the total charge displaced, was very sensitive to temperature. The time constant of decay of Ca gating currents had a Q10 of about 3, whereas the total amount of charge displaced had a Q10 of 1.2. 7. The charge transfer during both Na and Ca gating currents and the litude of Na and Ca (but not K) ionic currents were reduced in solutions containing 1 mm-n-octanol.
Block of Voltage-Gated Calcium Channels by Peptide Toxins
Publisher: Springer US
Date: 2005
Are α9α10 nicotinic acetylcholine receptors a pain target for α-conotoxins?
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 05-09-2007
Abstract: The synthetic alpha-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.
Overexpressed Cav β3 Inhibits N-type (Cav 2.2) Calcium Channel Currents through a Hyperpolarizing Shift of Ultra-slow and Closed-state Inactivation
Publisher: Rockefeller University Press
Date: 15-03-2004
Abstract: It has been shown that β auxiliary subunits increase current litude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of β3 subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed β3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at “physiological” holding potentials (HPs) of −60 and −80 mV. At a HP of −80 mV, coinjection of various concentrations (0–12.5 ng) of the β3 with Cav2.2α1 and α2δ enhanced the maximum conductance of expressed channels at lower β3 concentrations but at higher concentrations (& .5 ng/cell) caused a marked inhibition. The β3-induced current suppression was reversed at a HP of −120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba2+ (40 mM) as a charge carrier also largely diminished the effect of β3 at −80 mV. Therefore, experimental conditions (HP, alent cation concentration, and β3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel β3 effect. Steady-state inactivation curves revealed that N-type channels exhibited “closed-state” inactivation without β3, and that β3 caused an ∼40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately −85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of β3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at −80 mV. Similar “ultra-slow” inactivation was observed for N-type channels without β3. Thus, β3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting “ultra-slow” and “closed-state” inactivation properties.
Ionic channels in vascular endothelial cells
Publisher: Elsevier BV
Date: 1994
DOI: 10.1016/1050-1738(94)90021-3
Abstract: Vascular endothelial cells respond to neurohumoral and physical stimuli to secrete mediators that modulate blood pressure and tissue perfusion. The initial response of endothelial cells usually involves an elevation of cytoplasmic Ca(2+), due to Ca(2+) release from intracellular stores and Ca(2+) influx from the extracellular space, and subsequent activation of Ca(2+)-dependent enzymes. The activation of cell-surface receptors by vasoactive substances stimulates Ca(2+) entry via plasmalemmal ion channels and the rate of Ca(2+) influx depends on the electrochemical gradient, which is modulated by the resting membrane potential. Membrane potential is regulated by a variety of voltage- and ligand-gated potassium channels that may function to repolarize the stimulated endothelial cell. This review focuses on ionic channels involved in receptor-mediated Ca(2+) entry and control of the membrane potential that are critical to stimulus-secretion coupling in vascular endothelium.
Is the A‐ring lactone of brevetoxin PbTX‐3 required for sodium channel orphan receptor binding and activity?
Publisher: Wiley
Date: 07-1994
Abstract: Brevetoxin PbTx-3 and non-toxic derivative 4 were investigated for their abilities to bind to the specific brevetoxin receptor site on rat brain synaptosomes and to modulate the normal function of voltage-gated sodium channels as determined by patch cl ing of cultured neurons. Compounds 4 and 5 are produced from PbTx-3 by opening of the A-ring lactone to the saturated and unsaturated diols using sodium borohydride in ethanol. Natural PbTx-3 exhibited tighter binding to rat brain synaptosomes by at least 3 orders of magnitude as determined by competitive radioligand binding experiments, and was also more effective at activating voltage-gated channels. Patch cl ing revealed the 3 orders of magnitude greater potency of PbTx-3 toxin over 5, although each produced delayed sodium channel opening and a pronounced delay in inactivation. Conformational modeling of the Brevetoxin B backbone indicates that the two molecules are identical except for the region of the A-Ring lactone. Thus, we conclude that the brevetoxin PbTx-3 backbone requires electrophilic functionality in the region of the lactone in PbTx-3, and that opening of the ring in 5 is sufficient to substantially reduce both binding and activity.
Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA
Publisher: Elsevier BV
Date: 10-2003
Isolation, Structure, and Activity of GID, a Novel α4/7-Conotoxin with an Extended N-terminal Sequence
Publisher: Elsevier BV
Date: 2003
Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia
Publisher: Elsevier BV
Date: 12-1999
DOI: 10.1016/S0306-4522(99)00505-9
Abstract: Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential litude in the rat submandibular ganglion. The litude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the litude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals.
Interaction of internal anions with potassium channels of the squid giant axon.
Publisher: Rockefeller University Press
Date: 10-1983
DOI: 10.1085/JGP.82.4.429
Abstract: The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the litude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.
Inhibition of cholinergic pathways in Drosophila melanogaster by α-conotoxins
Publisher: Wiley
Date: 02-12-2015
DOI: 10.1096/FJ.14-262733
Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells
Publisher: Springer Science and Business Media LLC
Date: 08-1989
DOI: 10.1007/BF00585046
Abstract: A geophysical seismic survey was conducted in the summer of 2001 off the northeastern coast of Sakhalin Island, Russia. The area of seismic exploration was immediately adjacent to the Piltun feeding grounds of the endangered western gray whale (Eschrichtius robustus). This study investigates relative abundance, behavior, and movement patterns of gray whales in relation to occurrence and proximity to the seismic survey by employing scan s ling, focal follow, and theodolite tracking methodologies. These data were analyzed in relation to temporal, environmental, and seismic related variables to evaluate potential disturbance reactions of gray whales to the seismic survey. The relative numbers of whales and pods recorded from five shore-based stations were not significantly different during periods when seismic surveys were occurring compared to periods when no seismic surveys were occurring and to the post-seismic period. Univariate analyses indicated no significant statistical correlation between seismic survey variables and any of the eleven movement and behavior variables. Multiple regression analyses indicated that, after accounting for temporal and environmental variables, 6 of 11 movement and behavior variables (linearity, acceleration, mean direction, blows per surfacing, and surface- e blow rate) were not significantly associated with seismic survey variables, and 5 of 11 variables (leg speed, reorientation rate, distance-from-shore, blow interval, and e time) were significantly associated with seismic survey variables. In summary, after accounting for environmental variables, no correlation was found between seismic survey variables and the linearity of whale movements, changes in whale swimming speed between theodolite fixes, mean direction of whale movement, mean number of whale exhalations per minute at the surface, mean time at the surface, and mean number of exhalations per minute during a whales surface-to- e cycle. In contrast, at higher received sound energy exposure levels, whales traveled faster, changed directions of movement less, were recorded further from shore, and stayed under water longer between respirations.
Are nicotinic acetylcholine receptors coupled to G proteins?
Publisher: Wiley
Date: 08-10-2013
Abstract: It was, until recently, accepted that the two classes of acetylcholine (ACh) receptors are distinct in an important sense: muscarinic ACh receptors signal via heterotrimeric GTP binding proteins (G proteins), whereas nicotinic ACh receptors (nAChRs) open to allow flux of Na+, Ca2+, and K+ ions into the cell after activation. Here we present evidence of direct coupling between G proteins and nAChRs in neurons. Based on proteomic, biophysical, and functional evidence, we hypothesize that binding to G proteins modulates the activity and signaling of nAChRs in cells. It is important to note that while this hypothesis is new for the nAChR, it is consistent with known interactions between G proteins and structurally related ligand-gated ion channels. Therefore, it underscores an evolutionarily conserved metabotropic mechanism of G protein signaling via nAChR channels.
Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2.
Publisher: Elsevier BV
Date: 04-2007
Solving the α-conotoxin folding problem: Efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antaqonists
Publisher: American Chemical Society (ACS)
Date: 17-02-2010
DOI: 10.1021/JA910602H
Abstract: Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for erse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an hiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.
Coronaridine congeners decrease neuropathic pain in mice and inhibit α9α10 nicotinic acetylcholine receptors and CaV2.2 channels.
