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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 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 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.

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.)

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.

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.

Endogenous steroids can modulate the activity of transmitter-gated channels by directly interacting with the receptor. 17β-Estradiol potentiates activation of neuronal nicotinic α4β2 receptors by interacting with a 4 aa sequence at the extreme C terminus of the α4 subunit, but it is not known whether potentiation requires that the sequence be placed on a specific subunit (e.g., an α4 subunit that is involved in forming an acetylcholine-binding site). By using concatemers of subunits and chimeric subunits, we have found that the C-terminal domain can be moved from the α4 to the β2 subunit and still result in potentiation. In addition, the sequence can be placed on a subunit that contributes to an acetylcholine-binding site or on the structural subunit. The data indicate that this estradiol-binding element is a discrete sequence and suggest that the effect of 17β-estradiol is mediated by actions on single subunits and that the overall consequences for gating occur because of the summation of independent energetic contributions to overall gating of this receptor.

Background and Purpose: The binding of transmitter to specialized binding pockets leads to rearrangements in the structure of the receptor eventually resulting in channel opening. We used voltage-clamp fluorometry to investigate the pharmacological basis and biophysical processes that underlie structural changes at the transmitter binding site of the rat α1β2γ2L GABA(A) receptor., Experimental Approach: Simultaneous electrophysiological and site-specific fluorescence measurements were conducted on receptors expressed in Xenopus oocytes and labelled with an environmentally-sensitive fluorophore, Alexa 546 maleimide, at the α1L127C site., Key Results: Receptors activated by GABA demonstrate a concentration-dependent increase in fluorescence intensity, indicating that the environment surrounding the fluorophore becomes less polar upon activation. Qualitatively similar responses were observed with other GABA site ligands such as piperidine-4-sulphonic acid, muscimol, β-alanine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. Fluorescence changes were not affected by the direction of current flow. During long applications of GABA significant desensitization developed, which was not accompanied by additional changes in fluorescence. Pentobarbital was an efficacious agonist of the labelled mutant receptor but did not cause changes in fluorescence. Direct activation by etomidate or the steroid allopregnanolone also did not result in fluorescence changes. Functional potentiation of GABA-activated receptors by allopregnanolone or etomidate enhanced both the GABA-elicited functional response and the fluorescence change. In contrast, potentiation by pentobarbital was not accompanied by an enhanced fluorescence response., Conclusions and Implications: The data indicate that there is no direct correlation between current flow or position of the activation gate and the structural changes as detected by Alexa 546-labelled α1L127Cβ2γ2L GABA(A) receptors. Channel potentiation by pentobarbital qualitatively differs from potentiation by etomidate or allopregnanolone., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Results from genome-wide association studies of complex traits account for only a modest proportion of the trait variance predicted to be due to genetics. We hypothesize that joint analysis of polymorphisms may account for more variance. We evaluated this hypothesis on a case-control smoking phenotype by examining pairs of nicotinic receptor single-nucleotide polymorphisms (SNPs) using the Restricted Partition Method (RPM) on data from the Collaborative Genetic Study of Nicotine Dependence (COGEND). We found evidence of joint effects that increase explained variance. Four signals identified in COGEND were testable in independent American Cancer Society (ACS) data, and three of the four signals replicated. Our results highlight two important lessons: joint effects that increase the explained variance are not limited to loci displaying substantial main effects, and joint effects need not display a significant interaction term in a logistic regression model. These results suggest that the joint analyses of variants may indeed account for part of the genetic variance left unexplained by single SNP analyses. Methodologies that limit analyses of joint effects to variants that demonstrate association in single SNP analyses, or require a significant interaction term, will likely miss important joint effects.

