ORCID Profile
0000-0003-0353-7228
Current Organisations
Florey Institute of Neuroscience and Mental Health
,
University of Melbourne
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Publisher: American Thoracic Society
Date: 10-2012
Publisher: Wiley
Date: 03-2008
Publisher: Springer New York
Date: 24-10-2019
DOI: 10.1007/978-1-4939-9717-6_21
Abstract: P2X receptors are a structurally and functionally distinctive family of ligand-gated ion channels that play important roles in mediating extracellular adenosine 5'-triphosphate (ATP) signaling in erse physiological and pathophysiological processes. For several decades, the "manual" patch-cl technique was regarded as the gold standard assay for investigating ion channel properties. More recently, breakthroughs in the development of automated patch-cl technologies are enabling the study of ion channels, with much greater throughput capacities. These automated platforms, of which there are many, generate consistent, reliable, high-fidelity data. This chapter demonstrates the versatility of one of these technologies for ligand-gated ion channels, with a particular emphasis on protocols that address some of the issues of receptor desensitization that are commonly associated with P2X receptor-mediated currents.
Publisher: Wiley
Date: 05-2017
DOI: 10.1002/ANA.24929
Abstract: To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever. We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-cl to explore biophysical properties of wild-type and mutant K Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic-clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG-electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type K MEAK has a relatively homogeneous presentation, resembling Unverricht-Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type K
Publisher: Wiley
Date: 21-07-2016
DOI: 10.1111/EPI.13474
Abstract: Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-cl recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 μm) or phenytoin (50 μm). NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current litude for carbamazepine (31.6 ± 5.9%, n = 9 p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7 p < 0.001). Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength.
Publisher: Society for Neuroscience
Date: 18-08-2004
DOI: 10.1523/JNEUROSCI.1979-04.2004
Abstract: Fast inhibition in the nervous system is commonly mediated by GABA A receptors comprised of 2α/2β/1γ subunits. In contrast, GABA C receptors containing onlyρ subunits (ρ1-ρ3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the ρ1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the ρ1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA C receptor agonist cis- 4-aminocrotonic acid (100-800 μM) requires the ρ1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA A (bicuculline, 10 μM) and GABA C [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) 40-160 μM] receptors. Responses to GABA C agonists were also enhanced by the GABA A receptor modulator pentobarbitone (300 μM). Spontaneous and evoked IPSPs were reduced in litude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA A and GABA C subunits formed a heteromeric receptor. Immunohistochemistry indicated that ρ1 and α1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA C receptor agonists were enhanced by the GABA A receptor modulator zolpidem (500 n m ), which acts on the α1 subunit when the γ2 subunit is also present. Finally, coimmunoprecipitation indicated that the ρ1 subunit formed complexes that also containedα1 and γ2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA A and GABA C receptor subunits.
Publisher: Society for Neuroscience
Date: 02-02-2005
DOI: 10.1523/JNEUROSCI.3740-04.2005
Abstract: Homeostatic maintenance of widespread functions is critically dependent on the activity of the sympathetic nervous system. This activity is generated by the CNS acting on the sole output cells in the spinal cord, sympathetic preganglionic neurons (SPNs). SPNs are subject to control from both supraspinal and spinal inputs that exert effects through activation of direct or indirect pathways. A high proportion of indirect control is attributable to activation of spinal interneurons in a number of locations. However, little is known about the different groups of interneurons with respect to their neurochemistry or function. In this study, we report on a novel group of GABAergic interneurons located in the spinal central autonomic area (CAA) that directly inhibit SPN activity. In situ hybridization studies demonstrated a group of neurons that contained mRNA for glutamic acid decarboxylase (GAD) 65 and GAD 67 within the CAA. Combining in situ hybridization with trans-synaptic labeling from the adrenal gland using pseudorabies virus identified presympathetic GABAergic neurons in the CAA. Electrical stimulation of the CAA elicited monosynaptic IPSPs in SPNs located laterally in the intermediolateral cell column. IPSPs were GABAergic, because they reversed at the chloride reversal potential and were blocked by bicuculline. Chemical activation of neurons in the CAA hyperpolarized SPNs, an effect that was also bicuculline sensitive. We conclude that the CAA contains GABAergic interneurons that impinge directly onto SPNs to inhibit their activity and suggest that these newly identified interneurons may play an essential role in the regulation of sympathetic activity and thus homeostasis.
