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Zn2+ is concentrated into presynaptic vesicles at many central synapses and is released into the synaptic cleft by nerve terminal stimulation. There is strong evidence that synaptically released Zn2+ modulates glutamatergic neurotransmission, although there is debate concerning the peak concentration it reaches in the synaptic cleft. Glycine receptors (GlyRs), which mediate inhibitory neurotransmission in the spinal cord and brainstem, are potentiated by low nanomolar Zn2+ and inhibited by micromolar Zn2+. Mutations that selectively ablate Zn2+ potentiation result in hyperekplexia phenotypes suggesting that Znz2+ is a physiological regulator of glycinergic neurotransmission. There is, however, little evidence that Zn2+ is stored presynaptically at glycinergic terminals and an alternate possibility is that GlyRs are modulated by constitutively bound Zn2+. We sought to estimate the peak Zn2+ concentration in the glycinergic synaptic cleft as a means of evaluating whether it is likely to be synaptically released. We employed 'artificial' synapses because they permit the insertion of engineered a1b GlyRs with defined Zn2+ sensitivities into synapses. By comparing the effect of Zn2+ chelation on glycinergic IPSCs with the effects of defined Zn2+ and glycine concentrations applied rapidly to the same recombinant GlyRs in outside-out patches, we inferred that synaptic Zn2+ rises to at least 1 µM following a single presynaptic stimulation. Moreover, using the fast, high-affinity chelator, ZX1, we found no evidence for tonic Zn2+ bound constitutively to high affinity GlyR binding sites. We conclude that diffusible Zn2+ reaches 1 µM or higher and is therefore likely to be phasically released in artificial glycinergic synapses. [ABSTRACT FROM AUTHOR]

Background and Purpose GABAA receptors mediate neuronal inhibition in the brain. They are the primary targets for benzodiazepines, which are widely used to treat neurological disorders including anxiety, epilepsy and insomnia. The mechanism by which benzodiazepines enhance GABAA receptor activity has been extensively studied, but there is little mechanistic information on how non-benzodiazepine drugs that bind to the same site exert their effects. Eszopiclone and zolpidem are two non-benzodiazepine drugs for which no mechanism of action has yet been proposed, despite their clinical importance as sleeping aids. Here we investigate how both drugs enhance the activity of α1β2γ2 GABAA receptors. Experimental Approach We used rapid ligand application onto macropatches and single-channel kinetic analysis to assess rates of current deactivation. We also studied synaptic currents in primary neuronal cultures and in heterosynapses, whereby native GABAergic nerve terminals form synapses with HEK293 cells expressing α1β2γ2 GABAA receptors. Drug binding and modulation was quantified with the aid of an activation mechanism. Key Results At the single-channel level, the drugs prolonged the duration of receptor activation, with similar KD values of ∼80 nM. Channel activation was prolonged primarily by increasing the equilibrium constant between two connected shut states that precede channel opening. Conclusions and Implications As the derived mechanism successfully simulated the effects of eszopiclone and zolpidem on ensemble currents, we propose it as the definitive mechanism accounting for the effects of both drugs. Importantly, eszopiclone and zolpidem enhanced GABAA receptor currents via a mechanism that differs from that proposed for benzodiazepines. [ABSTRACT FROM AUTHOR]

The α1β2γ2 and α3β3γ2 are two isoforms of y-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. α1β2γ2 channels exhibited briefer active periods than α3β3γ2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at α3β3γ2 channels was ∼2.6 μM compared with ∼19 μM for α1β2γ2 channels, suggesting that GABA binds to the α3β3γ2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the α3β3γ2 channels. The longer active periods, higher affinity, and preference for conducting states are consis- tent with the slower decay of inhibitory currents at synapses that contain α3β3γ2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of GABAA receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate GABAA receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system. [ABSTRACT FROM AUTHOR]