Background and Purpose: A fundamental property of transmitter-gated ion channels is the probability a channel will be open (P(open)) when stimulated by a concentration of agonist that elicits a maximal response. This value is critical for interpreting steady-state concentration-response relationships in terms of channel activation, and for understanding the actions of drugs that potentiate responses. We used analysis of non-stationary noise to estimate the maximal probability the nicotinic alpha4beta2 receptor is open., Experimental Approach: HEK293 cells stably transfected to express human alpha4beta2 nicotinic receptors were studied using whole-cell voltage clamp. Nicotinic agonists (acetylcholine, nicotine, cytisine and 5-iodo A-85380) were applied, and the relationship between variance of the elicited whole-cell current and mean current was analysed., Key Results: The variance did not increase linearly with the mean current. For acetylcholine and nicotine the relationship between variance and mean indicates that the maximal P(open) is greater than 0.8. The number of agonist-activatable channels was estimated to be about 1000 per cell. The mean single channel conductance at -60 mV was indistinguishable when currents were elicited by acetylcholine (18 pS), nicotine (17 pS) or 5-iodo A-85380 (17 pS), whereas the value for cytisine was larger (24 pS)., Conclusions and Implications: The neuronal nicotinic alpha4beta2 receptor has a maximal probability of being open that is greater than 0.8. This conclusion applies to the receptor containing three alpha4 and two beta2 subunits (the low-sensitivity stoichiometry), but may not apply to the receptor containing two alpha4 and three beta2 subunits (the high-sensitivity stoichiometry).

Endogenous neurosteroids and synthetic neuroactive steroid analogs are among the most potent and efficacious potentiators of the mammalian GABA-A receptor. The compounds interact with one or more sites on the receptor leading to an increase in the channel open probability through a set of changes in the open and closed time distributions. The endogenous neurosteroid allopregnanolone potentiates the α1β2γ2L GABA-A receptor by enhancing the mean duration and prevalence of the longest-lived open time component and by reducing the prevalence of the longest-lived intracluster closed time component. Thus the channel mean open time is increased and the mean closed time duration is decreased, resulting in potentiation of channel function. Some of the other previously characterized neurosteroids and steroid analogs act through similar mechanisms while others affect a subset of these parameters. The steroids modulate the GABA-A receptor through interactions with the membrane-spanning region of the receptor. However, the number of binding sites that mediate the actions of steroids is unclear. We discuss data supporting the notions of a single site vs. multiple sites mediating the potentiating actions of steroids.

Background and Purpose: Potentiating neurosteroids are some of the most efficacious modulators of the mammalian GABA(A) receptor. One of the crucial interactions may be between the C20 ketone group (D-ring substituent at C17) of the neurosteroid, and the N407 and Y410 residues in the M4 domain of the receptor. In this study, we examined the contribution of hydrogen bonding between 17beta-substituents on the steroid D-ring and the GABA(A) receptor to potentiation by neurosteroids., Experimental Approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing wild-type and mutant alpha1beta2gamma2L GABA(A) receptors., Key Results: A steroid with a 17beta-carbonitrile group (3alpha5alpha18nor17betaCN) was a potent and efficacious potentiator of the GABA(A) receptor. Potentiation was also shown by a cyclosteroid in which C21 and the C18 methyl group of (3alpha,5alpha)-3-hydroxypregnan-20-one are connected within a six-membered ring containing a double bond as a hydrogen bond acceptor (3alpha5alphaCDNC12), a steroid containing a 17beta-ethyl group on the D-ring (3alpha5alpha17betaEt) and a steroid lacking a 17beta-substituent on the D-ring (3alpha5alpha17H). Single-channel kinetic analysis indicates that the kinetic mechanism of action is the same for the neurosteroid 3alpha5alphaP, 3alpha5alpha18nor17betaCN, 3alpha5alphaCDNC12, 3alpha5alpha17betaEt and 3alpha5alpha17H. Interestingly, 3alpha5alpha17betaEt, at up to 3 microM, was incapable of potentiating the alpha1N407A/Y410F double mutant receptor., Conclusions and Implications: Hydrogen bonding between the steroid 17beta-substituent and the GABA(A) receptor is not a critical requirement for channel potentiation. The alpha1N407/Y410 residues are important for neurosteroid potentiation for reasons other than hydrogen bonding between steroid and receptor.

Genetic association studies have shown the importance of variants in the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit gene cluster on chromosome 15q24-25.1 for the risk of nicotine dependence, smoking, and lung cancer in populations of European descent. We have carried out a detailed study of this region using dense genotyping in both European-Americans and African-Americans. We genotyped 75 known single nucleotide polymorphisms (SNPs) and one sequencing-discovered SNP in an African-American sample (N = 710) and in a European-American sample (N = 2,062). Cases were nicotine-dependent and controls were nondependent smokers. The nonsynonymous CHRNA5 SNP rs16969968 is the most significant SNP associated with nicotine dependence in the full sample of 2,772 subjects [P = 4.49 x 10(-8); odds ratio (OR), 1.42; 95% confidence interval (CI), 1.25-1.61] as well as in African-Americans only (P = 0.015; OR, 2.04; 1.15-3.62) and in European-Americans only (P = 4.14 x 10(-7); OR, 1.40; 1.23-1.59). Other SNPs that have been shown to affect the mRNA levels of CHRNA5 in European-Americans are associated with nicotine dependence in African-Americans but not in European-Americans. The CHRNA3 SNP rs578776, which has a low correlation with rs16969968, is associated with nicotine dependence in European-Americans but not in African-Americans. Less common SNPs (frequency

