ORCID Profile
0000-0003-1523-4814
Current Organisation
University of Sydney
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Biophysics | Central Nervous System | Applied Mathematics | Biochemistry and Cell Biology | Membrane Biology | Biological Mathematics | Mathematical Software |
Disease distribution and transmission | Infectious diseases | Nervous system and disorders | Biological sciences | Cancer and related disorders | Physical sciences
Publisher: Wiley
Date: 07-2001
DOI: 10.1111/J.1469-7793.2001.00159.X
Abstract: 1. The effects of niflumic acid on the substrate-gated currents mediated by the glutamate transporter EAAT4 expressed in Xenopus laevis oocytes were examined using radiolabelled substrate flux measurements and two-electrode voltage cl techniques. 2. Niflumic acid significantly enhanced the substrate-gated currents in EAAT4, without affecting the affinity of the substrates towards EAAT4. At a concentration of 300 microM, niflumic acid caused a 19 +/- 5 % reduction in L-[(3)H]glutamate uptake and no significant effect on the uptake of DL-[(3)H]aspartate. Thus, enhancement of the substrate-gated currents in EAAT4 does not correlate with the rate of substrate transport and suggests that the niflumic acid-induced currents are not thermodynamically coupled to the transport of substrate. 3. Niflumic acid and arachidonic acid co-applied with substrate to EAAT4-expressing oocytes had similar functional consequences. However, niflumic acid still enhanced the L-glutamate-gated current to the same extent in the presence and absence of a saturating dose of arachidonic acid, which suggests that the sites of action of the two compounds are distinct. 4. The EAAT4-mediated currents for the two substrates, L-glutamate and L-aspartate, were not enhanced equally by addition of the same dose of niflumic acid and the ionic composition of the niflumic acid-induced currents was not the same for the two substrates. Protons carry the L-glutamate-gated niflumic acid-induced current and both protons and chloride ions carry the L-aspartate-gated niflumic acid-induced current. 5. These results show that niflumic acid can be used to probe the functional aspects of EAAT4 and that niflumic acid and other non-steroid anti-inflammatory drugs could be used as the basis for the development of novel modulators of glutamate transporters.
Publisher: Wiley
Date: 21-07-2010
Publisher: Wiley
Date: 24-01-2013
Publisher: American Society for Clinical Investigation
Date: 04-01-2011
DOI: 10.1172/JCI44474
Publisher: Elsevier BV
Date: 08-2014
DOI: 10.1016/J.TIPS.2014.05.006
Abstract: Opioids, local anesthetics, anticonvulsant drugs, antidepressants, and non-steroidal anti-inflammatory drugs (NSAIDs) are used to provide pain relief but they do not provide adequate pain relief in a large proportion of chronic pain patients and are often associated with unacceptable side effects. Inhibitory glycinergic neurotransmission is impaired in chronic pain states, and this provides a novel target for drug development. Inhibitors of the glycine transporter 2 (GlyT2) enhance inhibitory neurotransmission and show particular promise for the treatment of neuropathic pain. N-arachidonyl-glycine (NAGly) is an endogenous lipid that inhibits glycine transport by GlyT2 and also shows potential as an analgesic, which may be further exploited in drug development. In this review we discuss the role of glycine neurotransmission in chronic pain and future prospects for the use of glycine transport inhibitors in the treatment of pain.
Publisher: Elsevier BV
Date: 09-2004
Publisher: Wiley
Date: 13-01-2003
DOI: 10.1046/J.1471-4159.2003.01549.X
Abstract: The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo-oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1-methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212-2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.