The family of-γ-aminobutyric acid type A receptors (GABAARs) mediates two types of inhibit;ion in the mammalian brain. Phasic inhibition is mediated by synaptic GABAARs that are mainly comprised of α1, β2, and γ2 subunits, whereas tonic inhibition is mediated by extrasynaptic GABAARS comprised of α4/6 β2, and δ subunits. We investigated the activation properties of recombinant α4β2δ and α4β2δ GABAARs in response to GABA and 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experinlents indicated that THIP produced faster opening rates at α4,β2δ GABAARs (β ~ 1600 s-1) than at α1β2Sγ2S GABAARS (β ~ 460 s-1), whereas GABA activated α1β2γ2s GARAARs more rapidly (β ~ 1800 s-1) than α4β2δ GABAARS (β < 440 s-1). Single channel recordings of α1β2γ2S and α4β2δ GABAARS showed that both channels open to a main conductance state of ~25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited ~36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (Po ~ 0.3) at α4β2δ GABAARs and at least two "modes" of single channel bursting activity, lasting ~100 nis at α1β2γ2S GABAARs. The most prevalent bursting mode had a Po of ~0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" Po mode (~0.9) was characterized by two shut and three THIP states. Single channel activity elicited by THIP in α4β2δ and α1β2γ2S GABAARS occurred as a single population of bursts (Po~0.4-0.5) of moderate duration (~33 ms) that could be described by schemes containing two shut and two open states for both GABAARS. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance. [ABSTRACT FROM AUTHOR]

Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (≥100 µM), and to be a weak agonist at GABAA receptors (GABAA-Rs), which are usually activated at high concentrations (≥1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABAA-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10 -100 µM) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 µM taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 µM Zn2+, consistent with the pharmacological properties of extrasynaptic GABAA-Rs (α4β2δ subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABAA-R α4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant α4β2δ GABAA-Rs than at α1β2γ2 GABAA-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABAA -Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus. [ABSTRACT FROM AUTHOR]

The binding of the neurotransmitter GABA induces conformational changes in the GABAA receptor (GABAAR), leading to the opening of a gate that controls ion permeation through an integral transmembrane pore. A number of structural elements within each subunit, located near the membrane interface, are believed to undergo relative movements during this activation process. In this study, we explored the functional role of the β-10 strand (pre-M1 segment), which connects the extracellular domain to the transmembrane domain. In α1β2γ2s GABAARs, analysis of the 12 residues of the β-10 strand in the α1 subunit proximal to the first transmembrane domain identified two residues, α1V212 and α1K220, in which mutations produced rightward shifts in the GABA concentration–response relationship and also reduced the relative efficacy of the partial agonist, piperidine-4-sulphonic acid. Ultra-fast agonist techniques were applied to mutant α1(K220A)β2γ2s GABAARs and revealed that the macroscopic functional deficit in this mutant could be attributed to a slowing of the opening rate constant, from ∼1500 s−1 in wild-type (WT) channels to ∼730 s−1 in the mutant channels, and a reduction in the time spent in the active state for the mutant. These changes were accompanied by a decrease in agonist affinity, with half-maximal activation rates achieved at 0.77 mm GABA in WT and 1.4 mm GABA in the α1(K220A)β2γ2s channels. The β-10 strand (pre-M1 segment) emerges, from this and other studies, as a key functional component in the activation of the GABAAR. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh) is a neurotransmitter commonly found in all animal species. It was shown to mediate fast excitatory and inhibitory neurotransmission in the molluscan CNS. Since early intracellular recordings, it was shown that the receptors mediating these currents belong to the family of neuronal nicotinic acetylcholine receptors and that they can be distinguished on the basis of their pharmacology. We previously identified 12 Lymnaea cDNAs that were predicted to encode ion channel subunits of the family of the neuronal nicotinic acetylcholine receptors. These Lymnaea nAChRs can be subdivided in groups according to the residues supposedly contributing to the selectivity of ion conductance. Functional analysis in Xenopus oocytes revealed that two types of subunits with predicted distinct ion selectivities form homopentameric nicotinic ACh receptor (nAChR) subtypes conducting either cations or anions. Phylogenetic analysis of the nAChR gene sequences suggests that molluscan anionic nAChRs probably evolved from cationic ancestors through amino acid substitutions in the ion channel pore, a mechanism different from acetylcholine-gated channels in other invertebrates. [ABSTRACT FROM AUTHOR]

