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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. [ABSTRACT FROM AUTHOR]

Objective: N‐methyl‐d‐aspartate (NMDA) receptors are expressed at synaptic sites, where they mediate fast excitatory neurotransmission. NMDA receptors are critical to brain development and cognitive function. Natural variants to the GRIN1 gene, which encodes the obligatory GluN1 subunit of the NMDA receptor, are associated with severe neurological disorders that include epilepsy, intellectual disability, and developmental delay. Here, we investigated the pathogenicity of three missense variants to the GRIN1 gene, p. Ile148Val (GluN1‐3b[I481V]), p.Ala666Ser (GluN1‐3b[A666S]), and p.Tyr668His (GluN1‐3b[Y668H]). Methods: Wild‐type and variant‐containing NMDA receptors were expressed in HEK293 cells and primary hippocampal neurons. Patch‐clamp electrophysiology and pharmacology were used to profile the functional properties of the receptors. Receptor surface expression was evaluated using fluorescently tagged receptors and microscopy. Results: Our data demonstrate that the GluN1(I481V) variant is inhibited by the open pore blockers ketamine and memantine with reduce potency but otherwise has little effect on receptor function. By contrast, the other two variants exhibit gain‐of‐function molecular phenotypes. Glycine sensitivity was enhanced in receptors containing the GluN1(A666S) variant and the potency of pore block by memantine and ketamine was reduced, whereas that for MK‐801 was increased. The most pronounced functional deficits, however, were found in receptors containing the GluN1(Y668H) variant. GluN1(Y668H)/2A receptors showed impaired surface expression, were more sensitive to glycine and glutamate by an order of magnitude, and exhibited impaired block by extracellular magnesium ions, memantine, ketamine, and MK‐801. These variant receptors were also activated by either glutamate or glycine alone. Single‐receptor recordings revealed that this receptor variant opened to several conductance levels and activated more frequently than wild‐type GluN1/2A receptors. Significance: Our study reveals a critical functional locus of the receptor (GluN1[Y668]) that couples receptor gating to ion channel conductance, which when mutated may be associated with neurological disorder. [ABSTRACT FROM AUTHOR]

GABAA receptors (GABAARs) 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 GABAAR-associated transmembrane proteins. We previously identified Shisa7 as a GABAAR auxiliary subu-nit that modulates the trafficking, pharmacology, and deactivation properties of these receptors. However, whether Shisa7 also regulates GABAAR single-channel properties has yet to be determined. Here, we performed single-channel recordings of a2b3y2L GABAARs 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 GABAAR 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 GABAAR deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABAAR gating and reveal for the first time that GABAAR single-channel properties can be modulated by an auxiliary subu-nit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission. [ABSTRACT FROM AUTHOR]

The [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] and [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] two isoforms of [gamma]-aminobutyric acid type A ([GABA.sub.A]) 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. [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] channels exhibited briefer active periods than [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] 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 [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels was ~2.6 [micro]M compared with ~19 [micro]M for [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] channels, suggesting that GABA binds to the [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] 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 [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of [GABA.sub.A] receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate [GABA.sub.A] receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system. doi/ 10.1085/jgp.200910317