Publisher: Elsevier BV
Date: 09-2020
Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons
Publisher: Frontiers Media SA
Date: 04-12-2020
DOI: 10.3389/FNCEL.2020.600895
Abstract: Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons ( NGN2 iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2 iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2 iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.
Role of CysI–CysIII Disulfide Bond on the Structure and Activity of α-Conotoxins at Human Neuronal Nicotinic Acetylcholine Receptors
Publisher: American Chemical Society (ACS)
Date: 17-08-2017
Inhibitors of calcium buffering depress evoked transmitter release at the squid giant synapse.
Publisher: Wiley
Date: 12-1985
DOI: 10.1113/JPHYSIOL.1985.SP015893
Abstract: Evoked release of transmitter at the squid giant synapse was examined under conditions where the calcium ion concentration in the presynaptic terminal was manipulated by inhibitors of calcium sequestration. Simultaneous intracellular recordings of presynaptic and post-synaptic resting and action potentials were made during bath application of one of the following metabolic inhibitors: sodium cyanide (NaCN), carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) ruthenium red (RuR) and sodium-free (lithium) sea water. Cyanide and lithium sea water reversibly depressed the post-synaptic potential (p.s.p.) whilst RuR and FCCP blocked the evoked post-synaptic response irreversibly. The progressive reduction of p.s.p. litude was accompanied by a reversible increase in synaptic delay. The time course of block of the p.s.p. was similar for different agents and dependent on the rate of presynaptic activity (30-40 min at 0.01 Hz). Recovery of the post-synaptic action potential following block by cyanide and lithium sea water was obtained within 40 min and 5 min respectively. Synaptic depression by the metabolic inhibitors does not result from changes in presynaptic resting or action potentials, nor from a change in post-synaptic receptor sensitivity. The post-synaptic response to the local ionophoresis of L-glutamate was unchanged following inhibition of evoked release of transmitter by cyanide. Injections of EGTA into presynaptic terminals poisoned by cyanide produced transient increases in p.s.p. litude, suggesting that cyanide is having its effect through raising intracellular calcium rather than lowering ATP. Control experiments injecting EGTA into unpoisoned nerve terminals showed no apparent effect on evoked transmitter release.
Analgesic transient receptor potential vanilloid‐1‐active compounds inhibit native and recombinant T‐type calcium channels
Publisher: Wiley
Date: 16-05-2019
DOI: 10.1111/BPH.14676
Acetylcholine‐evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.
Publisher: Wiley
Date: 03-1991
DOI: 10.1113/JPHYSIOL.1991.SP018466
Abstract: 1. The properties of acetylcholine (ACh)-activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch cl recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh-induced currents are mediated by nicotinic receptor activation. 2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currents exhibited strong inward rectification and a reversal (zero current) potential of -3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of alent cations from the intracellular or extracellular solutions. Unitary ACh-activated currents exhibited a linear I-V relationship with slope conductances of 32 pS in cell-attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions. 3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 approximately 1 microM), as well as by the neuromuscular relaxant, d-tubocurarine (Kd = 3 microM). Inhibition of ACh-evoked currents by hexamethonium could not be described by a simple blocking model for drug-receptor interaction. 4. The litude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (PX/PNa) followed the ionic selectivity sequence Cs+ (1.06) greater than Na+ (1.0) greater than Ca2+ (0.93). Anion substitution experiments showed a relative anion permeability, PCl/PNa less than or equal to 0.05. 5. The nicotinic ACh-activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation.
Cyclic-RGDfK-Directed Docetaxel Loaded Nanomicelles for Angiogenic Tumor Targeting
Publisher: Springer New York
Date: 2018
DOI: 10.1007/7653_2015_63
Functional modulation of the human voltage-gated sodium channel NaV1.8 by auxiliary β subunits
Publisher: Informa UK Limited
Date: 29-12-2020
RegIIA
Publisher: Elsevier BV
Date: 02-2012
DOI: 10.1016/J.BCP.2011.11.006
Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinson's disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage cl recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3β4 nAChRs found to date and is also active at α3β2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3β4 nAChRs.
