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Background and Purpose: GABA(A) receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABA(A) receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems.Experimental Approach: We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes.Key Results: δ-β2 -α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn(2+) . We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations.Conclusions and Implications: The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors. [ABSTRACT FROM AUTHOR]

Synaptic receptors of the nicotinic receptor gene family are pentamers of subunits. This modular structure creates problems in studies of drug actions, related to the number of copies of a subunit that are present and their position. A separate issue concerns the mechanism of action of many anesthetics, which involves potentiation of responses to neurotransmitters. Potentiation requires an interaction between a transmitter and a potentiator, mediated through the target receptor. We have studied the mechanism by which neurosteroids potentiate transmitter responses, using concatemers of covalently linked subunits to control the number and position of subunits in the assembled receptor and to selectively introduce mutations into positionally defined copies of a subunit. We found that the steroid needs to interact with only one site to produce potentiation, that the native sites for steroid interaction have indistinguishable properties, and that steroid potentiation appears to result from a global effect on receptor function. [ABSTRACT FROM AUTHOR]

The γ-aminobutyric acid type A receptor is the major transmitter-gated inhibitory channel in the central nervous system. The receptor is a target for anesthetics, anticonvulsants, anxiolytics, and sedatives whose actions facilitate the flow of chloride ions through the channel and enhance the inhibitory tone in the brain. Both the kinetic and structural aspects of the actions of modulators of the γ-aminobutyric acid type A receptor are of great importance to understanding the molecular mechanisms of general anesthesia. In this review, the structural rearrangements that take place in the γ-aminobutyric acid type A receptor during channel activation and modulation are described, focusing on data obtained using voltage-clamp fluorometry. Voltage-clamp fluorometry entails the binding of an environmentally sensitive fluorophore molecule to a site of interest in the receptor, and measurement of changes in the fluorescence signal resulting from activation- or modulation-elicited structural changes. Detailed investigations can provide a map of structural changes that underlie or accompany the functional effects of modulators. [ABSTRACT FROM AUTHOR]

The γ-aminobutyric acid type A (GABA A ) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3 β -hydroxy steroids inhibit, while 3 α -hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α 1 β 3γ2L GABA A receptor by the endogenous neurosteroid 3 α -hydroxy-5 β -pregnan-20-one (3 α 5 β P) and the synthetic neuroactive steroid 3 α -hydroxy-5 α -androstane-17 β -carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3 α 5 β P and ACN potentiate the GABA A receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC 50 s of ∼13 μ M and maximal inhibitory effects of 70-90%. Receptor inhibition by 3 α 5 β P was sensitive to substitution of the α 1 transmembrane domain (TM) 2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3 α 5 β P inhibit the GABA A receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α 1 and β 3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3 α 5 β P and ACN. SIGNIFICANCE STATEMENT: The heteromeric GABA A receptor is inhibited by sulfated steroids and 3 β -hydroxy steroids, while 3 α -hydroxy steroids are considered to potentiate the receptor. We show here that 3 α -hydroxy steroids have inhibitory effects on the α 1 β 3γ2L receptor, which are observed in specific experimental settings and are expected to manifest under different physiological conditions., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

The properties of a potentiator are typically evaluated by measuring its ability to enhance the magnitude of the control response. Analysis of the ability of drugs to potentiate responses from receptor channels takes place in the context of particular models to extract parameters for functional effects. In the often-used coagonist model, the agonist generating control activity and the potentiator enhancing the control activity make additive energetic contributions to stabilize the active state of the receptor. The energetic contributions are fixed and, once known, enable calculation of predicted receptor behavior at any concentration combination of agonist and potentiator. Here, we have examined the applicability of the coagonist model by measuring the relationship between the magnitude of receptor potentiation and the level of background activity. Ternary αβγ GABA A receptors were activated by GABA or the allosteric agonist propofol, or by a gain-of-function mutation, and etiocholanolone- or propofol-mediated potentiation of peak responses was measured. We show that the free energy change contributed by the modulators etiocholanolone or propofol is reduced at higher levels of control activity, thereby being in disagreement with basic principles of the coagonist model. Possible mechanisms underlying this discrepancy are discussed., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The GABA A receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human α 1 β 3 γ 2L GABA A receptor. Using electrophysiology and quantitative model-based data analysis, we show that exposure to the steroid promotes occupancy of a nonconducting state that retains high affinity to the transmitter but whose properties differ from those of the classic, transmitter-induced desensitized state. From the analysis of the inhibitory actions of two combined steroids, we infer that PS and DHEAS act through shared or overlapping binding sites. SIGNIFICANCE STATEMENT: Previous work has proposed that sulfated neurosteroids inhibit the GABA A receptor by enhancing the rate of entry into the desensitized state. This study shows that the inhibitory steroids pregnenolone sulfate and dehydroepiandrosterone sulfate act through a common interaction site by stabilizing a distinct nonconducting state., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: In electrophysiological experiments, inhibition of a receptor-channel, such as the GABAA receptor, is measured by co-applying an agonist producing a predefined control response with an inhibitor to calculate the fraction of the control response remaining in the presence of the inhibitor. The properties of the inhibitor are determined by fitting the inhibition concentration- response relationship to the Hill equation to estimate the midpoint (IC50) of the inhibition curve Objective: We sought to estimate sensitivity of the fitted IC50 to the level of activity of the control response Methods: The inhibition concentration-response relationships were calculated for models with distinct mechanisms of inhibition. In Model I, the inhibitor acts allosterically to stabilize the resting state of the receptor. In Model II, the inhibitor competes with the agonist for a shared binding site. In Model III, the inhibitor stabilizes the desensitized state., Results: The simulations indicate that the fitted IC50 of the inhibition curve is sensitive to the degree of activity of the control response. In Models I and II, the IC50 of inhibition was increased as the probability of being in the active state (PA) of the control response increased. In Model III, the IC50 of inhibition was reduced at higher PA., Conclusion: We infer that the apparent potency of an inhibitor depends on the PA of the control response. While the calculations were carried out using the activation and inhibition properties that are representative of the GABAA receptor, the principles and conclusions apply to a wide variety of receptor- channels., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