We examined the actions of a carboxylated analogue of pregnanolone ((3alpha,5beta)-20-oxopregnane-3-carboxylic acid; 3alphaCOOH5betaP) on receptors composed of glycine receptor alpha3 subunits, expressed in Xenopus oocytes. This analogue both inhibits and potentiates this receptor; potentiation increases with more negative membrane potentials while block increases with less negative membrane potentials. We used a second analogue ((3alpha,5beta)-3-hydroxymethylpregnan-20-one; 3alphaCH(2)OH5betaP) to examine the mechanism for voltage-dependent potentiation. This analogue potentiates but does not block the glycine alpha3 receptor. Steady-state responses and current relaxations following voltage jumps support the idea that the voltage dependence of potentiation indirectly arises from a voltage dependence for channel activation by glycine, rather than an intrinsic voltage dependence for potentiation. Potentiation results from a slowing of the channel deactivation rate. In the absence of steroid, at a low [glycine] current relaxations after a voltage jump show two exponential components, with a weighted average time constant of approximately 425 ms (-50 mV, 22 degrees C). The rate for channel deactivation increases at more negative potentials (e-fold per 170 mV) whereas activation decreases (e-fold per 230 mV). The probability a channel is active at a high [glycine] is greater than 0.9. The addition of 10 microM 3alphaCH(2)OH5betaP decreases the deactivation rate by 6.3-fold (-50 mV). Voltage-dependent block by 3alphaCOOH5betaP is consistent with simple open-channel block, with voltage dependence reflecting interactions of the charge on the analogue with the electrical field. Block and unblock have equal but opposite dependence on membrane potential, and the charge on 3alphaCOOH5betaP senses approximately 70% of the membrane field at the blocking site. The apparent forward rate for block, however, is very slow (2 x 10(5) m(-1) s(-1)).

The gamma-aminobutyric acid type A (GABA(A)) receptor assembles from individual subunits to form ligand-gated ion channels. Human (h) beta3 subunits assemble to form homomeric surface receptors in somatic cells, but hbeta1 subunits do not. We have identified three distinct sets of amino acid residues in the N-terminal extracellular domain of the hbeta1 subunit, which when mutated to the homologous residue in hbeta3 allow expression as a functional homomeric receptor. The three sets likely result in three modes of assembly. Mode 1 expression results from a single amino acid change at residue hbeta1 Asp-37. Mode 2 expression results from mutations of residues between positions 44 and 73 together with residues between positions 169 and 173. Finally, mode 3 results from the mutations A45V and K196R. Examination of homology-based structural models indicates that many of the residues are unlikely to be involved in physical inter-subunit interactions, suggesting that a major alteration is stabilization of an assembly competent form of the subunit. These mutations do not, however, have a major effect on the surface expression of heteromeric receptors which include the alpha1 subunit.

Background and Purpose: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor., Experimental Approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors., Key Results: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex., Conclusions and Implications: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.