The family of [gamma]-aminobutyric acid type A receptors ([GABA.sub.A]Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic [GABA.sub.A]Rs that are mainly comprised of [[alpha].sub.1], [[beta].sub.2], and [[gamma].sub.2] subunits, whereas tonic inhibition is mediated by extrasynaptic [GABA.sub.A]Rs comprised of [[alpha].sub.4/6], [[beta].sub.2], and [delta] subunits. We investigated the activation properties of recombinant [[alpha].sub.4][[beta].sub.2] [delta] and [[alpha].sub.1][[beta].sub.2][gamma].sub.2s] [GABA.sub.A]Rs 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 experiments indicated that THIP produced faster opening rates at [[alpha].sub.4][[beta].sub.2][delta] [GABA.sub.A]Rs ([beta] ~ 1600 [s.sup.-1]) than at [[alpha].sub.1][[beta].sub.2] [gamma].sub.2s] [GABA.sub.A]Rs ([beta] ~ 460 [s.sup.-1]), whereas GABA activated [[alpha].sub.1][[beta].sub.2][gamma].sub.2s] [GABA.sub.A]Rs more rapidly ([beta] ~1800 [s.sup.-1]) than [[alpha].sub.4][[beta].sub.2][delta] [GABA.sub.A]Rs ([beta] < 440 [s.sup.-1). Single channel recordings of [[alpha].sub.1] [[beta].sub.2][gamma].sub.2s] and [[alpha].sub.4][[beta].sub.2] [delta] [GABA.sub.A]Rs showed that both channels open to a main conductance state of ~25 pS at -70 mV when activated by GABA and low concentrations of THIE whereas saturating concentrations of THIP elicited ~36 pS openings at both channels. Saturating concentrations of GABA elicited brief (

Background: Genetic variants in the subunits of the gamma-aminobutyric acid type A (GABAA) receptors are implicated in the onset of multiple pathologic conditions including genetic epilepsy. Previous work showed that pathogenic GABAA subunits promote misfolding and inefficient assembly of the GABAA receptors, limiting receptor expression and activity at the plasma membrane. However, GABAA receptors containing variant subunits can retain activity, indicating that enhancing the folding, assembly, and trafficking of these variant receptors offers a potential opportunity to mitigate pathology associated with genetic epilepsy. Results: Here, we demonstrate that pharmacologically enhancing endoplasmic reticulum (ER) proteostasis using small molecule activators of the ATF6 (Activating Transcription Factor 6) signaling arm of the unfolded protein response (UPR) increases the assembly, trafficking, and surface expression of variant GABAA receptors. These improvements are attributed to ATF6-dependent remodeling of the ER proteostasis environment, which increases protein levels of pro-folding ER proteostasis factors including the ER chaperone BiP (Immunoglobulin Binding Protein) and trafficking receptors, such as LMAN1 (Lectin Mannose-Binding 1) and enhances their interactions with GABAA receptors. Importantly, we further show that pharmacologic ATF6 activators increase the activity of GABAA receptors at the cell surface, revealing the potential for this strategy to restore receptor activity to levels that could mitigate disease pathogenesis. Conclusions: These results indicate that pharmacologic ATF6 activators offer an opportunity to restore GABAA receptor activity in diseases including genetic epilepsy and point to the potential for similar pharmacologic enhancement of ER proteostasis to improve trafficking of other disease-associated variant ion channels implicated in etiologically-diverse diseases. [ABSTRACT FROM AUTHOR]

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRarl subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRAl mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels. [ABSTRACT FROM AUTHOR]

Background and Purpose: Between half to 1 million people die annually from malaria. Anopheles gambiae mosquitoes are major malaria vectors. Unfortunately, resistance has emerged to the agents currently used to control A. gambiae, creating a demand for novel control measures. The pentameric glutamate-gated chloride channel (GluCl) expressed in the muscle and nerve cells of these organisms are a potentially important biological target for malaria control. The pharmacological properties of Anophiline GluCl receptors are, however, largely unknown. Accordingly, we compared the efficacy of four insecticides (lindane, fipronil, picrotoxin, and ivermectin) on two A. gambiae GluCl receptor splice variants with the aim of providing a molecular basis for designing novel anti-malaria treatments.Experimental Approach: The A. gambiae GluCl receptor b1 and c splice variants were expressed homomerically in Xenopus laevis oocytes and studied with electrophysiological techniques, using two-electrode voltage-clamp.Key Results: The b1 and c GluCl receptors were activated with similar potencies by glutamate and ivermectin. Fipronil was more potent than picrotoxin and lindane at inhibiting glutamate- and ivermectin-gated currents. Importantly, b1 GluCl receptors exhibited reduced sensitivity to picrotoxin and lindane. They also recovered from these effects to a greater extent than c GluCl receptors CONCLUSIONS AND IMPLICATIONS: The two splice variant subunits exhibited differential sensitivities to multiple, structurally divergent insecticides, without accompanying changes in the sensitivity to the endogenous neurotransmitter, glutamate, implying that drug resistance may be caused by alterations in relative subunit expression levels, without affecting physiological function. Our results strongly suggest that it should be feasible to develop novel subunit-specific pharmacological agents. [ABSTRACT FROM AUTHOR]

