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.

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.

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.

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.

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.

Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors.

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 ß4 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 ß4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into ß4 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.

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.

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.

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.

Multiple modes for conferring surface expression of homomeric beta1 GABAA receptors.

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.

Neuroactive steroids have multiple actions to potentiate GABAA receptors.

The effects of neuroactive steroids on the function of GABAA receptors were studied using cell-attached records of single channel activity recorded from HEK293 cells transfected with alpha1 beta2 gamma2L subunits. Activity was elicited with a half-maximal (50 microM) concentration of GABA. Two steroids were studied in detail: ACN ((3alpha,5alpha,17beta)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times. When clusters of openings were elicited in the absence of steroid, the open time distribution contained three components. ACN produced concentration-dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC50 about 180 nM ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC50 about 35 nM ACN). B285 also increased the prevalence of the longest duration open times (EC50 about 18 nM B285) but increased the duration only at concentrations close to 10 microM. The differences in the actions of these two steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two brief duration closed time components decreased with increasing ACN, with an EC50 of about 50 nM ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation-related closed state selectively, with comparable IC50 concentrations (about 40 nM ACN, 20 nM B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 microM ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the gamma2 subunit, but not the alpha1 or beta2 subunits, abolished the ability of ACN to increase the duration of OT3 but had no effect on the reduction of the rate of occurrence of the activation-related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABAA receptor.

Neuroactive steroid interactions with voltage-dependent anion channels: lack of relationship to GABA(A) receptor modulation and anesthesia.

Neuroactive steroids modulate the function of gamma-aminobutyric acid type A (GABA(A)) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3alpha,5beta)-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity-labeling reagent, we showed that voltage-dependent anion channel-1 (VDAC-1) was the predominant protein labeled in brain. Antisera to VDAC-1 were shown to coimmunoprecipitate GABA(A) receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABA(A) receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABA(A) receptor function and anesthesia. Photolabeling of 35-kDa protein with [(3)H]6-AziP was reduced 85% in brain membranes prepared from VDAC-1-deficient mice but was unaffected by deficiency of VDAC-3. The photolabeled 35-kDa protein in membranes from VDAC-1-deficient mice was identified by two-dimensional electrophoresis and electrospray ionization-tandem mass spectrometry as VDAC-2. The absence of VDAC-1 or VDAC-3 had no effect on the ability of neuroactive steroids to modulate GABA(A) receptor function as evidenced by radioligand ([(35)S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABA(A) receptor function normally in VDAC-2-deficient fibroblasts transfected with alpha(1)beta(2)gamma(2) GABA(A) receptor subunits. Finally, the neuroactive steroid pregnanolone [(3alpha,5beta)-3-hydroxypregnan-20-one] produced anesthesia (loss of righting reflex) in VDAC-1- and VDAC-3-deficient mice, and there was no difference in the recovery time between the VDAC-deficient mice and wild-type controls. These data indicate that neuroactive steroid binding to VDAC-1, -2, or -3 is unlikely to mediate GABA(A) receptor modulation or anesthesia.

Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain.

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.