Publisher: Wiley
Date: 19-01-2011
DOI: 10.1096/FJ.10-173450
Abstract: Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age-related neurodegenerative diseases. An early step in this process is the loss of intracellular K(+) via K(+) channels, and evidence indicates that K(v)2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up-regulation of the inducible enzyme heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K(v)2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K(v)2.1 in HEK293 cells increases their vulnerability to oxidant-induced apoptosis, and this is reversed by CO. In hippoc al neurons, CO selectively inhibits K(v)2.1, reverses the dramatic oxidant-induced increase in K(+) current density, and provides marked protection against oxidant-induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.
Publisher: Wiley
Date: 04-2008
Publisher: Elsevier BV
Date: 03-2000
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 30-03-2016
Publisher: Wiley
Date: 20-01-2005
Publisher: Society for Neuroscience
Date: 15-09-2001
Publisher: Springer Science and Business Media LLC
Date: 02-2009
Publisher: Elsevier BV
Date: 04-2000
Publisher: Society for Neuroscience
Date: 08-2007
DOI: 10.1523/JNEUROSCI.0638-07.2007
Abstract: Sensory afferent signals from neck muscles have been postulated to influence central cardiorespiratory control as components of postural reflexes, but neuronal pathways for this action have not been identified. The intermedius nucleus of the medulla (InM) is a target of neck muscle spindle afferents and is ideally located to influence such reflexes but is poorly investigated. To aid identification of the nucleus, we initially produced three-dimensional reconstructions of the InM in both mouse and rat. Neurochemical analysis including transgenic reporter mice expressing green fluorescent protein in GABA-synthesizing neurons, immunohistochemistry, and in situ hybridization revealed that the InM is neurochemically erse, containing GABAegric and glutamatergic neurons with some degree of colocalization with parvalbumin, neuronal nitric oxide synthase, and calretinin. Projections from the InM to the nucleus tractus solitarius (NTS) were studied electrophysiologically in rat brainstem slices. Electrical stimulation of the NTS resulted in antidromically activated action potentials within InM neurons. In addition, electrical stimulation of the InM resulted in EPSPs that were mediated by excitatory amino acids and IPSPs mediated solely by GABA A receptors or by GABA A and glycine receptors. Chemical stimulation of the InM resulted in (1) a depolarization of NTS neurons that were blocked by NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[ f ]quinoxaline-7-sulfonoamide) or kynurenic acid and (2) a hyperpolarization of NTS neurons that were blocked by bicuculline. Thus, the InM contains neurochemically erse neurons and sends both excitatory and inhibitory projections to the NTS. These data provide a novel pathway that may underlie possible reflex changes in autonomic variables after neck muscle spindle afferent activation.
Publisher: Elsevier BV
Date: 2010
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2010
DOI: 10.1161/ATVBAHA.110.205666
Abstract: Objective— To determine whether calcium-permeable channels are targets for the oxidized phospholipids: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC). Methods and Results— Oxidized phospholipids are key factors in inflammation and associated diseases, including atherosclerosis however, the initial reception mechanisms for cellular responses to the factors are poorly understood. Low micromolar concentrations of PGPC and POVPC evoked increases in intracellular calcium in human embryonic kidney 293 cells that overexpressed human transient receptor potential canonical 5 (TRPC5) but not human TRP melastatin (TRPM) 2 or 3. The results of electrophysiological experiments confirmed stimulation of TRPC5. To investigate relevance to endogenous channels, we studied proliferating vascular smooth muscle cells from patients undergoing coronary artery bypass surgery. PGPC and POVPC elicited calcium entry that was inhibited by anti-TRPC5 or anti-TRPC1 antibodies or dominant-negative mutant TRPC5. Calcium release did not occur. The effect was functionally relevant because it enhanced cell migration. The actions of PGPC and POVPC depended on G i/o proteins but not on previously identified G protein–coupled receptors for oxidized phospholipids. Conclusion— Stimulation of calcium-permeable TRPC5-containing channels may be an early event in cellular responses to oxidized phospholipids that couples to cell migration and requires an unidentified G protein–coupled receptor.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 10-10-2008
DOI: 10.1161/CIRCRESAHA.108.182931
Abstract: Stromal interaction molecule 1 (STIM1) is a predicted single membrane–spanning protein involved in store-operated calcium entry and interacting with ion channels including TRPC1. Here, we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a nonselective cationic current in response to store depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA. Calcium entry evoked by store depletion was partially inhibited by STIM1 short interfering RNA, whereas calcium release was unaffected. STIM1 short interfering RNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1, and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localization studies revealed TRPC1 alone, as well as with STIM1. The data suggest a complex situation in which there is not only plasma membrane–spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current but also TRPC1-STIM1 interaction, a TRPC1-dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation.