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor. [ABSTRACT FROM AUTHOR]

The GABAA receptor (GABAAR) α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dβ2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat) was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dβ2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR 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 γ2K289M 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 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1β2γ2N40S, α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABAARs. 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 α1β2γ2K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2K289M GABAARs and SAHA-treated α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G GABAARs 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 α1A295D epilepsy mutation, the present study extends its potential utility to a new subunit and four new mutations. [ABSTRACT FROM AUTHOR]

α5-containing GABA A Rs are potential therapeutic targets for clinical conditions including age-related dementia, stroke, schizophrenia, Down syndrome, anaesthetic-induced amnesia, anxiety and pain. α5-containing GABA A Rs are expressed in layer 5 cortical neurons and hippocampal pyramidal neurons where they mediate both tonic currents and slow inhibitory postsynaptic currents (IPSCs). A range of drugs has been developed to specifically modulate these receptors. The main α5-containing GABA A Rs that are likely to exist in vivo are the α5β1γ2, α5β2γ2 and α5β3γ2 isoforms. We currently have few clues as to how these isoforms are distributed between synaptic and extrasynaptic compartments or their relative roles in controlling neuronal excitability. Accordingly, the aim of this study was to define the basic biophysical and pharmacological properties of IPSCs mediated by the three isoforms in a hippocampal neuron-HEK293 cell co-culture assay. The IPSC decay time constants were slow (α5β1γ2L: 45 ms; α5β1γ2L: 80 ms; α5β3γ2L: 184 ms) and were largely dominated by the intrinsic channel deactivation rates. By comparing IPSC rise times, we inferred that α5β1γ2L GABA A Rs are located postsynaptically whereas the other two are predominantly perisynaptic. α5β3γ2L GABA A Rs alone mediated tonic currents. We quantified the effects of four α5-specific inverse agonists (TB-21007, MRK-016, α5IA and L-655708) on IPSCs mediated by the three isoforms. All compounds selectively inhibited IPSC amplitudes and accelerated IPSC decay rates, albeit with distinct isoform specificities. MRK-016 also significantly accelerated IPSC rise times. These results provide a reference for future studies seeking to identify and characterize the properties of IPSCs mediated by α5-containing GABA A R isoforms in neurons. [ABSTRACT FROM AUTHOR]

GABA-A receptors (GABAARs) are pentameric ligand-gated ion channels that are assembled mainly from α (α1-6), β (β1-3) and γ (γ1-3) subunits. Although GABAARs containing γ2L subunits mediate most of the inhibitory neurotransmission in the brain, significant expression of γ1 subunits is seen in the amygdala, pallidum and substantia nigra. However, the location and function of γ1-containing GABAARs in these regions remains unclear. In "artificial" synapses, where the subunit composition of postsynaptic receptors is specifically controlled, γ1 incorporation slows the synaptic current decay rate without affecting channel deactivation, suggesting that γ1-containing receptors are not clustered and therefore activated by diffuse neurotransmitter. However, we show that γ1-containing receptors are localized at neuronal synapses and form clusters in both synaptic and extrasynaptic regions. In addition, they exhibit rapid membrane diffusion and a higher frequency of exchange between synaptic and perisynaptic populations compared to g2L-containing GABAARs. A point mutation in the large intracellular domain and a pharmacological analysis reveal that when a single non-conserved γ2L residue is mutated to its γ1 counterpart (T349L), the synaptic current decay is slowed from γ2L-to γ1-like without changing the clustering or diffusion properties of the receptors. In addition, previous fast perfusion and single channel kinetic experiments revealed no difference in the intrinsic closing rates of γ2Land γ1-containing receptors when expressed in HEK293 cells. These observations together with Monte Carlo simulations of synaptic function confirm that decreased clustering does not control γ1-containing GABAAR kinetics. Rather, they suggest that γ1- and γ2L-containing receptors exhibit differential synaptic current decay rates due to differential gating dynamics when localized at the synapse. [ABSTRACT FROM AUTHOR]