Publisher: Elsevier BV
Date: 07-2020
Publisher: Elsevier BV
Date: 07-2014
DOI: 10.1016/J.NEUINT.2013.08.012
Abstract: Glycine plays a key role in regulating inhibitory neurotransmission in the spinal cord and concentrations of glycine in the CNS are regulated by two subtypes of high affinity glycine transporters, GlyT1 and GlyT2. In this mini review we will discuss a series of lipid inhibitors of GlyT2 that show promise as analgesics in the treatment of neuropathic and inflammatory pain. N-arachidonyl-glycine inhibits the rate of transport by GlyT2, but has very little or no activity on GlyT1. We will discuss structure-activity studies of the actions of related lipids on GlyT2 and also the characterization of a more potent lipid inhibitor of GlyT2, oleoyl-l-carnitine. Both N-arachidonyl-glycine and oleoyl-l-carnitine show specificity for GlyT2 over GlyT1, which has allowed the use of chimeric GlyT1/GlyT2 transporters to begin characterizing the molecular basis for specificity and mechanism of action of these lipid inhibitors. Although our understanding of the molecular basis for lipid inhibition is still in its infancy, it appears that extracellular loop 4 of GlyT2 plays an important role in the inhibitory mechanism.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 02-07-2022
Abstract: Aberrations in spinal glycinergic signaling are a feature of pain chronification. Normalizing these changes by inhibiting glycine transporter (GlyT)-2 is a promising treatment strategy. However, existing GlyT2 inhibitors (e.g., ORG25543) are limited by narrow therapeutic windows and severe dose-limiting side effects, such as convulsions, and are therefore poor candidates for clinical development. Here, intraperitoneally administered oleoyl-
Publisher: Cold Spring Harbor Laboratory
Date: 09-03-2020
DOI: 10.1101/2020.03.08.982900
Abstract: 3. Reproducibility of research data is a significant problem with more than 60% of biological and medical researchers reporting they have failed to reproduce published data. General acceptance of incorrect results can mean that future data is incorrectly interpreted and progress significantly interrupted. Thus, replication studies play an essential role in corroborating research findings and validating future research objectives. Here, we attempted to replicate data demonstrating the neurotransmitter glutamate, as well as NMDA and AP5, acts as positive allosteric modulators of the inhibitory glycine receptor. Notably, it was shown that the litude of miniature glycinergic currents recorded in spinal cord slices were reversibly enhanced when extracellular glutamate concentrations were increased by the glutamate transporter antagonist TBOA. This finding indicates that endogenous fluctuations in extracellular [glutamate] permits cross-talk between excitatory and inhibitory synapses and likely plays a role in setting the spinal inhibitory glycinergic tone and modulating baseline neurotransmission. We re-evaluated the data in primary cultured spinal cord neurons, spinal cord slice and Xenopus laevis oocytes expressing recombinant glycine receptors. Despite extensive efforts, we were unable to reproduce the finding that glutamate, AP5 or NMDA positively modulate glycine receptor currents. We paid careful attention to key aspects of the original study design, ensured rapid drug exposure by using fast-flow application and took into account receptor saturation and protocol deviations such as animal species. This study refutes the finding that glycine receptors are directly modulated by glutamate spill-over and suggests that glycinergic tone is independent of changes in excitatory activity. 4. Glutamate spill-over onto inhibitory synapses has been reported to positively modulate glycine receptors and alter the inhibitory tone of the spinal cord. This finding has important implications for baseline spinal transmission and could play a role when chronic pain develops. However, we failed to replicate these results and did not observe any modulation of native or recombinant glycine receptor-mediated currents by AP5, NMDA or glutamate. This indicates that inhibitory glycine receptors operate independently of fluctuations in extracellular [glutamate]. 5. N/A
Publisher: Springer Science and Business Media LLC
Date: 15-05-2009
DOI: 10.1007/S11064-009-9983-Y
Abstract: Three fenamates--niflumic, flufenamic and mefenamic acids--were tested for effects on substrate-induced currents of glutamate and glycine transporters (EAAT1, EAAT2, GLYT1b and GLYT2a) expressed in Xenopus laevis oocytes. All fenamates inhibited EAAT1 currents 100 microM flufenamic acid produced the most inhibition, decreasing the I (max) by 53 +/- 4% (P < 0.001). EAAT2 currents were less sensitive, but 100 microM flufenamic acid inhibited the I (max) by 34 +/- 5% (P = 0.006). All fenamates inhibited GLYT1b currents 100 microM flufenamic acid produced the most inhibition, decreasing the I (max) by 61 +/- 1% (P < 0.001). At 100 microM, effects on the GLYT2a I (max) were mixed: 13 +/- 2% inhibition by flufenamic acid (P = 0.002), 30 +/- 6% enhancement by niflumic acid (P = 0.002), and no effect by mefenamic acid. Minor effects on substrate affinity suggested non-competitive mechanisms. These data could contribute to the development of selective transport modulators.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 30-09-2022
Publisher: Wiley
Date: 22-06-2010
Publisher: Springer Science and Business Media LLC
Date: 2002
Abstract: (2S,1'S,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG III) was a substrate of Na(+)-dependent glutamate transporters (GluT) in Xenopus laevis oocytes (IC50 to approximately 13 and to approximately 2 microM for, respec tively, EAAT 1 and EAAT 2) and caused an apparent inhibition of [3H]L-glutamate uptake in "mini-slices" of guinea pig cerebral cortex (IC50 to approximately 12 microM). In slices (350 microM) of guinea pig cerebral cortex, 5 microM L-CCG III increased both the flux of label through pyruvate carboxylase and the fractional enrichment of glutamate, GABA, glutamine and lactate, but had no effect on total metabolite pool sizes. At 50 microM L-CCG III decreased incorporation of 13C from [3-13C]-pyruvate into glutamate C4, glutamine C4, lactate C3 and alanine C3. The total metabolite pool sizes were also decreased with no change in the fractional enrichment. Furthermore, L-CCG III was accumulated in the tissue, probably via GluT. At lower concentration, L-CCG III would compete with L-glutamate for GluT and the changes probably reflect a compensation for the "missing" L-glutamate. At 50 microM, intracellular L-CCG III could reach > 10 mM and metabolism might be affected directly.