GABA A receptors (GABA A Rs) play a crucial role in inhibition in the central nervous system. GABA A Rs containing the δ subunit mediate tonic inhibition, have distinctive pharmacological properties and are associated with disorders of the nervous system. To explore this receptor sub-class, we recently developed mice with δ-containing receptors rendered resistant to the common non-competitive antagonist picrotoxin (PTX). Resistance was achieved with a knock-in point mutation (T269Y; T6'Y) in the mouse genome. Here we characterize pharmacological and biophysical features of GABA A Rs containing the mutated subunit to contextualize results from the KI mice. Recombinant receptors containing δ T6'Y plus WT α4 and WT β2 subunits exhibited 3-fold lower EC 50 values for GABA but not THIP. GABA EC 50 values in native receptors containing the mutated subunit were in the low micromolar range, in contrast with some published results that have suggested nM sensitivity of recombinant receptors. Rectification properties of δ-containing GABA A Rs were similar to γ2-containing receptors. Receptors containing δ T6'Y had marginally weaker sensitivity to positive allosteric modulators, likely a secondary consequence of differing GABA sensitivity. Overexpression of δT6'Y in neurons resulted in robust PTX-insensitive IPSCs, suggesting that δ-containing receptors are readily recruited by synaptically released GABA. Overall, our results give context to the use of δ receptors with the T6'Y mutation to explore the roles of δ-containing receptors in inhibition., Competing Interests: CZ serves on the Scientific Advisory Board to Sage Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shu, Lu, Bracamontes, Steinbach, Zorumski and Mennerick.)

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABA A receptor. The sites are located in the membrane-spanning domains of the receptor at the β - α subunit interface (site I) and within the α (site II) and β subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α 1 β 2 γ 2L GABA A receptor by the endogenous neurosteroid allopregnanolone (3 α 5 α P). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3 α 5 α P, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3 α 5 α P act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT: Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric γ -aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Synaptic GABA A receptors are alternately exposed to short pulses of a high, millimolar concentration of GABA and prolonged periods of low, micromolar concentration of the transmitter. Prior work has indicated that exposure to micromolar concentrations of GABA can both activate the postsynaptic receptors generating sustained low-amplitude current and desensitize the receptors, thereby reducing the peak amplitude of subsequent synaptic response. However, the precise relationship between tonic activation and reduction of peak response is not known. Here, we have measured the effect of prolonged exposure to GABA or the combination of GABA and the neurosteroid allopregnanolone, which was intended to desensitize a fraction of receptors, on a subsequent response to a high concentration of agonist in human α 1 β 3 γ 2L receptors expressed in Xenopus oocytes. We show that the reduction in the peak amplitude of the post-exposure test response correlates with the open probability of the preceding desensitizing response. Curve fitting of the inhibitory relationship yielded an IC 50 of 12.5 µM and a Hill coefficient of -1.61. The activation and desensitization data were mechanistically analyzed in the framework of a three-state Resting-Active-Desensitized model. Using the estimated affinity, efficacy, and desensitization parameters, we calculated the amount of desensitization that would accumulate during a long (2-minute) application of GABA or GABA plus allopregnanolone. The results indicate that accumulation of desensitization depends on the level of activity rather than agonist or potentiator concentration per se. We estimate that in the presence of 1 µM GABA, approximately 5% of α 1 β 3 γ 2L receptors are functionally eliminated because of desensitization. SIGNIFICANCE STATEMENT: We present an analytical approach to quantify and predict the loss of activatable GABA A receptors due to desensitization in the presence of transmitter and the steroid allopregnanolone. The findings indicate that the peak amplitude of the synaptic response is influenced by ambient GABA and that changes in ambient concentrations of the transmitter and other GABAergic agents can modify tonically and phasically activated synaptic receptors in opposite directions., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria and a potent orthosteric agonist of the GABA A receptor. The binding of [ 3 H]muscimol has been used to evaluate the distribution of GABA A receptors in the brain, and studies of modulation of [ 3 H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABA A receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor. Here, we employed the Monod-Wyman-Changeux concerted transition model to analyze muscimol binding isotherms. We show that the binding isotherms from recombinant α 1 β 3 GABA A receptors can be qualitatively predicted using electrophysiological data pertaining to properties of receptor activation and desensitization in the presence of muscimol. The model predicts enhancement of [ 3 H]muscimol binding in the presence of the steroids allopregnanolone and pregnenolone sulfate, although the steroids interact with distinct sites and either enhance (allopregnanolone) or reduce (pregnenolone sulfate) receptor function. We infer that the concerted transition model can be used to link radioligand binding and electrophysiological data. SIGNIFICANCE STATEMENT: The study employs a three-state resting-active-desensitized model to link radioligand binding and electrophysiological data. We show that the binding isotherms can be qualitatively predicted using parameters estimated in electrophysiological experiments and that the model accurately predicts the enhancement of [ 3 H]muscimol binding in the presence of the potentiating steroid allopregnanolone and the inhibitory steroid pregnenolone sulfate., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