Glycine receptors (GlyRs) containing the α2 subunit regulate cortical interneuron migration. Disruption of the GlyR α2 subunit gene (Glrα2) in mice leads to disrupted dorsal cortical progenitor homeostasis, leading to a depletion of projection neurons and moderate microcephaly in newborn mice. In humans, rare variants in GLRα2, which is located on the X chromosome, are associated with autism spectrum disorder (ASD) in the hemizygous state in males. These include a microdeletion (GLRα2Dex8-9) and missense mutations in GLRα2 (p.N109S and p.R126Q) that impair cell-surface expression of GlyR α2, and either abolish or markedly reduce sensitivity to glycine. We report the functional characterization of a third missense variant in GLRα2 (p.R323L), associated with autism, macrocephaly, epilepsy and hypothyroidism in a female proband. Using heterosynapse and macroscopic current recording techniques, we reveal that GlyR α2R323L exhibits reduced glycine sensitivity, but significantly increased inhibitory postsynaptic current (IPSC) rise and decay times. Site-directed mutagenesis revealed that the nature of the amino acid switch at position 323 is critical for impairment of GlyR function. Single-channel recordings revealed that the conductance of α2R323Lb channels was higher than α2b channels. Longer mean opening durations induced by p.R323L may be due to a change in the gating pathway that enhances the stability of the GlyR open state. The slower synaptic decay times, longer duration active periods and increase in conductance demonstrates that the GlyR α2 p.R323L mutation results in an overall gain of function, and that GlyR α2 mutations can be pathogenic in the heterozygous state in females. [ABSTRACT FROM AUTHOR]

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]

Anion/cation selectivity is a critical property of ion channels and underpins their physiological function. Recently, there have been numerous mutagenesis studies, which have mapped sites within the ion channel-forming segments of ligand-gated ion channels that are determinants of the ion selectivity. Site-directed mutations to specific amino acids within or flanking the M2 transmembrane segments of the anion-selective glycine, GABAA and GABAC receptors and the cation-selective nicotinic acetylcholine and serotonin (type 3) receptors have revealed discrete, equivalent regions within the ion channel that form the principal selectivity filter, leading to plausible molecular mechanisms and mathematical models to describe how ions preferentially permeate these channels. In particular, the dominant factor determining anion/cation selectivity seems to be the sign and exposure of charged amino acids lining the selectivity filter region of the open channel. In addition, the minimum pore diameter, which can be influenced by the presence of a local proline residue, also makes a contribution to such ion selectivity in LGICs with smaller diameters increasing anion/cation selectivity and larger ones decreasing it. [Copyright &y& Elsevier]

The glycine receptor-channel (GlyR) mediates neuronal inhibition by selectively allowing the passage of Cl− ions through its channel. The pore region for ion selectivity is localised to the constricted internal end of the M2 transmembrane domain. This paper investigates the contribution of the P-2′ residue in determining pore diameter and ion charge selectivity of the GlyR. The deletion of this proline has been shown to decrease the anion/cation permeability ratio, with PCl/PNa decreasing from ∼27 to ∼4. We show that the P-2′ deletion by itself produces a GlyR with a larger pore diameter (∼0.69 nm) than the wild type value (∼0.54 nm). This confirms that the P-2′ residue reduces pore size, which suggests that, in addition to electrostatic effects, pore size also contributes to ion-charge selectivity. [Copyright &y& Elsevier]

Presents a study which investigated the single channel conductance and rectification properties of cation selective mutant glycine receptors. Background on ligand-gated ion channels; Materials and methods used; Results and discussion.

