Aminomethanesulfonic acid illuminates the boundary between full and partial agonists of the pentameric glycine receptor.

To clarify the determinants of agonist efficacy in pentameric ligand-gated ion channels, we examined a new compound, aminomethanesulfonic acid (AMS), a molecule intermediate in structure between glycine and taurine. Despite wide availability, to date there are no reports of AMS action on glycine receptors, perhaps because AMS is unstable at physiological pH. Here, we show that at pH 5, AMS is an efficacious agonist, eliciting in zebrafish α1 glycine receptors a maximum single-channel open probability of 0.85, much greater than that of ß-alanine (0.54) or taurine (0.12), and second only to that of glycine itself (0.96). Thermodynamic cycle analysis of the efficacy of these closely related agonists shows supra-additive interaction between changes in the length of the agonist molecule and the size of the anionic moiety. Single particle cryo-electron microscopy structures of AMS-bound glycine receptors show that the AMS-bound agonist pocket is as compact as with glycine, and three-dimensional classification demonstrates that the channel populates the open and the desensitized states, like glycine, but not the closed intermediate state associated with the weaker partial agonists, ß-alanine and taurine. Because AMS is on the cusp between full and partial agonists, it provides a new tool to help us understand agonist action in the pentameric superfamily of ligand-gated ion channels.

Acidic pH reduces agonist efficacy and responses to synaptic-like glycine applications in zebrafish α1 and rat α1ß recombinant glycine receptors.

Many pentameric ligand-gated ion channels are modulated by extracellular pH. Glycine receptors (GlyRs) share this property, but it is not well understood how they are affected by pH changes. Whole cell experiments on HEK293 cells expressing zebrafish homomeric α1 GlyR confirmed previous reports that acidic pH (6.4) reduces GlyR sensitivity to glycine, whereas alkaline pH (8.4) has small or negligible effects. In addition to that, at pH 6.4 we observed a reduction in the maximum responses to the partial agonists ß-alanine and taurine relative to the full agonist glycine. In cell-attached single-channel recording, low pH reduced agonist efficacy, as the maximum open probability decreased from 0.97, 0.91 and 0.66 to 0.93, 0.57 and 0.34 for glycine, ß-alanine and taurine, respectively, reflecting a threefold decrease in efficacy equilibrium constants for all three agonists. We also tested the effect of pH 6.4 in conditions that replicate those at the native synapse, recording outside-out currents elicited by fast application of millisecond pulses of agonists on α1 and α1ß GlyR, at a range of intracellular chloride concentrations. Acidic pH reduced the area under the curve of the currents, by reducing peak amplitude, slowing activation and speeding deactivation. Our results show that acidification of the extracellular pH by one unit, as may occur in pathological conditions such as ischaemia, impairs GlyR gating and is likely to reduce the effectiveness of glycinergic synaptic inhibition. KEY POINTS: Extracellular pH in the central nervous system (CNS) is known to shift towards acidic values during pathophysiological conditions such as ischaemia and seizures. Acidic extracellular pH is known to affect GABAergic inhibitory synapses, but its effect on signals mediated by glycine receptors (GlyR) is not well characterised. Moderate acidic conditions (pH 6.4) reduce the maximum single channel open probability of recombinant homomeric GlyRs produced by the neurotransmitter glycine or other agonists, such as ß-alanine and taurine. When glycine was applied with a piezoelectric stepper to outside out patches, to simulate its fast rise and short duration at the synapse, responses became shorter and smaller at pH 6.4. The effect was also observed with physiologically low intracellular chloride and in mammalian heteromeric GlyRs. This suggests that acidic pH is likely to reduce the strength of inhibitory signalling at glycinergic synapses.

Heterogeneity of cell membrane structure studied by single molecule tracking.

Heterogeneity in cell membrane structure, typified by microdomains with different biophysical and biochemical properties, is thought to impact on a variety of cell functions. Integral membrane proteins act as nanometre-sized probes of the lipid environment and their thermally-driven movements can be used to report local variations in membrane properties. In the current study, we have used total internal reflection fluorescence microscopy (TIRFM) combined with super-resolution tracking of multiple individual molecules, in order to create high-resolution maps of local membrane viscosity. We used a quadrat sampling method and show how statistical tests for membrane heterogeneity can be conducted by analysing the paths of many molecules that pass through the same unit area of membrane. We describe experiments performed on cultured primary cells, stable cell lines and ex vivo tissue slices using a variety of membrane proteins, under different imaging conditions. In some cell types, we find no evidence for heterogeneity in mobility across the plasma membrane, but in others we find statistically significant differences with some regions of membrane showing significantly higher viscosity than others.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Ion channels.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

The intracellular domain of homomeric glycine receptors modulates agonist efficacy.