The ρ 1 GABA A receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ 1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABA A receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5 β -reduced steroids, sulfated or carboxylated steroids, and β -estradiol, whereas many 5 α -reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ 1 GABA A receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3 α 5 β P), 3 α 5 β P sulfate, and β -estradiol. The site for 3 α 5 β P can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABA A receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Pentameric GABA A receptors mediate a large share of CNS inhibition. The γ2 subunit is a typical constituent. At least 11 mutations in the γ2 subunit cause human epilepsies, making the role of γ2-containing receptors in brain function of keen basic and translational interest. How small changes to inhibition may cause brain abnormalities, including seizure disorders, is unclear. In mice, we perturbed fast inhibition with a point mutation T272Y (T6'Y in the second membrane-spanning domain) to the γ2 subunit. The mutation imparts resistance to the GABA A receptor antagonist picrotoxin, allowing verification of mutant subunit incorporation. We confirmed picrotoxin resistance and biophysical properties in recombinant receptors. T6'Y γ2-containing receptors also exhibited faster deactivation but unaltered steady-state properties. Adult T6'Y knockin mice exhibited myoclonic seizures and abnormal cortical EEG, including abnormal hippocampal-associated theta oscillations. In hippocampal slices, picrotoxin-insensitive inhibitory synaptic currents exhibited fast decay. Excitatory/inhibitory balance was elevated by an amount expected from the IPSC alteration. Partial pharmacological correction of γ2-mediated IPSCs with diazepam restored total EEG power toward baseline, but had little effect on the abnormal low-frequency peak in the EEG. The results suggest that at least part of the abnormality in brain function arises from the acute effects of truncated inhibition.