Antagonism of Saxitoxin and Tetrodotoxin Block by Internal Monovalent Cations in Squid Axon
Publisher: Cold Spring Harbor Laboratory
Date: 22-12-2021
DOI: 10.1101/2021.12.21.473550
Abstract: The block of voltage-dependent sodium channels by saxitoxin (STX) and tetrodotoxin (TTX) was investigated in voltage-cl ed squid giant axons internally perfused with a variety of permeant monovalent cations. Substitution of internal Na + by either NH 4 + or N 2 H 5 + resulted in a reduction of outward current through sodium channels under control conditions. In contrast, anomalous increases in both inward and outward currents were seen for the same ions if some of the channels were blocked by STX or TTX, suggesting a relief of block by these internal cations. External NH 4 + was without effect on the apparent magnitude of toxin block. Likewise, internal inorganic monovalent cations were without effect, suggesting that proton donation by NH 4 + might be involved in reducing toxin block. Consistent with this hypothesis, decreases in internal pH mimicked internal perfusion with NH 4 + in reducing toxin block. The interaction between internally applied protons and externally applied toxin molecules appears to be competitive, as transient increases in sodium channel current were observed during step increases in intracellular pH in the presence of a fixed STX concentration. In addition to these effects on toxin block, low internal pH produced a voltage-dependent block of sodium channels and enhanced steady-state inactivation. Elevation of external buffer capacity only marginally diminished the modulation of STX block by internal NH 4 + , suggesting that alkalinization of the periaxonal space and a resultant decrease in the cationic STX concentration during NH 4 + perfusion may play only a minor role in the effect. These observations indicate that internal monovalent cations can exert trans-channel influences on external toxin binding sites on sodium channels.
Ciguatoxin (CTX-1) modulates single tetrodotoxin-sensitive sodium channels in rat parasympathetic neurones
Publisher: Elsevier BV
Date: 07-1998
DOI: 10.1016/S0304-3940(98)00575-8
Abstract: The actions of the marine neurotoxin, ciguatoxin-1 (CTX-1), were investigated in isolated parasympathetic neurones from neonatal rat intracardiac ganglia using patch-cl recording techniques. Under current cl conditions, bath application of 1-10 nM CTX-1 caused gradual membrane depolarization and tonic action potential firing. Action potential firing ceased with depolarization beyond approximately -35 mV and application of 300 nM tetrodotoxin (TTX) repolarized the cell to its control resting potential. In cell-attached membrane patches, 1-10 nM CTX-1 in the patch pipette markedly increased the open probability of single TTX-sensitive Na+ channels in response to depolarizing voltage steps but did not alter the unitary conductance (10 pS) or reversal potential. Under steady-state conditions, CTX-1 caused spontaneous opening of single Na+ channels which did not inactivate at hyperpolarized membrane potentials. CTX-1 increases neuronal excitability by shifting the voltage of activation of TTX-sensitive Na+ channels to more negative potentials.
Investigation of CACNA1I Cav3.3 Dysfunction in Hemiplegic Migraine.
Publisher: Frontiers Media SA
Date: 19-07-2022
DOI: 10.3389/FNMOL.2022.892820
Abstract: Familial hemiplegic migraine (FHM) is a severe neurogenetic disorder for which three causal genes, CACNA1A , SCN1A , and ATP1A2 , have been implicated. However, more than 80% of referred diagnostic cases of hemiplegic migraine (HM) are negative for exonic mutations in these known FHM genes, suggesting the involvement of other genes. Using whole-exome sequencing data from 187 mutation-negative HM cases, we identified rare variants in the CACNA1I gene encoding the T-type calcium channel Cav3.3. Burden testing of CACNA1I variants showed a statistically significant increase in allelic burden in the HM case group compared to gnomAD (OR = 2.30, P = 0.00005) and the UK Biobank (OR = 2.32, P = 0.0004) databases. Dysfunction in T-type calcium channels, including Cav3.3, has been implicated in a range of neurological conditions, suggesting a potential role in HM. Using patch-cl electrophysiology, we compared the biophysical properties of five Cav3.3 variants (p.R111G, p.M128L, p.D302G, p.R307H, and p.Q1158H) to wild-type (WT) channels expressed in HEK293T cells. We observed numerous functional alterations across the channels with Cav3.3-Q1158H showing the greatest differences compared to WT channels, including reduced current density, right-shifted voltage dependence of activation and inactivation, and slower current kinetics. Interestingly, we also found significant differences in the conductance properties exhibited by the Cav3.3-R307H and -Q1158H variants compared to WT channels under conditions of acidosis and alkalosis. In light of these data, we suggest that rare variants in CACNA1I may contribute to HM etiology.