Publisher: Wiley
Date: 31-01-2020
Publisher: Springer Science and Business Media LLC
Date: 2008
DOI: 10.1038/NATURE06414
Publisher: Springer US
Date: 30-10-2020
Publisher: Elsevier BV
Date: 2013
Publisher: Wiley
Date: 09-11-2016
DOI: 10.1111/JNC.13858
Abstract: Toluene is a commonly abused inhalant that is easily accessible to adolescents. Despite the increasing incidence of use, our understanding of its long-term impact remains limited. Here, we used a range of techniques to examine the acute and chronic effects of toluene exposure on glutameteric and GABAergic function, and on indices of psychological function in adult rats after adolescent exposure. Metabolomics conducted on cortical tissue established that acute exposure to toluene produces alterations in cellular metabolism indicative of a glutamatergic and GABAergic profile. Similarly, in vitro electrophysiology in Xenopus oocytes found that acute toluene exposure reduced NMDA receptor signalling. Finally, in an adolescent rodent model of chronic intermittent exposure to toluene (10 000 ppm), we found that, while toluene exposure did not affect initial learning, it induced a deficit in updating that learning when response-outcome relationships were reversed or degraded in an instrumental conditioning paradigm. There were also group differences when more effort was required to obtain the reward toluene-exposed animals were less sensitive to progressive ratio schedules and to delayed discounting. These behavioural deficits were accompanied by changes in subunit expression of both NMDA and GABA receptors in adulthood, up to 10 weeks after the final exposure to toluene in the hippoc us, prefrontal cortex and ventromedial striatum regions with recognized roles in behavioural flexibility and decision-making. Collectively, our data suggest that exposure to toluene is sufficient to induce adaptive changes in glutamatergic and GABAergic systems and in adaptive behaviour that may underlie the deficits observed following adolescent inhalant abuse, including susceptibility to further drug-use.
Publisher: Hindawi Limited
Date: 13-04-2017
DOI: 10.1002/HUMU.23218
Publisher: Springer Science and Business Media LLC
Date: 11-2002
DOI: 10.1038/420042A
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.BBRC.2008.07.096
Abstract: Reciprocal functional inhibition between P2X and GABA(A/C) receptors represents a novel mechanism fine-tuning neuronal excitability. However, the participating receptors and underlying mechanisms are not fully understood. P2X(4) receptor is widely found in neurons that express GABA(C) rho1 receptor. Thus, we co-expressed P2X(4) and rho1 receptors in HEK293 cells and, using patch-cl recording, examined whether they have mutual functional inhibition. Currents evoked by simultaneous application of ATP and GABA (I(ATP+GABA)) were significantly smaller compared to the addition of I(ATP) and I(GABA). Furthermore, I(ATP) were strongly suppressed during rho1 receptor activation. Similarly, I(GABA) were greatly attenuated during P2X(4) receptor activation. Such mutual inhibition was absent in cells only expressing P2X(4) or rho1 receptor. Taken together, these functional data support negative cross-talk between P2X(4) and rho1 receptors.
Publisher: Public Library of Science (PLoS)
Date: 20-03-2015
Publisher: Wiley
Date: 10-2008
DOI: 10.1038/BJP.2008.283
Publisher: Wiley
Date: 04-2014
DOI: 10.1002/ANA.24128
Publisher: Wiley
Date: 06-06-2002
DOI: 10.1046/J.1471-4159.2002.00920.X
Abstract: Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippoc al slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippoc us. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippoc us, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.