Hyperekplexia is a rare human neuromotor disorder caused by mutations that impair the efficacy of glycinergic inhibitory neurotransmission. Loss-of-function mutations in the GLRA1 or GLRB genes, which encode the α1 and β glycine receptor (GlyR) subunits, are the major cause. Paradoxically, gain-offunction GLRA1 mutations also cause hyperekplexia, although the mechanism is unknown. Here we identify two new gain-offunction mutations (I43F and W170S) and characterize these along with known gain-of-function mutations (Q226E, V280M, and R414H) to identify how they cause hyperekplexia. Using artificial synapses, we show that all mutations prolong the decay of inhibitory postsynaptic currents (IPSCs) and induce spontaneous GlyR activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as the depleted chloride gradient should also lead to pain sensitization and to a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients with GLRA1 hyperekplexia mutations. We also rule out small increases in IPSC decay times (as caused by W170S and R414H) as a possible mechanism given that the clinically important drug, tropisetron, significantly increases glycinergic IPSC decay times without causing motor side effects. A recent study on cultured spinal neurons concluded that an elevated intracellular chloride concentration late during development ablates α1β glycinergic synapses but spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is primarily responsible for the hyperekplexia phenotype. [ABSTRACT FROM AUTHOR]

Synaptic GABAA receptors (GABAARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABAAR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABAAR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABAARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly (<1 ms) and deactivated as a function of the α-subunit, with α2-containing GABAARs consistently deactivating ~10-fold more slowly. Single channel analysis revealed that the slower current decay of α2-containing GABAARs was due to longer burst durations at low GABAA concentrations, corresponding to ~4-fold higher affinity for GABA. Synaptic currents revealed a different pattern of activation and deactivation to that of macropatch data. The inclusion of α2 and γ1 subunits slowed both the activation and deactivation rates, suggesting that receptors containing these subunits cluster more diffusely at synapses. Switching the intracellular domains of the γ2 and γ1 subunits substantiated this inference. Because this region determines post-synaptic localization, we hypothesize that GABAARs containing γ1 and γ2 use different mechanisms for synaptic clustering. [ABSTRACT FROM AUTHOR]

Alcohol is the most consumed and abused psychoactive drug globally, but the molecular mechanisms driving alcohol action and its associated behaviors in the brain remain enigmatic. Here, we have discovered a transmembrane protein TMEM132B that is a GABA A receptor (GABA A R) auxiliary subunit. Functionally, TMEM132B promotes GABA A R expression at the cell surface, slows receptor deactivation, and enhances the allosteric effects of alcohol on the receptor. In TMEM132B knockout (KO) mice or TMEM132B I499A knockin (KI) mice in which the TMEM132B-GABA A R interaction is specifically abolished, GABAergic transmission is decreased and alcohol-induced potentiation of GABA A R-mediated currents is diminished in hippocampal neurons. Behaviorally, the anxiolytic and sedative/hypnotic effects of alcohol are markedly reduced, and compulsive, binge-like alcohol consumption is significantly increased. Taken together, these data reveal a GABA A R auxiliary subunit, identify the TMEM132B-GABA A R complex as a major alcohol target in the brain, and provide mechanistic insights into alcohol-related behaviors., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Erythromelalgia (EM), is a familial pain syndrome characterized by episodic 'burning' pain, warmth, and erythema. EM is caused by monoallelic variants in SCN9A, which encodes the voltage-gated sodium channel (NaV) NaV1.7. Over 25 different SCN9A mutations attributed to EM have been described to date, all identified in the SCN9A transcript utilizing exon 6N. Here we report a novel SCN9A missense variant identified in seven related individuals with stereotypic episodes of bilateral lower limb pain presenting in childhood. The variant, XM&#95;011511617.3:c.659G>C;p.(Arg220Pro), resides in the exon 6A of SCN9A, an exon previously shown to be selectively incorporated by developmentally regulated alternative splicing. The mutation is located in the voltage-sensing S4 segment of domain I, which is important for regulating channel activation. Functional analysis showed the p.Arg220Pro mutation altered voltage-dependent activation and delayed channel inactivation, consistent with a NaV1.7 gain-of-function molecular phenotype. These results demonstrate that alternatively spliced isoforms of SCN9A should be included in all genomic testing of EM., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Genetic variants associated with developmental and epileptic encephalopathies have been identified in the GABRB3 gene that encodes the β3 subunit of GABAA receptors. Typically, variants alter receptor sensitivity to GABA resulting in either gain- or loss-of-function, which correlates with patient phenotypes. However, it is unclear how another important receptor property, desensitization, contributes to the greater clinical severity of gain-of-function variants. Desensitization properties of 20 gain-of-function GABRB3 variant receptors were evaluated using two-electrode voltage-clamp electrophysiology. The parameters measured included current decay rates and steady-state currents. Selected variants with increased or reduced desensitization were also evaluated using whole-cell electrophysiology in transfected mammalian cell lines. Of the 20 gain-of-function variants assessed, 13 were found to alter receptor desensitization properties. Seven variants reduced desensitization at equilibrium, which acts to worsen gain-of-function traits. Six variants accelerated current decay kinetics, which limits gain-of-function traits. All affected patients displayed severe clinical phenotypes with intellectual disability and difficult-to-treat epilepsy. Nevertheless, variants that reduced desensitization at equilibrium were associated with more severe clinical outcomes. This included younger age of first seizure onset (median 0.5 months), movement disorders (dystonia and dyskinesia), epilepsy of infancy with migrating focal seizures (EIMFS) and risk of early mortality. Variants that accelerated current decay kinetics were associated with slightly milder phenotypes with later seizure onset (median 4 months), unclassifiable developmental and epileptic encephalopathies or Lennox-Gastaut syndrome and no movement disorders. Our study reveals that gain-of-function GABRB3 variants can increase or decrease receptor desensitization properties and that there is a correlation with the degree of disease severity. Variants that reduced the desensitization at equilibrium were clustered in the transmembrane regions that constitute the channel pore and correlated with greater disease severity, while variants that accelerated current decay were clustered in the coupling loops responsible for receptor activation and correlated with lesser severity., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