Molecular engineering of conotoxins: The importance of loop size to α-conotoxin structure and function
Publisher: American Chemical Society (ACS)
Date: 28-08-2008
DOI: 10.1021/JM800278K
Abstract: Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.
Correction: Analgesic α-Conotoxin Binding Site on the Human GABAB Receptor (Molecular Pharmacology (2022) 102 (4) (196–208) DOI: 10.1124/molpharm.122.000543)
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 21-11-2022
Analgesic α-Conotoxin Binding Site on the Human GABAB Receptor.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 09-08-2022
DOI: 10.1124/MOLPHARM.122.000543
Abstract: The analgesic
Targeting of N-Type Calcium Channels via GABAB-Receptor Activation by α-Conotoxin Vc1.1 Variants Displaying Improved Analgesic Activity
Publisher: American Chemical Society (ACS)
Date: 25-10-2018
DOI: 10.1021/ACS.JMEDCHEM.8B01343
Abstract: α-Conotoxins exhibiting analgesic activity, such as Vc1.1, have been shown to inhibit α9α10 nicotinic acetylcholine receptors (nAChRs) and GABA
A novel α-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity
Publisher: Springer Science and Business Media LLC
Date: 17-01-2018
DOI: 10.1038/S41598-017-18479-4
Abstract: We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus , which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3β4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABA B receptor activation. In HEK293 cells expressing Ca V 2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba 2+ currents in a voltage- and state-dependent manner. Inhibition of Ca V 2.2 by Eu1.6 was concentration-dependent (IC 50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5–5.0 μg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.
A 4/8 Subtype α-Conotoxin Vt1.27 Inhibits N-Type Calcium Channels With Potent Anti-Allodynic Effect
Publisher: Frontiers Media SA
Date: 29-04-2022
DOI: 10.3389/FPHAR.2022.881732
Abstract: A novel 4/8 subtype α-conotoxin, Vt1.27 (NCCMFHTCPIDYSRFNC-NH 2 ), was identified from Conus vitulinus in the South China Sea by RACE methods. The peptide was synthesized and structurally characterized. Similar to other α-conotoxins that target neuronal nicotinic acetylcholine receptor (nAChR) subtypes, Vt1.27 inhibited the rat α3β2 nAChR subtype (IC 50 = 1160 nM) and was inactive at voltage-gated sodium and potassium channels in rat sensory neurons. However, Vt1.27 inhibited high voltage-activated N-type (Ca V 2.2) calcium channels expressed in HEK293T cells with an IC 50 of 398 nM. An alanine scan of the peptide showed that residues Phe 5 , Pro 9 , Ile 10 , and Ser 13 contribute significantly to the inhibitory activity of Vt1.27. The molecular dockings indicate that Vt1.27 inhibits the transmembrane region of Ca V 2.2, which is different from that of ω-conotoxins. Furthermore, Vt1.27 exhibited potent anti-allodynic effect in rat partial sciatic nerve injury (PNL) and chronic constriction injury (CCI) pain models at 10 nmol/kg level with the intramuscular injection. The pain threshold elevation of Vt1.27 groups was higher than that of α-conotoxin Vc1.1 in CCI rat models. These findings expand our knowledge of targets of α-conotoxins and potentially provide a potent, anti-allodynic peptide for the treatment of neuropathic pain.
Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons
Publisher: Elsevier BV
Date: 1992
DOI: 10.1016/S0928-4257(05)80009-9
Abstract: Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch cl recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current litude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.