Publisher: Springer Science and Business Media LLC
Date: 10-06-2022
DOI: 10.1186/S42238-022-00136-X
Abstract: Purified cannabidiol (CBD), a non-psychoactive phytocannabinoid, has gained regulatory approval to treat intractable childhood epilepsies. Despite this, artisanal and commercial CBD-dominant hemp-based products continue to be used by epilepsy patients. Notably, the CBD doses used in these latter products are much lower than that found to be effective in reducing seizures in clinical trials with purified CBD. This might be because these CBD-dominant hemp products contain other bioactive compounds, including phytocannabinoids and terpenes, which may exert unique effects on epilepsy-relevant drug targets. Voltage-gated sodium (Na V ) channels are vital for initiation of neuronal action potential propagation and genetic mutations in these channels result in epilepsy phenotypes. Recent studies suggest that Na V channels are inhibited by purified CBD. However, the effect of cannabis-based products on the function of Na V channels is unknown. Using automated-planar patch-cl technology, we profile a hemp-derived nutraceutical product (NP) against human Na V 1.1–Na V 1.8 expressed in mammalian cells to examine effects on the biophysical properties of channel conductance, steady-state fast inactivation and recovery from fast inactivation. NP modifies peak current litude of the Na V 1.1–Na V 1.7 subtypes and has variable effects on the biophysical properties for all channel subtypes tested. NP potently inhibits Na V channels revealing half-maximal inhibitory concentration (IC 50 ) values of between 1.6 and 4.2 μg NP/mL. Purified CBD inhibits Na V 1.1, Na V 1.2, Na V 1.6 and Na V 1.7 to reveal IC 50 values in the micromolar range. The CBD content of the product equates to IC 50 values (93–245 nM), which are at least an order of magnitude lower than purified CBD. Unlike NP, hemp seed oil vehicle alone did not inhibit Na V channels, suggesting that the inhibitory effects of NP are independent of hemp seed oil. This CBD-dominant NP potently inhibits Na V channels. Future study of the in idual elements of NP, including phytocannabinoids and terpenes, may reveal a potent in idual component or that its components interact to modulate Na V channels.
Publisher: Wiley
Date: 18-11-2015
DOI: 10.1002/ANA.24520
Publisher: Elsevier BV
Date: 09-2003
DOI: 10.1016/S0006-8993(03)03112-3
Abstract: The angiotensin II type 1 receptor (AT1R) in the central nervous system (CNS) plays a pivotal role in determining blood pressure. However, the relationship of the receptor to neurones in the spinal cord which are the final CNS contribution to sympathetic outflow is unknown. Here we first use RT-PCR to show that AT1A, AT1B and AT2 receptors are expressed in thoracic spinal cord of the rat. Using light microscopic immunohistochemistry we find that the AT1 receptor in the thoracic spinal cord is located on neurones and ependymal cells. Neurones with extensive immunostaining of somata and dendrites were located in the intermediolateral cell column (IML) and lamina X (the central autonomic area), regions associated with autonomic outflow, as well as in lamina V. Retrograde labelling and dual immunolabelling with nNOS revealed that those AT1R-immunopositive cells in the IML were sympathetic preganglionic neurones, while those in lamina X were unlikely to be. Punctate labelling resembling that of axonal fibres and terminals was evident in lamina II of the dorsal horn and throughout the cord. Electron microscopy in the IML and lamina X revealed that these puncta were presynaptic terminals, but also astrocyte processes. Immunolabelling was also evident beneath the plasma membrane in neuronal somata. These data show that the AT1R in the spinal cord is ideally located to influence autonomic outflow and hence participate in the CNS determination of blood pressure.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 15-03-2011
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 14-05-2010
DOI: 10.1161/CIRCRESAHA.110.219329
Abstract: Rationale : Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in β cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system. Objective : To determine the relevance and regulation of TRPM3 in vascular biology. Methods and Results : TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. β-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity. Conclusions : The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.
Publisher: Wiley
Date: 07-06-2017
DOI: 10.1002/PRP2.319
Publisher: Frontiers Media SA
Date: 20-02-2023
DOI: 10.3389/FPHYS.2023.1081186
Abstract: Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabi arinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (Na V ) channels play a pivotal role in initiation and propagation of the neuronal action potential and Na V 1.1, Na V 1.2, Na V 1.6 and Na V 1.7 are associated with the intractable epilepsies and pain conditions. Methods: In this study, using automated-planar patch-cl technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD. Results: CBD and CBGA inhibited peak current litude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited Na V 1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on Na V 1.1, Na V 1.2, and Na V 1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for Na V 1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced Na V 1.1 and Na V 1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V 0.5 inact), and for Na V 1.7 channel conductance was reduced. CBGA also reduced Na V 1.1 and Na V 1.7 channel availability by shifting the voltage-dependence of activation (V 0.5 act) to a more depolarized potential, and for Na V 1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for Na V 1.2, where V 0.5 inact was unaffected. Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.