Publisher: Elsevier BV
Date: 2021
Publisher: Life Science Alliance, LLC
Date: 23-01-2023
Abstract: Membrane cholesterol binds to and modulates the function of various SLC6 neurotransmitter transporters, including stabilizing the outward-facing conformation of the dopamine and serotonin transporters. Here, we investigate how cholesterol binds to GlyT2 (SLC6A5), modulates glycine transport rate, and influences bioactive lipid inhibition of GlyT2. Bioactive lipid inhibitors are analgesics that bind to an allosteric site accessible from the extracellular solution when GlyT2 adopts an outward-facing conformation. Using molecular dynamics simulations, mutagenesis, and cholesterol depletion experiments, we show that bioactive lipid inhibition of glycine transport is modulated by the recruitment of membrane cholesterol to a binding site formed by transmembrane helices 1, 5, and 7. Recruitment involves cholesterol flipping from its membrane orientation, and insertion of the 3′ hydroxyl group into the cholesterol binding cavity, close to the allosteric site. The synergy between cholesterol and allosteric inhibitors provides a novel mechanism of inhibition and a potential avenue for the development of potent GlyT2 inhibitors as alternative therapeutics for the treatment of neuropathic pain and therapeutics that target other SLC6 transporters.
Publisher: Elsevier BV
Date: 06-2014
Publisher: Elsevier BV
Date: 03-2013
Publisher: Public Library of Science (PLoS)
Date: 13-03-2012
Publisher: Cold Spring Harbor Laboratory
Date: 10-09-2020
DOI: 10.1101/2020.09.10.290908
Abstract: The human glycine transporter 2 (GlyT2 or SLC6A5) has emerged as a promising drug target for the development of new analgesics to manage chronic pain. N-acyl amino acids inhibit GlyT2 through binding to an allosteric binding site to produce analgesia in vivo with minimal overt side effects. In this paper we use a combination of medicinal chemistry, electrophysiology, and computational modelling to explore the molecular basis of GlyT2 inhibition at the allosteric site. We show how N-acyl amino acid head group stereochemistry, tail length and double bond position promote enhanced inhibition by deep penetration into the binding pocket. This work provides new insights into the interaction of lipids with transport proteins and will aid in future rational design of novel GlyT2 inhibitors.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 13-10-2020
Publisher: Humana Press
Date: 2010
DOI: 10.1007/978-1-60761-700-6_15
Abstract: One of the major goals in membrane transporter research is to understand how transporter proteins work at the molecular level. Ideally, this research would be carried out with a detailed knowledge of the three-dimensional structure of the protein. However, in the absence of atomic resolution structures for many membrane transporters other molecular tools need to be employed. In vitro site-directed mutagenesis is one method that has the capacity to provide both structural information and identification of the role of in idual residues and/or regions of a protein that are involved in function.
