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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]

Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of [alpha]1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2'[DELTA]) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels. KEY WORDS: ligand-gated ion channels * rings of charge * ion permeation * ion selectivity * M2 domain

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective [alpha]1 homomeric glycine receptor ([alpha]1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1'E GlyR) was sufficient to convert the channels to select cations over anions with [P.sub.Cl]/[P.sub.Na] = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2'[DELTA] mutant GlyR retained its anion selectivity ([P.sub.Cl]/[P.sub.Na] = 3.81), but it was much reduced compared with the wild-type (WT) GlyR ([P.sub.Cl]/[P.sub.Na] = 27.9). When the A-1'E and the P-2'[DELTA] mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced ([P.sub.Cl]/[P.sub.Na] = 0.13). The SDM GlyR was also [Ca.sup.2+] permeable ([P.sub.Ca]/[P.sub.Na] = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more [Ca.sup.2+]-permeable channel ([P.sub.Ca]/[P.sub.Na] = 0.73), without drastically altering monovalent charge selectivity ([P.sub.Cl]/[P.sub.Na] = 0.23). The SDM+R19'E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced [Ca.sup.2+] permeability ([P.sub.Ca]/[P.sub.Na] = 0.92), with little effect on monovalent selectivity ([P.sub.Cl]/[P.sub.Na] = 0.19). Estimates of the minimum pore diameter of the A-1'E, SDM, SDM+R19'A, and SDM+R19'E GlyRs revealed that these pores are larger than the [alpha]1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity. KEY WORDS: ligand-gated ion channels * electrostatics * pore diameter * permeability * selectivity filter

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]

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]

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]