Publisher: Proceedings of the National Academy of Sciences
Date: 03-08-2018
Abstract: Spider venom is a rich source of peptides, many targeting ion channels. We assessed a venom peptide, Hm1a, as a potential targeted therapy for Dravet syndrome, the genetic epilepsy linked to a mutation in the gene encoding the sodium channel alpha subunit Na V 1.1. Cell-based assays showed Hm1a was selective for hNa V 1.1 over other sodium and potassium channels. Utilizing a mouse model of Dravet syndrome, Hm1a restored inhibitory neuron function and significantly reduced seizures and mortality in heterozygote mice. Evidence from the structure of Hm1a and modeling suggest Hm1a interacts with Na V 1.1 inactivation domains, providing a structural correlate of the functional mechanisms. This proof-of-concept study provides a promising strategy for future drug development in genetic epilepsy and other neurogenetic disorders.
Publisher: Elsevier BV
Date: 04-2006
DOI: 10.1016/J.BRAINRES.2006.01.019
Abstract: Hyperpolarization-activated cyclic nucleotide-gated (HCN) non-selective cation channels in neurons carry currents proposed to perform erse functions, including the hyperpolarization activated Ih current. The 4 HCN subunits have unique but overlapping patterns of expression in the CNS. Here, we examined the distribution of HCN1 channel subunits in the brainstem and spinal cord using immunohistochemistry. At all levels of the spinal cord dorsal horn, HCN1 immunoreactivity (HCN1-IR) was predominantly absent from laminae I and II, while a dense band of punctate labeling was visible in lamina III. Labeled neurons were identified in close vicinity to the central canal, in the lateral spinal nucleus, in the ventral horn and occasionally in lamina II and III. Those in the ventral horn were identified as alpha motor neurons using retrograde tracing and/or double or triple immunostaining with neuronal markers neurofilament 200 (NF200) and choline acetyltransferase. HCN1-IR neurons in the brainstem included neurons in sensory pathways such as the dorsal column nuclei, the area postrema, the spinal trigeminal nucleus as well as identified motor neurons in motor nuclei. In the nucleus ambiguus, a mixed visceral/motor nucleus, HCN1-IR was present only in NF200-IR cells, suggesting that it is expressed in motor but not autonomic preganglionic neurons. HCN1-IR motor neurons in the nucleus ambiguus also expressed the neurokinin 1 receptor and were labeled retrogradely from the larnyx. At the light microscopic level, the NTS and inferior olive contained punctate labeling, which ultrastructural examination revealed to be present in predominantly synaptic terminals or dendrites respectively. These data therefore described the first localization of the HCN1 subunit in the spinal cord and extend previous reports from the brainstem.
Publisher: Humana Press
Date: 2013
DOI: 10.1007/978-1-62703-351-0_13
Abstract: Ion channels are integral membrane proteins that regulate the flow of ions across the plasma membrane and the membranes of intracellular organelles of both excitable and non-excitable cells. Ion channels are vital to a wide variety of biological processes and are prominent components of the nervous system and cardiovascular system, as well as controlling many metabolic functions. Furthermore, ion channels are known to be involved in many disease states and as such have become popular therapeutic targets. For many years now manual patch-cl ing has been regarded as one of the best approaches for assaying ion channel function, through direct measurement of ion flow across these membrane proteins. Over the last decade there have been many remarkable breakthroughs in the development of technologies enabling the study of ion channels. One of these breakthroughs is the development of automated planar patch-cl technology. Automated platforms have demonstrated the ability to generate high-quality data with high throughput capabilities, at great efficiency and reliability. Additional features such as simultaneous intracellular and extracellular perfusion of the cell membrane, current cl operation, fast compound application, an increasing rate of parallelization, and more recently temperature control have been introduced. Furthermore, in addition to the well-established studies of over-expressed ion channel proteins in cell lines, new generations of planar patch-cl systems have enabled successful studies of native and primary mammalian cells. This technology is becoming increasingly popular and extensively used both within areas of drug discovery as well as academic research. Many platforms have been developed including NPC-16 Patchliner(®) and SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich), CytoPatch™ (Cytocentrics AG, Rostock), PatchXpress(®) 7000A, IonWorks(®) Quattro and IonWorks Barracuda™, (Molecular Devices, LLC) Dynaflow(®) HT (Cellectricon AB, Mölndal), QPatch HT (Sophion A/S, Copenhagen), IonFlux HT (Fluxion Bioscience Inc, USA), which have demonstrated the capability to generate recordings similar in quality to that of conventional patch cl ing. Here we describe features of Nanion's NPC-16 Patchliner(®) and processes and protocols suited for this particularly flexible and successful high-throughput automated platform, which is based on planar patch-cl technology. However, many of the protocols and notes given in this chapter can be applied to other automated patch-cl platforms, similarly.
Publisher: Elsevier BV
Date: 06-2001
Location: Australia
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Carol Milligan.