Publisher: American Chemical Society (ACS)
Date: 19-03-2020
Publisher: American Physiological Society
Date: 10-2013
DOI: 10.1152/PHYSREV.00007.2013
Abstract: l-Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and plays important roles in a wide variety of brain functions, but it is also a key player in the pathogenesis of many neurological disorders. The control of glutamate concentrations is critical to the normal functioning of the central nervous system, and in this review we discuss how glutamate transporters regulate glutamate concentrations to maintain dynamic signaling mechanisms between neurons. In 2004, the crystal structure of a prokaryotic homolog of the mammalian glutamate transporter family of proteins was crystallized and its structure determined. This has paved the way for a better understanding of the structural basis for glutamate transporter function. In this review we provide a broad perspective of this field of research, but focus primarily on the more recent studies with a particular emphasis on how our understanding of the structure of glutamate transporters has generated new insights.
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.BBRC.2009.05.014
Abstract: Membrane rafts, the highly-ordered, cholesterol-rich microdomains of the plasma membrane play important roles in cellular functions. In this study, GLYT1-CFP and GLYT2-CFP were constructed, followed by investigation of whether the tagged transporters associate with a fluorescence probe that labels membrane rafts (DilC16) by using Fluorescence Resonance Energy Transfer. A close association was observed between DiIC16 and GLYT1-CFP, but not for GLYT2-CFP. The glycine transport ability of GLYT1 is also highly dependent on the integrity of this area. Together, the results suggest that GLYT1 and membrane rafts are co-localized in the membrane, and that this influences the rate of glycine transport.
Publisher: Portland Press Ltd.
Date: 28-09-2011
DOI: 10.1042/BJ20110905
Abstract: Glutamate transport is coupled to the co-transport of 3 Na+ and 1 H+ followed by the counter-transport of 1 K+. In addition, glutamate and Na+ binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [14C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.
Publisher: Elsevier BV
Date: 07-2001
Publisher: American Chemical Society (ACS)
Date: 08-2007
DOI: 10.1021/BI700647F
Abstract: L-Glutamate is the predominant excitatory neurotransmitter in the brain, and its extracellular concentration is tightly controlled by the excitatory amino acid transporters (EAATs). The transport of 1 glutamate molecule is coupled to the cotransport of 3 Na+ and 1 H+ and the countertransport of 1 K+. In addition to substrate transport, the binding of glutamate and Na+ activates an anion current which is thermodynamically uncoupled from the transport process. We have identified three amino acid residues in EAAT1 (D272 in TM5, K384 and R385 in TM7) that influence the litude of the anion channel current relative to the transport current. Transporters containing the mutations R268A, D272A, D272K, K384A, K384D, R385A, and R385D were expressed in Xenopus laevis oocytes and their transport and anion channel functions measured using the two-electrode voltage cl techniques. The D272, K384, and R385 mutant transporters showed no change in transport properties but have increased levels of anion channel activity compared to wild-type transporters. These results identify additional residues of the EAAT1 transporter that may contribute to the gating mechanism of the anion channel of glutamate transporters and also provide hints as to how substrate binding leads to channel activation.
Publisher: Elsevier BV
Date: 05-2004
Publisher: Springer Science and Business Media LLC
Date: 24-01-2020
DOI: 10.1007/S11064-019-02934-X
Abstract: The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems-the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues Glt
Publisher: American Chemical Society (ACS)
Date: 04-02-2019
Publisher: Humana Press
Date: 2003
Publisher: Elsevier BV
Date: 02-2009
Publisher: Elsevier BV
Date: 05-2007
Publisher: Cold Spring Harbor Laboratory
Date: 03-09-2022
DOI: 10.1101/2022.08.31.506132
Abstract: Membrane cholesterol binds to and modulates the function of the specific SLC6 transporters. Here we investigate how cholesterol binds to and modulates the rate of glycine transport by the SLC6 glycine transporter GlyT2, and how this impacts lipid inhibition of GlyT2. Bioactive lipid inhibitors of GlyT2 are analgesics that bind to the lipid allosteric site of the outward facing GlyT2 conformation that is accessible from the extracellular solution. Using molecular dynamics simulations, mutagenesis and cholesterol depletion experiments, we show that bioactive lipid inhibition of glycine transport is modulated by the recruitment of membrane cholesterol to a cholesterol binding site formed by transmembrane helices 1, 5 and 7. Recruitment involves cholesterol flipping from its membrane orientation, and insertion of the 3’ hydroxyl group into the cholesterol binding cavity to interact with the base of the lipid allosteric site and the bound inhibitor. The recruitment of membrane cholesterol by allosteric GlyT2 inhibitors is a potential avenue for the development of high-potency, specific pain analgesics and could provide alternative therapeutics that target GlyT2 and other SLC6 neurotransmitter transporters.
