Three classes of propofol binding sites on GABAA receptors.

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator (PAM) of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. o-PD labels ß-H267, a pore-lining residue, whereas AziPm labels residues ß-M286, ß-M227 and α-I239 in the two membrane-facing interfaces (ß(+)/α(-) and α(+)/ß(-)) between α and ß subunits. This study used photoaffinity labeling of α1ß3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether ß3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects ß3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of ß3-M286, ß3-M227 and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in ß3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per ß-subunit. The native MS data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17' H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1ß3 GABAA receptors.

Multiple functional neurosteroid binding sites on GABAA receptors.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1ß3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and ß3 (labeled residue ß3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues ß3-L294 and G308) in the interface between the ß3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and ß3-α1 intersubunit sites are critical for neurosteroid action.

Chemogenetic Isolation Reveals Synaptic Contribution of δ GABAA Receptors in Mouse Dentate Granule Neurons.

Two major GABAA 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 GABAA receptors play key roles in transient and tonic inhibition. The prevailing view suggests that synaptic γ2-containing GABAA 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.

Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC.

Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the Gloeobacter ligand-gated ion channel (GLIC), a prototypic pLGIC. The neurosteroid-based reagents photolabeled two sites per GLIC subunit, both within the transmembrane domain; one site was an intrasubunit site, and the other was located in the interface between subunits. By using reagents with photoreactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Amino acid substitutions introduced at either site altered neurosteroid modulation of GLIC channel activity, demonstrating the functional role of both sites. These results provide a detailed molecular model of multisite neurosteroid modulation of GLIC, which may be applicable to other mammalian pLGICs.

Mutations in the main cytoplasmic loop of the GABA(A) receptor α4 and δ subunits have opposite effects on surface expression.

We examined the role of putative trafficking sequences in two GABA(A) receptor subunits: α4 and δ. These subunits assemble with a ß 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 ß2 subunit. Expression of the mutated α4 subunit resulted in increased surface expression of ß2 compared with wild-type α4, indicating enhanced forward trafficking. In contrast, mutated δ resulted in decreased surface expression of ß2 compared with wild-type δ and to α4 and ß2 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.

γ-aminobutyric acid type A α4, ß2, and δ subunits assemble to produce more than one functionally distinct receptor type.

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, ß1-3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4ßδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4ß2δ 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 ß2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, ß2, 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 α4ßδ receptors may assemble in a similar configuration in neurons and oocytes.

Direct measurements of neurosteroid binding to specific sites on GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

The Mechanism of Enantioselective Neurosteroid Actions on GABAA Receptors.

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that ent-ALLO potentiates GABA-elicited currents in α1ß3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-ALLO was prevented by the α1(Q242L) mutation in the intersubunit neurosteroid binding site between the ß3 and α1 subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that ent-ALLO binds weakly to the ß3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or ß3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the ß3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.

Agonist-specific conformational changes in the α1-γ2 subunit interface of the GABA A receptor.

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.

A neurosteroid potentiation site can be moved among GABAA receptor subunits.

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 ß2 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 ß2 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.

Functional characterization of the α5(Asn398) variant associated with risk for nicotine dependence in the α3ß4α5 nicotinic receptor.

Smoking is a major cause for premature death. Work aimed at identifying genetic factors that contribute to nicotine addiction has revealed several single nucleotide polymorphisms (SNPs) that are linked to smoking-related behaviors such as nicotine dependence and level of smoking. One of these SNPs leads to an aspartic acid-to-asparagine substitution in the nicotinic receptor α5 subunit at amino acid position 398 [rs16969968; α5(Asn398)]. The α5 subunit is expressed both in the brain and in the periphery. In the brain, it associates with the α4 and ß2 subunits to form α4ß2α5 receptors. In the periphery, the α5 subunit combines with the α3 and ß4 subunits to form the major ganglionic postsynaptic nicotinic receptor subtype. The α3ß4α5 receptor regulates a variety of autonomic responses such as control of cardiac rate, blood pressure, and perfusion. In this paradigm, the α5(Asn398) variant may act by regulating autonomic responses that may affect nicotine intake by humans. Here, we have investigated the effect of the α5(Asn398) variant on the function of the α3ß4α5 receptor. The wild-type or variant α5 subunits were coexpressed with the α3 and ß4 subunits in human embryonic kidney 293 cells. The properties of the receptors were studied using whole-cell and single-channel electrophysiology. The data indicate that the introduction of the α5(Asn398) mutation has little effect on the pharmacology of receptor activation, receptor desensitization, or single-channel properties. We propose that the effect of the α5(Asn398) variant on nicotine use is not mediated by an action on the physiological or pharmacological properties of the α3ß4α5 subtype.

Occupation of either site for the neurosteroid allopregnanolone potentiates the opening of the GABAA receptor induced from either transmitter binding site.

Potentiating neuroactive steroids are potent and efficacious modulators of the GABA(A) receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the α subunits of the receptor, whereas GABA binds to pockets in the interfaces between ß and α subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same ß-α pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same ß-α pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the α subunit from the same or the other ß-α pair. We conclude that steroids potentiate the opening of the GABA(A) receptor induced by either agonist binding site.

Pharmacological and Biophysical Characteristics of Picrotoxin-Resistant, δSubunit-Containing GABAA Receptors.

GABAA receptors (GABAARs) play a crucial role in inhibition in the central nervous system. GABAARs 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 GABAARs containing the mutated subunit to contextualize results from the KI mice. Recombinant receptors containing δ T6'Y plus WT α4 and WT ß2 subunits exhibited 3-fold lower EC50 values for GABA but not THIP. GABA EC50 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 GABAARs 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.

Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.

The rho 1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236-247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the rho 1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We selected representatives of the three groups (pregnanolone, tetrahydrodeoxycorticosterone, pregnanolone sulfate, allopregnanolone sulfate, and beta-estradiol) and probed how these steroids, as well as the nonsteroidal inhibitor picrotoxinin, modify GABA-elicited fluorescence changes from the Alexa 546 C5 maleimide fluorophore attached to residues in the extracellular region of the receptor. The fluorophore responds with changes in quantum yield to changes in the environment, allowing it to probe for structural changes taking place during channel activation or modulation. The results indicate that the modulators have specific effects on fluorescence changes suggesting that distinct conformational changes accompany inhibition. The findings are consistent with the steroids acting as allosteric inhibitors of the rho 1 GABA receptor and support the hypothesis that divergent mechanisms underlie the action of inhibitory steroids on the rho 1 GABA receptor.

A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors.

In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the α1ß3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3ß-epimer epi-allopregnanolone, binds to the canonical ß3(+)-α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the ß3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

Steroid interaction with a single potentiating site is sufficient to modulate GABA-A receptor function.

Neuroactive steroids are efficacious potentiators of GABA-A receptors. Recent work has identified a site in the alpha1 subunit of the GABA-A receptor, that is essential for potentiation by steroids. However, each receptor contains two copies of the alpha1 subunit. We generated concatemers of subunits so that the alpha1 subunits could be mutated separately and examined the consequences of mutations that remove potentiation by most neurosteroids (alpha1 Q241L, alpha1 Q241W). Concatemers were expressed in Xenopus laevis oocytes, and activation by GABA, potentiation by neurosteroids, and the agonist activity of piperidine-4-sulfonic acid (P4S) were determined. When the alpha1 Q241L mutation is present in alpha1 subunits the EC(50) for activation by GABA is shifted to higher concentration and potentiation by neurosteroids is diminished. When the alpha1 Q241W mutation is expressed, the EC(50) for GABA is shifted to lower concentration, the relative efficacy of P4S is increased, and potentiation by neurosteroids is diminished. Mutation of only one alpha1 subunit does not produce the full effect of mutating both sites. Overall, the data demonstrate that at a macroscopic level, the presence of a single wild-type steroid-binding site is sufficient to mediate responses to steroid, but both must be mutated to completely remove the effects of steroids. Differences between the two sites seem to be relatively subtle.

Activation and modulation of concatemeric GABA-A receptors expressed in human embryonic kidney cells.

We have employed whole-cell and single-channel electrophysiology to examine the kinetic and pharmacological properties of GABA-A receptors consisting of gamma2L-beta2-alpha1 and beta2-alpha1 subunit concatemeric constructs expressed in human embryonic kidney cells. Concatemeric receptors activated by GABA exhibited the same single-channel conductance, channel opening rate constant, and basic open- and closed-time properties as receptors containing free subunits. However, the whole-cell GABA dose-response and the single-channel effective opening rate curves were shifted to higher GABA concentrations, suggesting that the concatemeric receptors have a lower affinity to GABA. Pharmacological tests demonstrated that the concatemeric receptors were potentiated by pentobarbital, diazepam, and the neurosteroid (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP), and were insensitive to Zn(2+). Selective introduction of the alpha1Q241L mutation, previously shown to abolish alpha1beta2gamma2L channel potentiation by neurosteroids, into one of the two concatemeric constructs had a relatively small effect on receptor activation by GABA or macroscopic potentiation by the neurosteroid 3alpha5alphaP. Single-channel measurements showed that the kinetic mechanism of action of the steroid is unchanged when the mutation is introduced to the gamma2L-beta2-alpha1 concatemer. We infer that a single wild-type alpha subunit is capable of mediating the full set of kinetic effects in the presence of steroids. Introduction of the alpha1Q241W mutation, previously shown to mimic the effect of the steroid on alpha1beta2gamma2L channels, selectively into either concatemeric construct altered the mode of activity elicited by P4S, but the presence of mutations in both alpha subunits was required to affect open-time distributions. The data indicate that the alpha1Q241W mutation acts as a partial steroid modulator.

The molecular determinants of neurosteroid binding in the GABA(A) receptor.

Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.

Common binding sites for cholesterol and neurosteroids on a pentameric ligand-gated ion channel.

Cholesterol is an essential component of cell membranes, and is required for mammalian pentameric ligand-gated ion channel (pLGIC) function. Computational studies suggest direct interactions between cholesterol and pLGICs but experimental evidence identifying specific binding sites is limited. In this study, we mapped cholesterol binding to Gloeobacter ligand-gated ion channel (GLIC), a model pLGIC chosen for its high level of expression, existing crystal structure, and previous use as a prototypic pLGIC. Using two cholesterol analogue photolabeling reagents with the photoreactive moiety on opposite ends of the sterol, we identified two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both the inter- and intrasubunit sites, cholesterol is oriented such that the 3­OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces. We then compared this binding to that of the cholesterol metabolite, allopregnanolone, a neurosteroid that allosterically modulates pLGICs. The same binding pockets were identified for allopregnanolone and cholesterol, but the binding orientation of the two ligands was markedly different, with the 3­OH group of allopregnanolone pointing to the intra- and extracellular termini of the transmembrane domains rather than to their centers. We also found that cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. These data indicate that cholesterol and neurosteroids bind to common hydrophobic pockets in the model pLGIC, GLIC, but that their effects depend on the orientation and specific molecular interactions unique to each sterol.