GABA A receptors (GABA A Rs) mediate the majority of fast inhibitory transmission throughout the brain. Although it is widely known that pore-forming subunits critically determine receptor function, it is unclear whether their single-channel properties are modulated by GABA A R-associated transmembrane proteins. We previously identified Shisa7 as a GABA A R auxiliary subunit that modulates the trafficking, pharmacology, and deactivation properties of these receptors. However, whether Shisa7 also regulates GABA A R single-channel properties has yet to be determined. Here, we performed single-channel recordings of α2β3γ2L GABA A Rs cotransfected with Shisa7 in HEK293T cells and found that while Shisa7 does not change channel slope conductance, it reduced the frequency of receptor openings. Importantly, Shisa7 modulates GABA A R gating by decreasing the duration and open probability within bursts. Through kinetic analysis of individual dwell time components, activation modeling, and macroscopic simulations, we demonstrate that Shisa7 accelerates GABA A R deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABA A R gating and reveal for the first time that GABA A R single-channel properties can be modulated by an auxiliary subunit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission. SIGNIFICANCE STATEMENT Although GABA A receptor (GABA A R) single-channel properties are largely determined by pore-forming subunits, it remains unknown whether they are also controlled by GABA A R-associated transmembrane proteins. Here, we show that Shisa7, a recently identified GABA A R auxiliary subunit, modulates GABA A R activation by altering single-channel burst kinetics. These results reveal that Shisa7 primarily decreases the duration and open probability of receptor burst activity during gating, leading to accelerated GABA A R deactivation. These experiments are the first to assess the gating properties of GABA A Rs in the presence of an auxiliary subunit and provides a kinetic basis for how Shisa7 modifies temporal attributes of GABAergic transmission at the single-channel level., (Copyright © 2022 the authors.)

The GABA A receptor (GABA A R) α1 subunit A295D epilepsy mutation reduces the surface expression of α1 A295D β2γ2 GABA A Rs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat) was recently shown to correct the misfolding of α1 A295D subunits and thereby enhance the functional surface expression of α1 A295D β2γ2 GABA A Rs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABA A R subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G) known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2 K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic α1β2γ2 GABA A R isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABA A Rs of undefined subunit composition. Transfection with α1β2γ2 N40S , α1β2γ2 R43Q , α1β2γ2 P44S and α1β2γ2 R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABA A Rs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABA A Rs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1β2γ2 K289M GABA A Rs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2 K289M GABA A Rs and SAHA-treated α1β2γ2 R43Q , α1β2γ2 P44S and α1β2γ2 R138G GABA A Rs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile seizures (FS). Given that SAHA is approved by therapeutic regulatory agencies for human use, we propose that it may be worth investigating as a treatment for epilepsies caused by the N40S, R43Q, P44S and R138G mutations. Although SAHA has already been proposed as a therapeutic for patients harbouring the α1 A295D epilepsy mutation, the present study extends its potential utility to a new subunit and four new mutations.