Publisher: Elsevier BV
Date: 10-2010
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.NEUROBIOLAGING.2010.03.008
Abstract: A characteristic of Alzheimer's disease (AD) is that neuron populations in the temporal, frontal, and parietal cortices are selectively vulnerable. Several neurotransmitters have been proposed to play roles in neural destruction as AD progresses, including glutamate. Failure to clear the synaptic cleft of glutamate can overstimulate postsynaptic glutamate receptors, promoting neuronal death. Excitatory amino acid transporter 2 (EAAT2), which is concentrated in perisynaptic astrocytes, performs 90% of glutamate uptake in mammalian central nervous system. Alternative splicing of EAAT2 mRNA could regulate glutamate transport in normal and disease states. We report disease- and pathology-specific variations in EAAT2 splice variant expression in AD brain obtained at autopsy. While wild type EAAT2 showed a global reduction in expression, brain regions susceptible to neuronal loss demonstrated greater expression of transcripts that reduced glutamate transport in an in vitro assay. Functional splice variant EAAT2b showed no significant variation with disease state. These results have implications for the treatment of AD as modulators of EAAT2 splicing and/or glutamate uptake would augment current therapies aimed at blocking glutamate receptors.
Publisher: Elsevier BV
Date: 04-2002
Publisher: American Association for the Advancement of Science (AAAS)
Date: 19-04-2005
Abstract: Traditionally, transporters and ion channels have been thought of as structurally and functionally distinct proteins. However, neurotransmitter transporters can be both. Vandenberg and Ryan discuss recent developments in understanding how and why these transporters contain ion channels.
Publisher: American Chemical Society (ACS)
Date: 15-06-2017
DOI: 10.1021/ACSCHEMNEURO.7B00105
Abstract: It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC
Publisher: Wiley
Date: 08-08-2006
DOI: 10.1111/J.1471-4159.2006.04107.X
Abstract: N-arachidonyl-glycine is one of a series of N-arachidonyl-amino acids that are derived from arachidonic acid. N-arachidonyl-glycine is produced in a wide range of tissues with greatest abundance in the spinal cord. Here we report that N-arachidonyl-glycine is a reversible and non-competitive inhibitor of glycine transport by GLYT2a, but has little effect on glycine transport by GLYT1b or gamma-amino butyric acid transport by GAT1. It has previously been reported that the activity of GLYT2a is down-regulated by protein kinase C and therefore we investigated whether the actions of N-arachidonyl-glycine on GLYT2a are mediated by second messenger systems that lead to the activation of protein kinase C. However, the protein kinase C inhibitor, staurosporine, had no effect on the actions of N-arachidonyl-glycine on GLYT2a. Thus, the actions of N-arachidonyl-glycine are likely to be mediated by a direct interaction with the transporter. We have further defined the pharmacophore by investigating the actions of other N-arachidonyl amino acids as well as the closely related compounds arachidonic acid, anandamide and R1-methanandamide. Arachidonic acid, anandamide and R1-methanandamide have no effect on glycine transport, but N-arachidonyl-l-alanine has similar efficacy at GLYT2a to N-arachidonyl-glycine, and N-arachidonyl-gamma-amino butyric acid is less efficacious. These observations define a novel recognition site for the N-arachidonyl amino acids.
Publisher: Elsevier BV
Date: 09-2005
Publisher: American Chemical Society (ACS)
Date: 09-11-2018
DOI: 10.1021/ACSCHEMNEURO.7B00407
Abstract: Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuT
Publisher: eLife Sciences Publications, Ltd
Date: 17-10-2019
DOI: 10.7554/ELIFE.47150
Abstract: The treatment of chronic pain is poorly managed by current analgesics, and there is a need for new classes of drugs. We recently developed a series of bioactive lipids that inhibit the human glycine transporter GlyT2 (SLC6A5) and provide analgesia in animal models of pain. Here, we have used functional analysis of mutant transporters combined with molecular dynamics simulations of lipid-transporter interactions to understand how these bioactive lipids interact with GlyT2. This study identifies a novel extracellular allosteric modulator site formed by a crevice between transmembrane domains 5, 7, and 8, and extracellular loop 4 of GlyT2. Knowledge of this site could be exploited further in the development of drugs to treat pain, and to identify other allosteric modulators of the SLC6 family of transporters.