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, ß-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.

Mechanism of gating and partial agonist action in the glycine receptor.

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.

The startle disease mutation α1S270T predicts shortening of glycinergic synaptic currents.

KEY POINTS: Loss-of-function mutations in proteins found at glycinergic synapses, most commonly in the α1 subunit of the glycine receptor (GlyR), cause the startle disease/hyperekplexia channelopathy in man. It was recently proposed that the receptors responsible are presynaptic homomeric GlyRs, rather than postsynaptic heteromeric GlyRs (which mediate glycinergic synaptic transmission), because heteromeric GlyRs are less affected by many startle mutations than homomers. We examined the α1 startle mutation S270T, at the extracellular end of the M2 transmembrane helix. Recombinant heteromeric GlyRs were less impaired than homomers by this mutation when we measured their response to equilibrium applications of glycine. However, currents elicited by synaptic-like millisecond applications of glycine to outside-out patches were much shorter (7- to 10-fold) in all mutant receptors, both homomeric and heteromeric. Thus, the synaptic function of heteromeric receptors is likely to be impaired by the mutation. ABSTRACT: Human startle disease is caused by mutations in glycine receptor (GlyR) subunits or in other proteins associated with glycinergic synapses. Many startle mutations are known, but it is hard to correlate the degree of impairment at molecular level with the severity of symptoms in patients. It was recently proposed that the disease is caused by disruption in the function of presynaptic homomeric GlyRs (rather than postsynaptic heteromeric GlyRs), because homomeric GlyRs are more sensitive to loss-of-function mutations than heteromers. Our patch-clamp recordings from heterologously expressed GlyRs characterised in detail the functional consequences of the α1S270T startle mutation, which is located at the extracellular end of the pore lining M2 transmembrane segment (18'). This mutation profoundly decreased the maximum single-channel open probability of homomeric GlyRs (to 0.16; cf. 0.99 for wild type) but reduced only marginally that of heteromeric GlyRs (0.96; cf. 0.99 for wild type). However, both heteromeric and homomeric mutant GlyRs became less sensitive to the neurotransmitter glycine. Responses evoked by brief, quasi-synaptic pulses of glycine onto outside-out patches were impaired in mutant receptors, as deactivation was approximately 10- and 7-fold faster for homomeric and heteromeric GlyRs, respectively. Our data suggest that the α1S270T mutation is likely to affect the opening step in GlyR activation. The faster decay of synaptic currents mediated by mutant heteromeric GlyRs is expected to reduce charge transfer at the synapse, despite the high equilibrium open probability of these mutant channels.

A tale of ligands big and small: an update on how pentameric ligand-gated ion channels interact with agonists and proteins.

Pentameric ligand-gated ion channels (pLGICs, also known as Cys-loop receptors) are a large family of ion channels expressed in all Bilateria and in several groups of bacteria and archaea. They are activated by small-molecule neurotransmitters to mediate fast transmission at many central and peripheral nervous system synapses and are the target of several drugs and insecticides. Here we review recent advances in the field, focussing on new insights on the structure of the agonist-binding site and on newly discovered protein-protein interactions involving pLGICs.

The Startle Disease Mutation E103K Impairs Activation of Human Homomeric α1 Glycine Receptors by Disrupting an Intersubunit Salt Bridge across the Agonist Binding Site.

Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules.

High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor.

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype ß1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

Bayesian Statistical Inference in Ion-Channel Models with Exact Missed Event Correction.

The stochastic behavior of single ion channels is most often described as an aggregated continuous-time Markov process with discrete states. For ligand-gated channels each state can represent a different conformation of the channel protein or a different number of bound ligands. Single-channel recordings show only whether the channel is open or shut: states of equal conductance are aggregated, so transitions between them have to be inferred indirectly. The requirement to filter noise from the raw signal further complicates the modeling process, as it limits the time resolution of the data. The consequence of the reduced bandwidth is that openings or shuttings that are shorter than the resolution cannot be observed; these are known as missed events. Postulated models fitted using filtered data must therefore explicitly account for missed events to avoid bias in the estimation of rate parameters and therefore assess parameter identifiability accurately. In this article, we present the first, to our knowledge, Bayesian modeling of ion-channels with exact missed events correction. Bayesian analysis represents uncertain knowledge of the true value of model parameters by considering these parameters as random variables. This allows us to gain a full appreciation of parameter identifiability and uncertainty when estimating values for model parameters. However, Bayesian inference is particularly challenging in this context as the correction for missed events increases the computational complexity of the model likelihood. Nonetheless, we successfully implemented a two-step Markov chain Monte Carlo method that we called "BICME", which performs Bayesian inference in models of realistic complexity. The method is demonstrated on synthetic and real single-channel data from muscle nicotinic acetylcholine channels. We show that parameter uncertainty can be characterized more accurately than with maximum-likelihood methods. Our code for performing inference in these ion channel models is publicly available.