Presents a study which determined whether cation-selective mutations in the M2-domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity. Background on selectivity; Materials and methods used; Results and discussion.

NMDA receptor (NMDAR)-dependent Ca2+ influx underpins multiple forms of synaptic plasticity. Most synaptic NMDAR currents in the adult forebrain are mediated by GluN2A-containing receptors, which are rapidly inserted into synapses during long-term potentiation (LTP); however, the underlying molecular mechanisms remain poorly understood. In this study, we show that GluN2A is phosphorylated at Ser-1459 by Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) in response to glycine stimulation that mimics LTP in primary neurons. Phosphorylation of Ser-1459 promotes GluN2A interaction with the sorting nexin 27 (SNX27)-retromer complex, thereby enhancing the endosomal recycling of NMDARs. Loss of SNX27 or CaMKIIα function blocks the glycine-induced increase in GluN2A-NMDARs on the neuronal membrane. Interestingly, mutations of Ser-1459, including the rare S1459G human epilepsy variant, prolong the decay times of NMDAR-mediated synaptic currents in heterosynapses by increasing the duration of channel opening. These findings not only identify a critical role of Ser-1459 phosphorylation in regulating the function of NMDARs, but they also explain how the S1459G variant dysregulates NMDAR function. [Display omitted] • CaMKIIα phosphorylates GluN2A at Ser-1459 in response to glycine stimulation • CaMKIIα and SNX27 are required for the glycine-induced increase in surface GluN2A • GluN2A Ser-1459 is a critical residue that controls the gating of NMDA receptors • The epilepsy-associated GluN2A S1459G variant prolongs open channel duration Yong et al. identify that activity-dependent phosphorylation of Ser-1459 in the GluN2A C-terminal domain by CaMKIIα promotes its interaction with the SNX27-retromer complex, thereby enhancing the surface expression of NMDARs during synaptic potentiation. Mutations of Ser-1459 prolong the decay times of NMDAR-mediated synaptic currents by increasing the duration of channel opening. [ABSTRACT FROM AUTHOR]

Mutations in synaptic NMDA receptors (NMDARs) are associated with epilepsy and neurodevelopmental disorders. The effects of several such mutations have been investigated in recombinantly-expressed NMDARs under conditions of steady-state activation. Such experiments provide only limited insight into how mutations affect NMDAR-mediated excitatory synaptic currents (EPSCs). The present study aimed to characterize the effects of the GluN2AN615K, GluN2BN615I and GluN2BV618G gain-of-function mutations on EPSCs mediated by diheteromeric GluN1/2A and GluN1/2B receptors and triheteromeric GluN1/2A/2B receptors, as these are the most abundant synaptic NMDARs in vivo. Subunit composition was controlled by studying 'artificial' synapses formed between cultured neurons (which provide presynaptic terminals) and HEK293 cells that express the NMDAR subunits of interest plus the synapse-promoting molecule, neuroligin-1B. When incorporated into diheteromeric receptors, all three mutations ablated voltage-dependent Mg2+ block of EPSCs, as previously shown. In addition, we were surprised to find that increasing external Mg2+ from 0 to 1 mM strongly enhanced the magnitude of EPSCs mediated by mutant diheteromers. In contrast, triheteromeric receptors exhibited normal voltage-dependent Mg2+ block. The GluN2AN615K mutation also slowed the decay of GluN1/2A/2B- but not GluN1/2A-mediated EPSCs. The GluN2BN615I mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs. The GluN2BV618G mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs, although these effects were partly compensated by a faster EPSC decay rate. The mutations also diminished the potency of the anti-epileptic pore-blocker, memantine, thus explaining the lack of memantine efficacy in patients with GluN2BN615I or GluN2BV618G mutations. Given these effects, the three mutations would be expected to enhance the cation influx rate and thereby contribute to epilepsy phenotypes. • All three mutations eliminated voltage-dependent Mg2+ block of diheteromeric EPSCs • Increasing Mg2+ from 0 to 1 mM enhanced the size of mutant diheteromeric EPSCs • Mutant triheteromeric EPSCs exhibited normal voltage-dependent Mg2+ block • They also exhibited larger magnitudes and/or altered decay rates • These mutations would enhance neuronal excitability, promoting epilepsy phenotypes [ABSTRACT FROM AUTHOR]