Publisher: Springer Science and Business Media LLC
Date: 17-02-2021
Publisher: Wiley
Date: 22-06-2010
DOI: 10.1111/J.1471-4159.2010.06796.X
Abstract: Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and extracellular glutamate levels are controlled by a family of transporters known as excitatory amino acid transporters (EAATs). The EAATs transport glutamate and aspartate with similar micromolar affinities and this transport is coupled to the movement of Na(+), K(+), and H(+). The crystal structure of a prokaryotic homologue of the EAATs, aspartate transporter from Pyrococcus horokoshii (Glt(Ph)), has yielded important insights into the architecture of this transporter family. Glt(Ph) is a Na(+)-dependent transporter that has significantly higher affinity for aspartate over glutamate and is not coupled to H(+) or K(+). The highly conserved carboxy-terminal domains of the EAATs and Glt(Ph) contain the substrate and ion binding sites, however, there are a couple of striking differences in this region that we have investigated to better understand the transport mechanism. An arginine residue is in close proximity to the substrate binding site of both Glt(Ph) and the EAATs, but is located in transmembrane domain (TM) 8 in the EAATs and hairpin loop 1 (HP1) of Glt(Ph). Here we report that the position of this arginine residue can explain some of the functional differences observed between the EAATs and Glt(Ph). Moving the arginine residue from TM8 to HP1 in EAAT1 results in a transporter that has significantly increased affinity for both glutamate and aspartate and is K(+) independent. Conversely, moving the arginine residue from HP1 to TM8 in Glt(Ph) results in a transporter that has reduced affinity for aspartate.
Publisher: Elsevier BV
Date: 12-2009
Publisher: American Chemical Society (ACS)
Date: 19-07-2023
Publisher: American Physiological Society
Date: 09-2004
DOI: 10.1152/AJPREGU.00051.2004
Abstract: The tachykinin peptide bufokinin, isolated from the cane toad intestine, is important in intestinal and cardiovascular regulation in the toad. In this study, three tachykinin NK 1 -like receptor isoforms, bNK 1 -A, bNK 1 -B, and bNK 1 -C, encoding proteins of 309, 390, and 371 amino acids, respectively, were cloned from the toad brain and intestine. These isoforms differ only at the intracellular COOH terminus. The bNK 1 -A and bNK 1 -B isoforms are similar to the truncated and full-length forms of the mammalian NK 1 receptor, whereas bNK 1 -C is unique and does not correspond to any previously described receptor. RT-PCR studies demonstrated that three isoform transcripts are widely distributed in the toad with high expression in gut, spinal cord, brain, lung, and skeletal muscle. When expressed in COS-7 cells, bufokinin showed similar high affinity (IC 50 0.6–0.8 nM) in competing for 125 I-labeled Bolton-Hunter bufokinin binding at all receptors, but the binding affinities of substance P (SP) and neurokinin A (NKA) were very different at each isoform. When expressed in Xenopus oocytes, the truncated isoform, bNK 1 -A, was inactive, whereas bNK 1 -B and bNK 1 -C produced changes in chloride current when stimulated by tachykinins (minimum concentrations: bufokinin, 0.1 nM SP, 1 nM and NKA, 10 nM). A marked desensitization of the response was seen to subsequent applications of tachykinins, as experienced by the mammalian NK 1 receptor. In summary, our study describing three isoforms of NK 1 -like receptor from the toad suggests that the alternative splicing of NK 1 receptor is a physiologically conserved mechanism and raises a fundamental question as to the physiological role of each isoform.
Publisher: Wiley
Date: 2005
DOI: 10.1111/J.1440-1681.2005.04164.X
Abstract: 1. Glutamate transporters (or excitatory amino acid transporters (EAAT)) are responsible for removing synaptically released glutamate from the extracellular space. The failure of EAAT to carry out this role will lead to excessive stimulation of glutamatergic receptors, causing excitotoxicity and cell death. 2. Glutamate is cotransported into the cell with three Na + and one H + , followed by the counter‐transport of one K + . In addition, glutamate and Na + binding activates an uncoupled chloride conductance. Thus, glutamate transporters can function as both a transporter and an ion channel. At present, there is no clear understanding of the structural basis for the dual functions of glutamate transporters and, in the present review, we shall discuss some recent studies that have started to address this question. 3. It is possible to modulate one function of glutamate transporters without affecting the other, which suggests that the two functions have separate molecular determinants, and a number of models have been suggested to account for the dual functions of the EAAT that predict both single and dual pores for transporter function. 4. It appears that the two functions of glutamate transporters arise from separate transmembrane domains. The C‐terminal region of the transporters forms the glutamate translocation domain, whereas the second transmembrane domain in the N‐terminal half of the protein plays a crucial role in chloride channel function. Although the two functions arise from separate molecular determinants, the two functional domains are likely to be in close proximity. The significance of these observations will be discussed in terms of likely functional models for the transport and channel processes.