Altered development in GABA co-release shapes glycinergic synaptic currents in cultured spinal slices of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

KEY POINTS: Increased environmental risk factors in conjunction with genetic susceptibility have been proposed with respect to the remarkable variations in mortality in amyotrophic lateral sclerosis (ALS). In vitro models allow the investigation of the genetically modified counter-regulator of motoneuron toxicity and may help in addressing ALS therapy. Spinal organotypic slice cultures from a mutant form of human superoxide dismutase 1 (SOD1G93A) mouse model of ALS allow the detection of altered glycinergic inhibition in spinal microcircuits. This altered inhibition improved spinal cord excitability, affecting motor outputs in early SOD1(G93A) pathogenesis. ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurological disease characterized by a progressive degeneration of motoneurons (MNs). In a previous study, we developed organotypic spinal cultures from an ALS mouse model expressing a mutant form of human superoxide dismutase 1 (SOD1(G93A) ). We reported the presence of a significant synaptic rearrangement expressed by these embryonic cultured networks, which may lead to the altered development of spinal synaptic signalling, which is potentially linked to the adult disease phenotype. Recent studies on the same ALS mouse model reported a selective loss of glycinergic innervation in cultured MNs, suggestive of a contribution of synaptic inhibition to MN dysfunction and degeneration. In the present study, we further exploit organotypic cultures from wild-type and SOD1(G93A) mice to investigate the development of glycine-receptor-mediated synaptic currents recorded from the interneurons of the premotor ventral circuits. We performed single cell electrophysiology, immunocytochemistry and confocal microscopy and suggest that GABA co-release may speed the decay of glycine responses altering both temporal precision and signal integration in SOD1(G93A) developing networks at the postsynaptic site. Our hypothesis is supported by the finding of an increased MN bursting activity in immature SOD1(G93A) spinal cords and by immunofluorescence microscopy detection of a longer persistence of GABA in SOD1(G93A) glycinergic terminals in cultured and ex vivo spinal slices.

Mechanism of activation of the prokaryotic channel ELIC by propylamine: a single-channel study.

Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5-50 mM) in outside-out patches at -60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50-600 ms, 1-50 mM, -100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the "Flip" model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The "Primed" model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼ 102 (∼ 20 for α1ß glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.

Agonist and antagonist binding in human glycine receptors.

The human glycine receptor (hGlyR) is an anion-permeable ligand-gated channel that is part of a larger superfamily of receptors called the Cys-loop family. hGlyRs are particularly amenable to single-channel recordings, thus making them a model experimental system for understanding the Cys-loop receptor family in general. Understanding the relationship between agonist binding and efficacy in Cys-loop receptors should improve our future prospects for making specific agonists or antagonists. However, at present, there is no high-resolution structure for the complete hGlyR, and thus, modeling is needed to provide a physical framework on which to interpret single-channel data. The structure of the glutamate-gated chloride channel from Caenorhabditis elegans shows a much higher level of sequence identity to human hGlyR than previous templates such as AChBP or the bacterial channels, GLIC and ELIC. Thus, we constructed a model of the hGlyR and validated it against previously reported mutagenesis data. We used molecular dynamics to refine the model and to explore binding of both an agonist (glycine) and an antagonist (strychnine). The model shows excellent agreement with previous data but also suggests some unique features: (i) a water molecule that forms part of the binding site and allows us to account for some previous results that were difficult to reconcile, (ii) an interaction of the glycine agonist with S129, and (iii) an effect from E211, both of which we confirmed with new site-directed mutagenesis and patch clamp recordings. Finally, examination of the simulations suggests that strychnine binding induces movement to a conformational state distinct from the glycine-bound or apo state, not only within the ligand-binding domain but also in the transmembrane domain.

Bayesian approaches for mechanistic ion channel modeling.

We consider the Bayesian analysis of mechanistic models describing the dynamic behavior of ligand-gated ion channels. The opening of the transmembrane pore in an ion channel is brought about by conformational changes in the protein, which results in a flow of ions through the pore. Remarkably, given the diameter of the pore, the flow of ions from a small number of channels or indeed from a single ion channel molecule can be recorded experimentally. This produces a large time-series of high-resolution experimental data, which can be used to investigate the gating process of these channels. We give a brief overview of the achievements and limitations of alternative maximum-likelihood approaches to this type of modeling, before investigating the statistical issues associated with analyzing stochastic model reaction mechanisms from a Bayesian perspective. Finally, we compare a number of Markov chain Monte Carlo algorithms that may be used to tackle this challenging inference problem.

The kinetic properties of the α3 rat glycine receptor make it suitable for mediating fast synaptic inhibition.