Caffeine‐evoked, calcium‐sensitive membrane currents in rabbit aortic endothelial cells
Publisher: Wiley
Date: 05-1995
DOI: 10.1111/J.1476-5381.1995.TB16330.X
Abstract: 1. Single cell photometry and whole-cell patch cl recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and litude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current litude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the litude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and litude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current. 6. Externally applied tetraethylammonium ions (TEA, 3-10mM) reversibly inhibited unitary Ca2+-activated K+ currents and STOCs in endothelial cells but failed to inhibit completely the outward current evoked by 20 mM caffeine.7. Bath application of 0.1 mM zinc ion (Zn2+), a chloride channel blocker, did not affect unitary currents or STOCs but reduced the litude of the caffeine-evoked current by >75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the litude of the caffeine-induced outward current. Bath application of 0.1 mM Zn2+ and 10 mM TEA completely blocked the caffeine-evoked outward current in endothelial cells.8. Caffeine-induced Ca2+ release from intracellular stores evokes a transient rise in [Ca2+1, which is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine sensitive current by TEA and Zn2+ suggests that Ca2+-activated K+ and Cl- conductances contribute to the caffeine response in rabbit aortic endothelial cells.
Alkyne-Bridged α-Conotoxin Vc1.1 Potently Reverses Mechanical Allodynia in Neuropathic Pain Models
Publisher: American Chemical Society (ACS)
Date: 16-03-2021
Brevetoxin versus Brevenal Modulation of Human Nav1 Channels
Publisher: MDPI AG
Date: 07-07-2023
DOI: 10.3390/MD21070396
Abstract: Brevetoxins (PbTx) and brevenal are marine ladder-frame polyethers. PbTx binds to and activates voltage-gated sodium (Nav) channels in native tissues, whereas brevenal antagonizes these actions. However, the effects of PbTx and brevenal on recombinant Nav channel function have not been systematically analyzed. In this study, the PbTx-3 and brevenal modulation of tissue-representative Nav channel subtypes Nav1.2, Nav1.4, Nav1.5, and Nav1.7 were examined using automated patch-cl . While PbTx-3 and brevenal elicit concentration-dependent and subtype-specific modulatory effects, PbTx-3 is -fold more potent than brevenal. Consistent with effects observed in native tissues, Nav1.2 and Nav1.4 channels were PbTx-3- and brevenal-sensitive, whereas Nav1.5 and Nav1.7 appeared resistant. Interestingly, the incorporation of brevenal in the intracellular solution caused Nav channels to become less sensitive to PbTx-3 actions. Furthermore, we generated a computational model of PbTx-2 bound to the lipid-exposed side of the interface between domains I and IV of Nav1.2. Our results are consistent with competitive antagonism between brevetoxins and brevenal, setting a basis for future mutational analyses of Nav channels’ interaction with brevetoxins and brevenal. Our findings provide valuable insights into the functional modulation of Nav channels by brevetoxins and brevenal, which may have implications for the development of new Nav channel modulators with potential therapeutic applications.
Auxiliary subunit regulation of high‐voltage activated calcium channels expressed in mammalian cells
Publisher: Wiley
Date: 21-06-2004
Nicotinic acetylcholine receptor subtype expression, function, and pharmacology: Therapeutic potential of α-conotoxins
Publisher: Elsevier BV
Date: 05-2023
NMDA receptor subunit‐dependent modulation by conantokin‐G and Ala(7)‐conantokin‐G
Publisher: Wiley
Date: 29-11-2005
DOI: 10.1111/J.1471-4159.2005.03574.X
Abstract: The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage cl technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.
Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins
Publisher: Elsevier BV
Date: 11-2016
Novel analgesic ω-conotoxins from the vermivorous cone snail Conus moncuri provide new insights into the evolution of conopeptides
Publisher: Springer Science and Business Media LLC
Date: 07-09-2018
DOI: 10.1038/S41598-018-31245-4
Abstract: Cone snails are a erse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Ca v 2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri , the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Ca v 2.2 in fluorimetric assays and rat Ca v 2.2 in patch cl studies, and both potently displaced radiolabeled ω-conotoxin GVIA ( 125 I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC 50 2–9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Ca v 2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae .
Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation
Publisher: Frontiers Media SA
Date: 28-10-2016
Ciguatoxin-induced oscillations in membrane potential and action potential firing in rat parasympathetic neurons
Publisher: Wiley
Date: 07-2002
DOI: 10.1046/J.1460-9568.2002.02071.X
Abstract: The actions of ciguatoxins from the Pacific (P-CTX-1) and Caribbean (C-CTX-1) regions were investigated in isolated parasympathetic neurons from rat intracardiac ganglia using patch-cl recording techniques. Under current-cl conditions, bath application of P-CTX-1 (1-10 nm) or C-CTX-1 (10-30 nm) caused a gradual depolarization that was accompanied by oscillation of the membrane potential leading to tonic action potential firing. Membrane potential oscillations were observed between -45 and -60 mV and had an litude of 10-20 mV and a mean frequency of 10 Hz. Oscillation frequency was temperature-dependent with a Q10 of 2.0. Membrane oscillations were temporarily inhibited by hyperpolarizing current pulses and potentiated by weak depolarizing current pulses. The litude of oscillations was reduced upon lowering the external Na+ concentration and inhibited by tetrodotoxin (TTX), tetracaine or Zn2+. Tetraethylammonium, 4-aminopyridine, Cs+, Cd2+, Ba2+, 1,4,4'-diothiocyanato-2,2'-stilbenedisulphonic acid (DIDS) and ouabain had no effect on the CTX-1-induced membrane depolarization and oscillations. Brevetoxin (PbTx-3, 100 nm), in contrast to CTX-1, caused a membrane depolarization that was not associated with oscillation of the membrane potential. Under voltage-cl conditions, P-CTX-1 inhibited the peak litude of the voltage-dependent Na+ current and shifted the activation curve to more negative potentials, but membrane oscillations were not seen in this configuration. These results suggest that ciguatoxins cause oscillation of the membrane potential in mammalian autonomic neurons by modifying the activation and inactivation properties of a population of TTX-sensitive Na+ channels.
Developmental changes in hyperpolarization-activated currents Ih and IK(IR) in isolated rat intracardiac neurons
Publisher: American Physiological Society
Date: 07-2001
Abstract: The hyperpolarization-activated nonselective cation current, I h , was investigated in neonatal and adult rat intracardiac neurons. I h was observed in all neurons studied and displayed slow time-dependent rectification. I h was isolated by blockade with external Cs + (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I h was approximately twofold greater in neurons from neonatal (−4.1 pA F at −130 mV) as compared with adult (−2.3 pA F) rats however, the reversal potential and activation parameters were unchanged. The reversal potential and litude of I h was sensitive to changes in external Na + and K + concentrations. An inwardly rectifying K + current, I K(IR) , was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba 2+ . I K(IR) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of −0.6 pA F at −130 mV. I K(IR) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K + rectifier described in other mammalian autonomic neurons. I K(IR) was sensitive to changes in external K + , whereby raising the external K + concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na + had no effect on the reversal potential or litude of I K(IR) . I K(IR) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I h may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.