Publisher: Elsevier BV
Date: 08-2014
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.BCP.2008.07.037
Abstract: Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord motor and pain sensory neurons. Recent studies demonstrated apparently contradictory (potentiating versus inhibitory) effects of the endocannabinoid anandamide on these receptors. The present study characterised the effects of cannabinoid agonists on alpha1, alpha1beta, alpha2 and alpha3 GlyRs recombinantly expressed in HEK293 cells with the aims of reconciling effects of cannabinoids on these receptor subtypes and to establish the potential of different GlyR isoforms as novel physiological or analgesic targets for cannabinoids. The compounds investigated were anandamide, HU-210, HU-308, WIN55,212-2 and the endogenous non-cannabinoid, N-arachidonyl-glycine. The latter compound was chosen due to the structural similarity with anandamide and known analgesic actions in the spinal cord. Recombinant alpha1 and alpha1beta GlyRs were potentiated by anandamide and HU-210 at submicromolar concentrations, whereas WIN55,212-2 had no effect and HU-308 produced only weak inhibition. By contrast, N-arachidonyl-glycine exerted complex effects including both potentiation and inhibition. Anandamide had no effect at alpha2 or alpha3 GlyRs although the other cannabinoids produced potent inhibition. On alpha2 GlyRs, the inhibitory potency sequence was HU-210=WIN55,212-2>HU-308>N-arachidonyl-glycine but on alpha3 GlyRs it was HU-210=WIN55212=HU-308>N-arachidonyl-glycine. These results suggest that alpha1, alpha2 and alpha3 containing GlyRs exhibit distinct pharmacological profiles for cannabinoids. We conclude that cannabinoid agonists may be useful as pharmacological tools for selectively inhibiting alpha2 and alpha3 GlyRs. Our results also establish GlyRs as potential novel targets for endogenous and exogenous cannabinoids.
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.NEUINT.2015.12.007
Abstract: Neurotransmitter transporters are targets for a wide range of therapeutically useful drugs. This is because they have the capacity to selectively manipulate the dynamics of neurotransmitter concentrations and thereby enhance or diminish signalling through particular brain pathways. High affinity glycine transporters (GlyTs) regulate extracellular concentrations of glycine and provide novel therapeutic targets for neurological disorders.
Publisher: Elsevier BV
Date: 05-2004
Publisher: Wiley
Date: 12-2001
Publisher: Elsevier BV
Date: 04-2015
Publisher: Springer Science and Business Media LLC
Date: 02-01-2018
DOI: 10.1038/S41467-017-02444-W
Abstract: Cancer cells undergo a shift in metabolism where they become reliant on nutrients such as the amino-acid glutamine. Glutamine enters the cell via the alanine/serine/cysteine transporter 2 (ASCT2) that is upregulated in several cancers to maintain an increased supply of this nutrient and are therefore an attractive target in cancer therapeutic development. ASCT2 belongs to the glutamate transporter (SLC1A) family but is the only transporter in this family able to transport glutamine. The structural basis for glutamine selectivity of ASCT2 is unknown. Here, we identify two amino-acid residues in the substrate-binding site that are responsible for conferring glutamine selectivity. We introduce corresponding mutations into a prokaryotic homologue of ASCT2 and solve four crystal structures, which reveal the structural basis for neutral amino acid and inhibitor binding in this family. This structural model of ASCT2 may provide a basis for future development of selective ASCT2 inhibitors to treat glutamine-dependent cancers.
Start Date: 02-2010
End Date: 12-2013
Amount: $420,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 02-2004
End Date: 11-2004
Amount: $20,000.00
Funder: Australian Research Council
View Funded Activity