Glycine receptors mediate fast synaptic inhibition in spinal cord and brainstem. Two α subunits are present in adult neurones, α1, which forms most of the synaptic glycine receptors, and α3. The physiological role of α3 is not known, despite the fact that α3 expression is concentrated in areas involved in nociceptive processing, such as the superficial dorsal horn. In the present study, we characterized the kinetic properties of rat homomeric α3 glycine receptors heterologously expressed in HEK293 cells. We analysed steady state single channel activity at a range of different glycine concentrations by fitting kinetic schemes and found that α3 channels resemble α1 receptors in their high maximum open probability (99.1% cf. 98% for α1), but differ in that maximum open probability is reached when all five binding sites are occupied by glycine (cf. three out of five sites for α1). α3 activation was best described by kinetic schemes that allow the channel to open also when partially liganded and that contain more than the minimum number of shut states, either as desensitized distal states (Jones and Westbrook scheme) or as pre-open gating intermediates (flip scheme). We recorded also synaptic-like α3 currents elicited by the rapid application of 1 ms pulses of high concentration glycine to outside-out patches. These currents had fast deactivation, with a time constant of decay of 9 ms. Thus, if native synaptic currents can be mediated by α3 glycine receptors, they are likely to be very close in their kinetics to α1-mediated synaptic events.

Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations.

The modulation of pentameric ligand-gated ion channels (pLGICs) by divalent cations is believed to play an important role in their regulation in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit channel activation. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent cations inhibit the activation of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentrations, its maximum response. Crystal structures of the channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibition is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding region. Here, divalent cations interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent ions binding to a similar region, even though the interacting residues are not conserved within the family. Our study provides structural and functional insight into the allosteric regulation of ELIC and is of potential relevance for the entire family.

The α1K276E startle disease mutation reveals multiple intermediate states in the gating of glycine receptors.

Loss-of-function mutations in human glycine receptors cause hyperekplexia, a rare inherited disease associated with an exaggerated startle response. We have studied a human disease mutation in the M2-M3 loop of the glycine receptor α1 subunit (K276E) using direct fitting of mechanisms to single-channel recordings with the program HJCFIT. Whole-cell recordings from HEK293 cells showed the mutation reduced the receptor glycine sensitivity. In single-channel recordings, rat homomeric α1 K276E receptors were barely active, even at 200 mM glycine. Coexpression of the ß subunit partially rescued channel function. Heteromeric mutant channels opened in brief bursts at 300 µM glycine (a concentration that is near-maximal for wild type) and reached a maximum one-channel open probability of about 45% at 100 mm glycine (compared to 96% for wild type). Distributions of apparent open times contained more than one component in high glycine and, therefore, could not be described by mechanisms with only one fully liganded open state. Fits to the data were much better with mechanisms in which opening can also occur from more than one fully liganded intermediate (e.g., "primed" models). Brief pulses of glycine (∼3 ms, 30 mM) applied to mutant channels in outside-out patches activated currents with a slower rise time (1.5 ms) than those of wild-type channels (0.2 ms) and a much faster decay. These features were predicted reasonably well by the mechanisms obtained from fitting single-channel data. Our results show that, by slowing and impairing channel gating, the K276E mutation facilitates the detection of closed reaction intermediates in the activation pathway of glycine channels.

Chloride ions in the pore of glycine and GABA channels shape the time course and voltage dependence of agonist currents.

In the vertebrate CNS, fast synaptic inhibition is mediated by GABA and glycine receptors. We recently reported that the time course of these synaptic currents is slower when intracellular chloride is high. Here we extend these findings to measure the effects of both extracellular and intracellular chloride on the deactivation of glycine and GABA currents at both negative and positive holding potentials. Currents were elicited by fast agonist application to outside-out patches from HEK-293 cells expressing rat glycine or GABA receptors. The slowing effect of high extracellular chloride on current decay was detectable only in low intracellular chloride (4 mm). Our main finding is that glycine and GABA receptors "sense" chloride concentrations because of interactions between the M2 pore-lining domain and the permeating ions. This hypothesis is supported by the observation that the sensitivity of channel gating to intracellular chloride is abolished if the channel is engineered to become cation selective or if positive charges in the external pore vestibule are eliminated by mutagenesis. The appropriate interaction between permeating ions and channel pore is also necessary to maintain the channel voltage sensitivity of gating, which prolongs current decay at depolarized potentials. Voltage dependence is abolished by the same mutations that suppress the effect of intracellular chloride and also by replacing chloride with another permeant ion, thiocyanate. These observations suggest that permeant chloride affects gating by a foot-in-the-door effect, binding to a channel site with asymmetrical access from the intracellular and extracellular sides of the membrane.