The synaptic α1β2γ2 GABA A receptor is activated phasically by presynaptically released GABA. The receptor is considered to be inactive between synaptic events when exposed to ambient GABA because of its low resting affinity to the transmitter. We tested the hypothesis that a combination of physiological and/or clinical positive allosteric modulators of the GABA A receptor with ambient GABA generates measurable steady-state activity. Recombinant α1β2γ2L GABA A receptors were expressed in Xenopus oocytes and activated by combinations of low concentrations of orthosteric (GABA, taurine) and allosteric (the steroid allopregnanolone, the anesthetic propofol) agonists, in the absence and presence of the inhibitory steroid pregnenolone sulfate. Steady-state activity was analyzed using the three-state cyclic Resting-Active-Desensitized model. We estimate that the steady-state open probability of the synaptic α1β2γ2L GABA A receptor in the presence of ambient GABA (1 μmol/L), taurine (10 μmol/L), and physiological levels of allopregnanolone (0.01 μmol/L) and pregnenolone sulfate (0.1 μmol/L) is 0.008. Coapplication of a clinical concentration of propofol (1 μmol/L) increases the steady-state open probability to 0.03. Comparison of total charge transfer for phasic and tonic activity indicates that steady-state activity can contribute strongly (~20 to >99%) to integrated activity from the synaptic GABA A receptor., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The two-state coagonist model has been successfully used to analyze and predict peak current responses of the γ -aminobutyric acid type A (GABA A ) receptor. The goal of the present study was to provide a model-based description of GABA A receptor activity under steady-state conditions after desensitization has occurred. We describe the derivation and properties of the cyclic three-state resting-active-desensitized (RAD) model. The relationship of the model to receptor behavior was tested using concatemeric α 1 β 2 γ 2 GABA A receptors expressed in Xenopus oocytes. The receptors were activated by the orthosteric agonists GABA or β -alanine, the allosteric agonist propofol, or combinations of GABA, propofol, pentobarbital, and the steroid allopregnanolone, and the observed steady-state responses were compared with those predicted by the model. A modified RAD model was employed to analyze and describe the actions on steady-state current of the inhibitory steroid pregnenolone sulfate. The findings indicate that the steady-state activity in the presence of multiple active agents that interact with distinct binding sites follows standard energetic additivity. The derived equations enable prediction of peak and steady-state activity in the presence of orthosteric and allosteric agonists, and the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: The study describes derivation and properties of a three-state resting-active-desensitized model. The model and associated equations can be used to analyze and predict peak and steady-state activity in the presence of one or more active agents., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The Monod-Wyman-Changeux (MWC) cyclic model was described as a kinetic scheme to explain enzyme function and modulation more than 50 years ago and was proposed as a model for understanding the activation of transmitter-gated channels soon afterward. More recently, the MWC model has been used to describe the activation of the GABA A receptor by the transmitter, GABA, and drugs that bind to separate sites on the receptor. It is most interesting that the MWC formalism can also describe the interactions among drugs that activate the receptor. In this review, we describe properties of the MWC model that have been explored experimentally using the GABA A receptor, summarize analytical expressions for activation and interaction for drugs, and briefly review experimental results., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Under both physiologic and clinical conditions GABA A receptors are exposed to multiple agonists, including the transmitter GABA, endogenous or exogenous neuroactive steroids, and various GABAergic anesthetic and sedative drugs. The functional output of the receptor reflects the interplay among all active agents. We have investigated the activation of the concatemeric α 1 β 2 γ 2L GABA A receptor by combinations of agonists. Simulations of receptor activity using the coagonist concerted transition model demonstrate that the response amplitude in the presence of agonist combinations is highly dependent on whether the paired agonists interact with the same or distinct sites. The experimental data for receptor activation by agonist combinations were in agreement with the established views of the overlap of binding sites for several pairs of orthosteric (GABA, β -alanine, and piperidine-4-sulfonic acid) and/or allosteric agents (propofol, pentobarbital, and several neuroactive steroids). Conversely, the degree of potentiation when two GABAergic agents are coapplied can be used to determine whether the compounds act by binding to the same or distinct sites. We show that common interaction sites mediate the actions of 5 α - and 5 β -reduced neuroactive steroids, and natural and enantiomeric steroids. Furthermore, the results indicate that the anesthetics propofol and pentobarbital interact with partially shared binding sites. We propose that the findings may be used to predict the efficacy of drug mixtures in combination therapy and thus have potential clinical relevance., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The co-agonist concerted transition model is a simple and practical solution to analyze various aspects of GABAA receptor function. Several model-based predictions have been verified experimentally in previous reports. We review here the practical implications of the model and demonstrate how it enables simplification of the experimental procedure and data analysis to characterize the effects of mutations or properties of novel ligands. Specifically, we show that the value of EC50 and the magnitude of current response are directly affected by basal activity, and that coapplication of a background agonist acting at a distinct site or use of a gain-of-function mutation can be employed to enable studies of weak activators or mutated receptors with impaired gating. We also show that the ability of one GABAergic agent to potentiate the activity elicited by another is a computable value that depends on the level of constitutive activity of the ion channel and the ability of each agonist to directly activate the receptor. Significantly, the model accurately accounts for situations where the paired agonists interact with the same site compared to distinct sites on the receptor., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Two major GABA A receptor classes mediate ionotropic GABA signaling, those containing a δ subunit and those with a γ2 subunit. The classical viewpoint equates γ2-containing receptors with IPSCs and δ-containing receptors with tonic inhibition because of differences in receptor localization, but significant questions remain because the populations cannot be pharmacologically separated. We removed this barrier using gene editing to confer a point mutation on the δ subunit in mice, rendering receptors containing the subunit picrotoxin resistant. By pharmacologically isolating δ-containing receptors, our results demonstrate their contribution to IPSCs in dentate granule neurons and weaker contributions to thalamocortical IPSCs. Despite documented extrasynaptic localization, we found that receptor localization does not preclude participation in isolated IPSCs, including mIPSCs. Further, phasic inhibition from δ subunit-containing receptors strongly inhibited summation of EPSPs, whereas tonic activity had little impact. In addition to any role that δ-containing receptors may play in canonical tonic inhibition, our results highlight a previously underestimated contribution of δ-containing receptors to phasic inhibition. SIGNIFICANCE STATEMENT GABA A receptors play key roles in transient and tonic inhibition. The prevailing view suggests that synaptic γ2-containing GABA A receptors drive phasic inhibition, whereas extrasynaptic δ-containing receptors mediate tonic inhibition. To re-evaluate the impact of δ receptors, we took a chemogenetic approach that offers a sensitive method to probe the synaptic contribution of δ-containing receptors. Our results reveal that localization does not strongly limit the contribution of δ receptors to IPSCs and that δ receptors make an unanticipated robust contribution to phasic inhibition., (Copyright © 2018 the authors 0270-6474/18/388128-18$15.00/0.)