Epilepsy is a spectrum of neurological disorders with many causal factors. The GABA type-A receptor (GABA A R) is a major genetic target for heritable human epilepsies. Here we examine the functional effects of three epilepsy-causing mutations to the α1 subunit (α1 T10'I , α1 D192N and α1 A295D ) on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic GABA A R isoform, α1β2γ2L. We employed a neuron - HEK293 cell heterosynapse preparation to record IPSCs mediated by mutant-containing GABA A Rs in isolation from other GABA A R isoforms. IPSCs were recorded in the presence of the anticonvulsant drugs, carbamazepine and midazolam, and at elevated temperatures (22, 37 and 40°C) to gain insight into mechanisms of febrile seizures. The mutant subunits were also transfected into cultured cortical neurons to investigate changes in synapse formation and neuronal morphology using fluorescence microscopy. We found that IPSCs mediated by α1 T10'I β2γ2L, α1 D192N β2γ2L GABA A Rs decayed faster than those mediated by α1β2γ2L receptors. IPSCs mediated by α1 D192N β2γ2L and α1 A295D β2γ2L receptors also exhibited a heightened temperature sensitivity. In addition, the α1 T10'I β2γ2L GABA A Rs were refractory to modulation by carbamazepine or midazolam. In agreement with previous studies, we found that α1 A295D β2γ2L GABA A Rs were retained intracellularly in HEK293 cells and neurons. However, pre-incubation with 100nM suberanilohydroxamic acid (SAHA) induced α1 A295D β2γ2L GABA A Rs to mediate IPSCs that were indistinguishable in magnitude and waveform from those mediated by α1β2γ2L receptors. Finally, mutation-specific changes to synaptic bouton size, synapse number and neurite branching were also observed. These results provide new insights into the mechanisms of epileptogenesis of α1 epilepsy mutations and suggest possible leads for improving treatments for patients harbouring these mutations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (> or = 100 microM), and to be a weak agonist at GABA(A) receptors (GABA(A)-Rs), which are usually activated at high concentrations (> or = 1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABA(A)-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10-100 microM) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 microM taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 microM Zn2+, consistent with the pharmacological properties of extrasynaptic GABA(A)-Rs (alpha4beta2delta subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABA(A)-R alpha4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant alpha4beta2delta GABA(A)-Rs than at alpha1beta2gamma2 GABA(A)-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABA(A)-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

The binding of the neurotransmitter GABA induces conformational changes in the GABAA receptor (GABAAR), leading to the opening of a gate that controls ion permeation through an integral transmembrane pore. A number of structural elements within each subunit, located near the membrane interface, are believed to undergo relative movements during this activation process. In this study, we explored the functional role of the beta-10 strand (pre-M1 segment), which connects the extracellular domain to the transmembrane domain. In alpha1beta2gamma2s GABAARs, analysis of the 12 residues of the beta-10 strand in the alpha1 subunit proximal to the first transmembrane domain identified two residues, alpha1V212 and alpha1K220, in which mutations produced rightward shifts in the GABA concentration-response relationship and also reduced the relative efficacy of the partial agonist, piperidine-4-sulphonic acid. Ultra-fast agonist techniques were applied to mutant alpha1(K220A)beta2gamma2s GABAARs and revealed that the macroscopic functional deficit in this mutant could be attributed to a slowing of the opening rate constant, from approximately 1500 s(-1) in wild-type (WT) channels to approximately 730 s(-1) in the mutant channels, and a reduction in the time spent in the active state for the mutant. These changes were accompanied by a decrease in agonist affinity, with half-maximal activation rates achieved at 0.77 mM GABA in WT and 1.4 mM GABA in the alpha1(K220A)beta2gamma2s channels. The beta-10 strand (pre-M1 segment) emerges, from this and other studies, as a key functional component in the activation of the GABAAR.