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1-1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation. [ABSTRACT FROM AUTHOR]

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemoelectrical transduction in the nervous system. The mechanism by which the energy of ligand binding leads to current-conducting receptors is poorly understood and may vary among family members. We addressed these questions by correlating the structural and energetic mechanisms by which a naturally occurring M1 domain mutation (α1Q-26'E) enhances receptor activation in homo- and heteromeric glycine receptors. We systematically altered the charge of spatially clustered residues at positions 19' and 24', in the M2 and M2-M3 linker domains, respectively, which are known to be critical to efficient receptor activation, on a background of α1Q-26'E. Changes in the durations of single receptor activations (clusters) and conductance were used to determine interaction coupling energies, which we correlated with conformational displacements as measured in pLGIC crystal structures. Presence of the α1Q-26'E enhanced cluster durations and reduced channel conductance in homo-and heteromeric receptors. Strong coupling between α1-26' and α119' across the subunit interface suggests an important role in receptor activation. A lack of coupling between α1-26' and α124' implies that 24' mutations disrupt activation via other interactions. A similar lack of energetic coupling between α126' and reciprocal mutations in the β subunit suggests that this subunit remains relatively static during receptor activation. However, the channel effects of α1Q-26'E on α1β receptors suggests at least one α1-α1 interface per pentamer. The coupling-energy change between α126' and α119' correlates with a local structural rearrangement essential for pLGIC activation, implying it comprises a key energetic pathway in activating glycine receptors and other pLGICs. [ABSTRACT FROM AUTHOR]

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 100 nM 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. [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]

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors. [ABSTRACT FROM AUTHOR]

Abstract: Startle disease is a rare, potentially fatal neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden unexpected auditory, visual or tactile stimuli. Mutations in the GlyR α1 subunit gene (GLRA1) are the major cause of this disorder, since remarkably few individuals with mutations in the GlyR β subunit gene (GLRB) have been found to date. Systematic DNA sequencing of GLRB in individuals with hyperekplexia revealed new missense mutations in GLRB, resulting in M177R, L285R and W310C substitutions. The recessive mutation M177R results in the insertion of a positively-charged residue into a hydrophobic pocket in the extracellular domain, resulting in an increased EC50 and decreased maximal responses of α1β GlyRs. The de novo mutation L285R results in the insertion of a positively-charged side chain into the pore-lining 9′ position. Mutations at this site are known to destabilize the channel closed state and produce spontaneously active channels. Consistent with this, we identified a leak conductance associated with spontaneous GlyR activity in cells expressing α1βL285R GlyRs. Peak currents were also reduced for α1βL285R GlyRs although glycine sensitivity was normal. W310C was predicted to interfere with hydrophobic side-chain stacking between M1, M2 and M3. We found that W310C had no effect on glycine sensitivity, but reduced maximal currents in α1β GlyRs in both homozygous (α1βW310C) and heterozygous (α1ββW310C) stoichiometries. Since mild startle symptoms were reported in W310C carriers, this may represent an example of incomplete dominance in startle disease, providing a potential genetic explanation for the ‘minor’ form of hyperekplexia. [Copyright &y& Elsevier]

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.)