The concerted transition model for multimeric proteins is a simple formulation for analyzing the behavior of transmitter-gated ion channels. We used the model to examine the relationship between the EC 50 for activation of the GABA type A (GABA A ) receptor by the transmitter GABA and basal activity employing concatemeric ternary GABA A receptors expressed in Xenopus oocytes. Basal activity, reflecting the receptor function in the absence of the transmitter, can be changed either by mutation to increase constitutive activity or by the addition of a second agonist (acting at a different site) to increase background activity. The model predicts that either mechanism for producing a change in basal activity will result in identical effects on the EC 50 We examined receptor activation by GABA while changing the level of basal activity with the allosterically acting anesthetics propofol, pentobarbital, or alfaxalone. We found that the relationship between EC 50 and basal activity was well described by the concerted transition model. Changes in the basal activity by gain-of-function mutations also resulted in predictable changes in the EC 50 Finally, we altered the number of GABA-binding sites by a mutation and again found that the relationship could be well described by the model. Overall, the results support the idea that interactions between the transmitter GABA and the allosteric agonists propofol, pentobarbital, or alfaxalone can be understood as reflecting additive and independent free energy changes, without assuming any specific interactions., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

GABA A receptors can be directly activated and potentiated by the intravenous anesthetic propofol. Previous photolabeling, modeling, and functional data have identified two binding domains through which propofol acts on the GABA A receptor. These domains are defined by the β (M286) residue at the β "+"- α "-" interface in the transmembrane region and the β (Y143) residue near the β "-" surface in the junction between the extracellular and transmembrane domains. In the ternary receptor, there are predicted to be two copies of each class of sites, for a total of four sites per receptor. We used β 2 α 1 γ 2L and β 2 α 1 concatemeric constructs to determine the functional effects of the β (Y143W) and β (M286W) mutations to gain insight into the number of functional binding sites for propofol and the energetic contributions stemming from propofol binding to the individual sites. A mutation of each of the four sites affected the response to propofol, indicating that each of the four sites is functional in the wild-type receptor. The mutations mainly impaired stabilization of the open state by propofol, i.e., reduced gating efficacy. The effects were similar for mutations at either site and were largely additive and independent of the presence of other Y143W or M286W mutations in the receptor. The two classes of sites appeared to differ in affinity for propofol, with the site affected by M286W having about a 2-fold higher affinity. Our analysis indicates there may be one or two additional functionally equivalent binding sites for propofol, other than those modified by substitutions at β (Y143) and β (M286)., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug interactions are often analyzed in terms of isobolograms. In the isobologram, the line connecting the axial points corresponding to the concentrations of two different drugs that produce an effect of the same magnitude is termed an isobole of additivity. Although the isobole of additivity can be a straight line in some special cases, previous work has proposed that it is curvilinear when the two drugs differ in their maximal effects or Hill slopes. Modulators of transmitter-gated ion channels have a wide range of maximal effects as well as Hill slopes, suggesting that the isoboles for drug actions on ion channel function are not linear. In this study, we have conducted an analysis of direct activation and potentiation of the human α 1 β 2 γ 2L GABA A receptor to demonstrate that: 1) curvilinear isoboles of additivity are predicted by a concerted transition model where the binding of each GABAergic drug additively and independently reduces the free energy of the open receptor compared with the closed receptor; and 2) experimental data for receptor activation using the agonist pair of GABA and propofol or potentiation of responses to a low concentration of GABA by the drug pair of alfaxalone and propofol agree very well with predictions. The approach assuming independent energetic contributions from GABAergic drugs enables, at least for the drug combinations tested, a straightforward method to accurately predict functional responses to any combination of concentrations., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Physostigmine can potentiate and inhibit neuronal nicotinic receptors, in addition to inhibiting the activity of acetylcholinesterase. We found that receptors containing three copies of the α 2 subunit are inhibited by low concentrations of physostigmine in contrast to receptors containing three copies of the α 4 subunit that are potentiated. We exploited this observation to determine the regions required for the actions of physostigmine. Chimeric constructs of the α 2 and α 4 subunits located two regions in the extracellular amino-terminal domain of the subunit: the E loop (a loop of the transmitter-binding domain) and a region closer to the amino-terminus that collectively could completely determine the different effects of physostigmine. Point mutations then identified a single residue, α 2(I92) versus α 4(R92), that, when combined with transfer of the E loop, could convert the inhibition seen with α 2 subunits to potentiation and the potentiation seen with α 4 subunits to inhibition. In addition, other point mutations could affect the extent of potentiation or inhibition, indicating that a more extensive set of interactions in the amino-terminal domain plays some role in the actions of physostigmine., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Physostigmine is a well known inhibitor of acetylcholinesterase, which can also activate, potentiate, and inhibit acetylcholine receptors, including neuronal nicotinic receptors comprising α4 and β2 subunits. We have found that the two stoichiometric forms of this receptor differ in the effects of physostigmine. The form containing three copies of α4 and two of β2 was potentiated at low concentrations of acetylcholine chloride (ACh) and physostigmine, whereas the form containing two copies of α4 and three of β2 was inhibited. Chimeric constructs of subunits indicated that the presence of inhibition or potentiation depended on the source of the extracellular ligand binding domain of the subunit. Further sets of chimeric constructs demonstrated that a portion of the ACh binding domain, the E loop, is a key determinant. Transferring the E loop from the β2 subunit to the α4 subunit resulted in strong inhibition, whereas the reciprocal transfer reduced inhibition. To control the number and position of the incorporated chimeric subunits, we expressed chimeric constructs with subunit dimers. Surprisingly, incorporation of a subunit with an altered E loop had similar effects whether it contributed either to an intersubunit interface containing a canonical ACh binding site or to an alternative interface. The observation that the α4 E loop is involved suggests that physostigmine interacts with regions of subunits that contribute to the ACh binding site, whereas the lack of interface specificity indicates that interaction with a particular ACh binding site is not the critical factor., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The neuropeptide PDF promotes the normal sequencing of circadian behavioral rhythms in Drosophila, but its signaling mechanisms are not well understood. We report daily rhythmicity in responsiveness to PDF in critical pacemakers called small LNvs. There is a daily change in potency, as great as 10-fold higher, around dawn. The rhythm persists in constant darkness and does not require endogenous ligand (PDF) signaling or rhythmic receptor gene transcription. Furthermore, rhythmic responsiveness reflects the properties of the pacemaker cell type, not the receptor. Dopamine responsiveness also cycles, in phase with that of PDF, in the same pacemakers, but does not cycle in large LNv. The activity of RalA GTPase in s-LNv regulates PDF responsiveness and behavioral locomotor rhythms. Additionally, cell-autonomous PDF signaling reversed the circadian behavioral effects of lowered RalA activity. Thus, RalA activity confers high PDF responsiveness, providing a daily gate around the dawn hours to promote functional PDF signaling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression.