Related Organisations
University College London
Location: United Kingdom of Great Britain and Northern Ireland
University Of Wollongong Illawarra Health And Medical Research Institute
Location: Australia
University Of Miami School Of Medicine
Location: United States of America
Related Funding Activities
G-protein Coupled Receptor-mediated Calcium Signalling In Parasympathetic Neurons
Start Date: 2004
End Date: 2006
Funder: Australian Research Council
View Funded ActivityIs Calcium Part Of The Mechanism Used In Glucose Signalling In Embryogenesis
Start Date: 2002
End Date: 2002
Funder: Australian Research Council
View Funded ActivityDefining Mechanisms Of Action Of Novel Alpha-conotoxins At Nicotinic Receptor-channels
Start Date: 2010
End Date: 2014
Funder: Australian Research Council
View Funded ActivityAlpha-Conotoxins: Selective Probes For Nicotinic Receptor Subtype Structure And Function
Start Date: 2007
End Date: 2009
Funder: Australian Research Council
View Funded ActivityDiscovery And Development Of Better Pain Treatments
Start Date: 2015
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityDiscovery And Development Of Better Pain Treatments
Start Date: 2015
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityA New G-protein Coupled Receptor Target For Conotoxins
Start Date: 2010
End Date: 2012
Funder: Australian Research Council
View Funded ActivityNew Modulators Of Voltage-gated Sodium Channel Subtypes From Australian Tarantula Venoms
Start Date: 2007
End Date: 2009
Funder: Australian Research Council
View Funded ActivityConotoxins: Novel Probes For Ion Channel Structure And Function
Start Date: 2002
End Date: 2006
Funder: Australian Research Council
View Funded ActivityNicotinic Receptor Structure And Function Probed With Conotoxins
Start Date: 2015
End Date: 2019
Funder: Australian Research Council
View Funded ActivityElectrophysiology Facility For Cell Phenotyping And Drug Discovery
Start Date: 2018
End Date: 2018
Funder: Australian Research Council
View Funded ActivityIONIC BASIS OF EXCITABILITY IN INTRINSIC CARDIAC NEURONS
Start Date: 1985
End Date: 1996
Funder: National Heart Lung and Blood Institute
View Funded ActivityAutomated Patch Clamp System
Start Date: 2007
End Date: 2007
Funder: Australian Research Council
View Funded ActivityThe Centre For Advanced Light Microscopy: Equipment For In-vivo Multiphoton Microscopy And High-throughput Confocal Microscopy
Start Date: 2004
End Date: 2004
Funder: Australian Research Council
View Funded ActivityMembrane Excitability And Cellular Calcium Regulation In The Peripheral Nervous System Under Different (patho)-physiological Conditions And In Inflammatory Disease
Start Date: 2008
End Date: 2008
Funder: Australian Research Council
View Funded ActivityA Cell Sorter Facility For Neuroscience And Related Biotechnology
Start Date: 2003
End Date: 2003
Funder: Australian Research Council
View Funded ActivityAlpha-Conotoxin Inhibition Of Chronic Visceral Pain
Start Date: 2013
End Date: 2016
Funder: National Health and Medical Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE0775613
Start Date: 02-2007
End Date: 02-2008
Amount: $467,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE0453524
Start Date: 06-2004
End Date: 06-2005
Amount: $321,456.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP1093177
Start Date: 2010
End Date: 12-2014
Amount: $360,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP150103990
Start Date: 2015
End Date: 06-2021
Amount: $634,100.00
Funder: Australian Research Council
View Funded ActivityLinkage - International - Grant ID: LX0211798
Start Date: 2002
End Date: 12-2003
Amount: $48,674.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP1093115
Start Date: 2010
End Date: 12-2015
Amount: $1,400,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP210102405
Start Date: 05-2021
End Date: 05-2024
Amount: $438,500.00
Funder: Australian Research Council
View Funded ActivityLinkage Projects - Grant ID: LP0774870
Start Date: 03-2008
End Date: 12-2010
Amount: $485,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP0452089
Start Date: 2004
End Date: 12-2007
Amount: $225,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE180100066
Start Date: 2018
End Date: 12-2018
Amount: $443,311.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE0347955
Start Date: 2002
End Date: 12-2003
Amount: $500,000.00
Funder: Australian Research Council
View Funded ActivityLinkage - International - Grant ID: LX0882427
Start Date: 2008
End Date: 12-2008
Amount: $131,306.00
Funder: Australian Research Council
View Funded ActivityIndustrial Transformation Training Centres - Grant ID: IC200100052
Start Date: 03-2021
End Date: 03-2026
Amount: $4,789,838.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE100100226
Start Date: 07-2010
End Date: 08-2011
Amount: $424,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP0770884
Start Date: 01-2009
End Date: 06-2011
Amount: $990,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP0208295
Start Date: 2002
End Date: 12-2007
Amount: $2,347,000.00
Funder: Australian Research Council
View Funded Activity