Neurosteroids are potent modulators of GABA-A receptors. We have examined the time course of development of potentiation of alpha1beta2gamma2L GABA-A receptors during coapplication of GABA and an endogenous neurosteroid (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP). The simultaneous application of 3alpha5alphaP with 5 microm GABA resulted in a biphasic rising phase of current with time constants of 50-60 ms for the rapid phase and 0.3-3 s for the slow phase. The properties of the rapid phase were similar at all steroid concentrations but the time constant of the slower phase became successively shorter as the steroid concentration was increased. Potentiation developed very rapidly (tau = 130 ms) when cells were preincubated with 300 nm 3alpha5alphaP before application of GABA + 3alpha5alphaP, and in outside-out patch recordings, suggesting that steroid diffusion to intracellular compartments competes with receptor potentiation by depleting the cell membrane of steroid. Very low steroid concentrations (3-5 nm) potentiated GABA responses but the effects took minutes to develop. Intracellular accumulation of a fluorescent steroid analogue followed a similar time course, suggesting that slow potentiation results from slow accumulation within plasma membrane rather than indirect effects, such as activation of second messenger systems. In cell-attached single-channel recordings, where 3alpha5alphaP is normally applied through the pipette solution, addition of steroid to the bath solution dramatically shifted the steroid potentiation concentration-effect curve to lower steroid concentrations. We propose that bath-supplied steroid compensates for the diffusion of pipette-supplied steroid out of the patch to the rest of the cell membrane and/or intracellular compartments. The findings suggest that previous studies overestimate the minimum concentration of steroid capable of potentiating GABA actions at GABA-A receptors. The results have implications for the physiological role of endogenous neurosteroids.

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.

The polypeptide snake toxin alpha-bungarotoxin (BTX) has been used in hundreds of studies on the structure, function, and development of the neuromuscular junction because it binds tightly and specifically to the nicotinic acetylcholine receptors (nAChRs) at this synapse. We show here that BTX also binds to and blocks a subset of GABA(A) receptors (GABA(A)Rs) that contain the GABA(A)R beta3 subunit. These results introduce a previously unrecognized tool for analysis of GABA(A)Rs but may complicate interpretation of some studies on neuronal nAChRs.

GABAA receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABAA receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABAA receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Biliary lipids (bile salts, phospholipids, cholesterol, plant sterols) were determined in 89 vertebrate species (cartilaginous and bony fish, reptiles, birds, and mammals), and individual phospholipid classes were measured in 35 species. All samples contained conjugated bile salts (C(27) bile alcohol sulfates and/or N-acyl amidates of C(27) and/or C(24) bile acids). Phospholipids were generally absent in the bile of cartilaginous fish and reptiles and were present in low amounts relative to bile salts in bony fish and most birds. In mammals, the phospholipid-bile salt ratio varied widely. The bile from species with low biliary phospholipid-bile salt ratios often contained a high proportion of sphingomyelin, confirmed by HPLC-MS. In species with a high phospholipid-bile salt ratio, the predominant biliary phospholipid was phosphatidylcholine (PC). The phospholipid-bile salt ratio correlated weakly with the calculated weighted hydrophobic index value. Cholesterol was present in the bile of virtually all species, with plant sterols uniformly being present in only trace amounts. The cholesterol-bile salt ratio tended to be higher in mammals than in non-mammals, but bile of all species was unsaturated. Thus, most nonmammalian vertebrates have relatively low levels of biliary phospholipid and cholesterol, suggesting that cholesterol is eliminated predominantly as bile salts. Mammals have a higher phospholipid and cholesterol to bile salt ratio, with the dominant phospholipid being PC.

Galantamine (Reminyl; Janssen Pharmaceutica, Titusville, NJ) belongs to a class of acetylcholinesterase inhibitors approved for symptomatic treatment of Alzheimer's disease. The drug presumably acts by raising and prolonging the profile of acetylcholine (ACh) via an inhibitory effect on the esterase. However, there is also evidence demonstrating that galantamine can activate the nicotinic ACh receptor or modulate its activation by ACh. In this study, we have examined the ability of galantamine to directly activate the muscle-type nicotinic ACh receptor or to modulate receptor activation by selected nicotinic agonists. Studies of direct activation by galantamine demonstrated that this ligand is a low-efficacy agonist of the muscle-type ACh receptor. Point mutations in the M2-M3 linker (alphaS269I) and the M2 transmembrane domain (epsilonT264P) had similar effects on receptor activation by galantamine and nicotinic agonists, suggesting that the general features of receptor activation by galantamine are similar to that in the presence of ACh. Experiments performed in the simultaneous presence of galantamine and various nicotinic ligands showed that channel activation by the nicotinic ligands studied (ACh, carbachol, and choline) was not affected by the presence of galantamine at concentrations up to 100 microm. In addition, galantamine did not reduce the initial rate of binding for 125I-alpha-bungarotoxin. These results demonstrate that galantamine does not interfere with the occupation of the nicotinic agonist binding site by ACh, carbachol, or choline. We conclude that galantamine activates the muscle-type ACh receptor by interacting with a binding site that is distinct from the site for nicotinic agonists.