Background: Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the "+" of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the "-" side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy., Method: We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the β subunit on activation of the α1β3 GABAA receptor by propofol., Results: We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol., Conclusion: We conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.

The endogenous steroid 17β-estradiol (βEST) potentiates activation of neuronal nicotinic receptors containing α4 subunits. Previous work has shown that the final 4 residues of the α4 subunit are required for potentiation. However, receptors containing the α2 subunit are not potentiated although it has these 4 residues, and only one amino acid difference in the C-terminal tail (FLAGMI vs. WLAGMI). Previous work had indicated that the tryptophan residue was involved in binding an analog of βEST, but not in potentiation by βEST. To determine the structural basis for the loss of potentiation we analyzed data from chimeric subunits, which indicated that the major factor underlying the difference between α2 and α4 is the tryptophan/phenylalanine difference, while the N-terminal extracellular domain is a less significant factor. When the tryptophan in α4 was mutated, both phenylalanine and tyrosine conferred lower potentiation while lysine and leucine did not. The reduction reflected a reduced maximal magnitude of potentiation, indicating that the tryptophan is involved in transduction of steroid effects. The regions of the α4 N-terminal extracellular domain involved in potentiation lie near the agonist-binding pocket, rather than close to the membrane or the C-terminal tail, and appear to be involved in transduction rather than binding. These observations indicate that the C-terminal region is involved in both steroid binding (AGMI residues) and transduction (W). The role of the N-terminus appears to be independent of the C-terminal tryptophan and likely reflects an influence on conformational changes caused during channel activation by agonist and potentiation by estradiol.

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1-3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Background and Purpose: Neurosteroids potentiate responses of the GABAA receptor to the endogenous agonist GABA. Here, we examined the ability of neurosteroids to potentiate responses to the allosteric activators etomidate, pentobarbital and propofol., Experimental Approach: Electrophysiological assays were conducted on rat α1β2γ2L GABAA receptors expressed in HEK 293 cells. The sedative activity of etomidate was studied in Xenopus tadpoles and mice. Effects of neurosteroids on etomidate-elicited inhibition of cortisol synthesis were determined in human adrenocortical cells., Key Results: The neurosteroid 5β-pregnan-3α-ol-20-one (3α5βP) potentiated activation of GABAA receptors by GABA and allosteric activators. Co-application of 1 μM 3α5βP induced a leftward shift (almost 100-fold) of the whole-cell macroscopic concentration-response relationship for gating by etomidate. Co-application of 100 nM 3α5βP reduced the EC50 for potentiation by etomidate of currents elicited by 0.5 μM GABA by about three-fold. In vivo, 3α5βP (1mg kg(-1) ) reduced the dose of etomidate required to produce loss of righting in mice (ED50 ) by almost 10-fold. In tadpoles, the presence of 50 or 100 nM 3α5βP shifted the EC50 for loss of righting about three- or ten-fold respectively. Exposure to 3α5βP did not influence inhibition of cortisol synthesis by etomidate., Conclusions and Implications: Potentiating neurosteroids act similarly on orthosterically and allosterically activated GABAA receptors. Co-application of neurosteroids with etomidate can significantly reduce dosage requirements for the anaesthetic, and is a potentially beneficial combination to reduce undesired side effects., (© 2014 The British Pharmacological Society.)

Rationale: While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified., Objectives: This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors., Results: Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific., Conclusions: CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.

We examined the role of putative trafficking sequences in two GABA(A) receptor subunits: α4 and δ. These subunits assemble with a β subunit to form a subtype of GABA(A) receptor involved in generating the "tonic" outward current. Both α4 and δ subunits contain dibasic retention motifs in homologous positions. When basic residues are mutated to alanine in the α4 subunit, surface expression of epitope-tagged δ subunits is increased. When basic residues in homologous regions of the δ subunit are mutated, however, surface expression is reduced. We focused on the mutants that had the maximal effects to increase (in α4) or reduce (in δ) surface expression. The total expression of δ subunits is significantly decreased by the δ mutation, suggesting an effect on subunit maturation. We also examined surface expression of the β2 subunit. Expression of the mutated α4 subunit resulted in increased surface expression of β2 compared with wild-type α4, indicating enhanced forward trafficking. In contrast, mutated δ resulted in decreased surface expression of β2 compared with wild-type δ and to α4 and β2 in the absence of any δ. This observation suggests that the mutated δ incorporates into multimeric receptors and reduces the overall forward trafficking of receptors. These observations indicate that the roles of trafficking motifs are complex, even when located in homologous positions in related subunits. The physiologic properties of receptors containing mutated subunits were not significantly affected, indicating that the mutations in the α4 subunit will be useful to enhance surface expression., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration-response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC50 (β = -0.67, r2 = 0.017, P = 2 × 10(-4)) or by response to low nicotine (β = -0.29, r2 = 0.02, P = 6 × 10(-5)) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (β = -0.04, r2 = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.

Heteropentameric neuronal nicotinic receptors assemble so that the canonical acetylcholine-binding sites are located at the interfaces between two pairs of subunits, while the fifth subunit does not participate in a canonical transmitter-binding site. Several subunits are considered to be unable to participate in forming a functional receptor when they occupy a position that would contribute to such a site, including the α5 subunit. The α5 subunit is of interest because of its apparent involvement in nicotine dependence and in the control of dopamine release. We have examined this question using α4 and β2 subunits in concatemeric constructs with the α5 subunit, expressed in Xenopus oocytes. Using dimeric constructs of α4 and β2 subunits expressed with free α5 and pentameric constructs incorporating a single copy of α5, we find that the α5 subunit can occupy the position of a nonbinding subunit, or replace a β2 subunit participating in a canonical binding site. The resulting receptors functionally resemble pentamers assembled with two copies of α4 and three copies of β2. Functional receptors apparently cannot be formed with α5 subunits in both canonical binding sites. These observations extend the present ideas on the possible positions in the pentamer that may be occupied by the α5 subunit, and suggest that additional physiologic or pharmacological subtypes of neuronal nicotinic receptors may be present in neurons.

Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels.

In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.

Background: The γ-aminobutyric acid (GABA) type A receptor is a target for several anesthetics, anticonvulsants, anxiolytics, and sedatives. Neurosteroids, barbiturates, and etomidate both potentiate responses to GABA and allosterically activate the receptor. We examined the ability of a benzodiazepine, diazepam, to potentiate responses to allosteric agonists., Methods: The GABA type A receptors were expressed in human embryonic kidney 293 cells and studied using whole-cell and single-channel patch clamp. The receptors were activated by the orthosteric agonist GABA and allosteric agonists pentobarbital, etomidate, and alfaxalone., Results: Diazepam is equally potent at enhancing responses to orthosteric and allosteric agonists. Diazepam EC50s were 25 ± 4, 26 ± 6, 33 ± 6, and 26 ± 3 nm for receptors activated by GABA, pentobarbital, etomidate, and alfaxalone, respectively (mean ± SD, 5-6 cells at each condition). Mutations to the benzodiazepine-binding site (α1(H101C), γ2(R144C), γ2(R197C)) reduced or removed potentiation for all agonists, and an inverse agonist at the benzodiazepine site reduced responses to all agonists. Single-channel data elicited by GABA demonstrate that in the presence of 1 μm diazepam the prevalence of the longest open-time component is increased from 13 ± 7 (mean ± SD, n = 5 patches) to 27 ± 8% (n = 3 patches) and the rate of channel closing is decreased from 129 ± 28 s(-1) to 47 ± 6 s(-1) (mean ± SD) CONCLUSIONS: We conclude that benzodiazepines do not act by enhancing affinity of the orthosteric site for GABA but rather by increasing channel gating efficacy. The results also demonstrate the presence of interactions between allosteric activators and potentiators, raising a possibility of effects on dosage requirements or changes in side effects.

Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA(A) receptors. It has been proposed that a conserved glutamine residue in the first membrane-spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single-channel kinetics are similar for wild-type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid.

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

The GABA(A) receptor undergoes conformational changes upon the binding of agonist that lead to the opening of the channel gate and a flow of small anions across the cell membrane. Besides the transmitter GABA, allosteric ligands such as the general anesthetics pentobarbital and etomidate can activate the receptor. Here, we have investigated the agonist specificity of structural changes in the extracellular domain of the receptor. We used the substituted cysteine accessibility method and focused on the γ2(S195C) site (loop F). We show that modification of the site with (2-sulfonatoethyl)methanethiosulfonate (MTSES) results in an enhanced response to GABA, indicating accessibility of the resting receptor to the modifying agent. Coapplication of GABA or muscimol, but not of gabazine, with MTSES prevented the effect, suggesting that GABA and muscimol elicit a conformational change that reduces access to the γ2(S195C) site. Exposure of the receptors to MTSES in the presence of the allosteric activators pentobarbital and etomidate resulted in an enhanced current response indicating accessibility and labeling of the γ2(S195C) site. However, comparison of the rates of modification indicated that labeling in the presence of etomidate was significantly faster than that in the presence of pentobarbital or gabazine or in resting receptors. We infer from the data that the structure of the α1-γ2 subunit interface undergoes agonist-specific conformational changes.

The enantiomer pair androsterone and ent-androsterone are positive allosteric modulators of γ-aminobutyric acid (GABA) type A receptors. Each enantiomer was shown to bind at the same receptor site. Binding orientations of the enantiomers at this site were deduced using enantiomer pairs containing OBn substituents at either C-7 or C-11. 11β-OBn-substituted steroids and 7α-OBn-substituted ent-steroids potently displace [(35)S]-tert-butylbicyclophosphorothionate, augment GABA currents, and anesthetize tadpoles. In contrast, 7β-OBn-substituted steroids and 11α-OBn-substituted ent-steroids have diminished actions. The results suggest that the binding orientations of the active analogues are inverted relative to each other with the 7α- and 11β-substituents similarly located on the edges of the molecules not in contact with the receptor surface. Analogue potentiation of the GABA current was abrogated by an α(1) subunit Q241L mutation, indicating that the active analogues act at the same sites in α(1)β(2)γ(2L) receptors previously associated with positive neurosteroid modulation.

Genome-wide association studies have identified common variation in the CHRNA5-CHRNA3-CHRNB4 and CHRNA6-CHRNB3 gene clusters that contribute to nicotine dependence. However, the role of rare variation in risk for nicotine dependence in these nicotinic receptor genes has not been studied. We undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes in African American and European American nicotine-dependent smokers and smokers without symptoms of dependence. Carrier status of individuals harboring rare missense variants at conserved sites in each of these genes was then compared in cases and controls to test for an association with nicotine dependence. Missense variants at conserved residues in CHRNB4 are associated with lower risk for nicotine dependence in African Americans and European Americans (AA P = 0.0025, odds-ratio (OR) = 0.31, 95% confidence-interval (CI) = 0.31-0.72; EA P = 0.023, OR = 0.69, 95% CI = 0.50-0.95). Furthermore, these individuals were found to smoke fewer cigarettes per day than non-carriers (AA P = 6.6 × 10(-5), EA P = 0.021). Given the possibility of stochastic differences in rare allele frequencies between groups replication of this association is necessary to confirm these findings. The functional effects of the two CHRNB4 variants contributing most to this association (T375I and T91I) and a missense variant in CHRNA3 (R37H) in strong linkage disequilibrium with T91I were examined in vitro. The minor allele of each polymorphism increased cellular response to nicotine (T375I P = 0.01, T91I P = 0.02, R37H P = 0.003), but the largest effect on in vitro receptor activity was seen in the presence of both CHRNB4 T91I and CHRNA3 R37H (P = 2 × 10(-6)).