GABA_Areceptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABA_Areceptor 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 GABA_Areceptor 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.

1 Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the γ-aminobutyric acid A (GABAA) receptor. We analysed the actions of PS on single-channel currents from recombinant GABAA receptors formed from α1, β2 and γ2L subunits. 2 Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 μm) and a saturating concentration (1 mm). PS reduced the duration of clusters of single-channel activity at either concentration of GABA. 3 PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules. 4 The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field. 5 Change in a specific residue near the intracellular end of the channel lining portion of the α1 subunit had a major effect on the rate of block. Mutation of the residue α1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the β2 subunit (β2 A252S) had no effect, nor did a complementary mutation in the γ2L subunit (γ2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

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 (GABA_A) 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 α₁β₃ GABA_A receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α₁ (labeled residues α₁-N⁴⁰⁸, Y⁴¹⁵) and β₃ (labeled residue β₃-Y⁴⁴²) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β₃-L²⁹⁴ and G³⁰⁸) in the interface between the β₃(+) and α₁(−) subunits of the GABA_A receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABA_A receptors. Electrophysiological studies of receptors with mutations based on these predictions (α₁-V²²⁷W, N⁴⁰⁸A/Y⁴¹¹F, and Q²⁴²L) indicate that both the α₁ intrasubunit and β₃-α₁ intersubunit sites are critical for neurosteroid action. Neurosteroids are cholesterol metabolites produced by neurons and glial cells that participate in central nervous system (CNS) development, regulate neuronal excitability, and modulate complex behaviors such as mood. Exogenously administered neurosteroid analogues are effective sedative hypnotics and are being developed as antidepressants and anticonvulsants. Gamma amino-butyric acid Type A (GABA_A) receptors, the principal ionotropic inhibitory neurotransmitter receptors in the brain, are the primary functional target of neurosteroids. Understanding the molecular details of neurosteroid interactions with GABA_A receptors is critical to understanding their mechanism of action and developing specific and effective therapeutic agents. In the current study, we developed a suite of neurosteroid analogue affinity labeling reagents, which we used to identify three distinct binding sites on GABA_A receptors and to determine the orientation of neurosteroid binding in each site. Electrophysiological studies performed on receptors with mutations designed to disrupt the identified binding sites showed that two of the three sites contribute to neurosteroid modulation of GABA_A currents. The distinct patterns of neurosteroid affinity, binding orientation, and effect provide the potential for the development of isoform-specific agonists, partial agonists, and antagonists with targeted therapeutic effects.

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues α1Asn407/Tyr410 in the M4 transmembrane domain and residue α1Gln241 in the M1 domain. We examined the role of residues in the α1 subunit M1 domain in the modulation of the rat α1β2γ2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the α1Q241W or α1Q241L mutations were insensitive to (3α,5α)-3-hydroxypregnan-20-one (3α5αP), albeit with different underlying mechanisms. The α1Q241S mutant was potentiated by 3α5αP, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the α1Q241L mutation had no effect on channel potentiation by (3α,5α)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, α1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5α-pregnan-20-one) potentiated the wild-type receptor but not the α1Q241L mutant. The findings are consistent with a model in which the α1Ser240 and α1Gln241 residues shape the surface to which steroid molecules bind.

The activation properties of GABA_A receptors containing α4β2γ2 and α4β2δ subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed transiently in HEK 293 cells, and the electrophysiological experiments were carried out using cell‐attached single‐channel patch clamp or whole‐cell macroscopic recordings. The data show that GABA is a stronger activator of α4β2γ2 receptors than α4β2δ receptors. Single‐channel clusters were recorded from α4β2γ2 receptors in the presence of 10–5000 μm GABA. The maximal intracluster open probability was 0.35, with a half‐maximal response elicited by 32 μm GABA. Simultaneous kinetic analysis of single‐channel currents obtained at various GABA concentrations yields a channel opening rate constant of 250 s⁻¹, and a K_D of 20 μm. In contrast, only isolated openings were observed in the presence of GABA for the α4β2δ receptor. Pentobarbital was a strong activator of both α4β2γ2 and α4β2δ receptors. The maximal cluster open probability, recorded in the presence of 100 μm pentobarbital, was 0.7. At higher pentobarbital concentrations, the cluster open probability was reduced, probably due to channel block. The results from single‐channel experiments were confirmed by macroscopic recordings from HEK cells in the presence of GABA or pentobarbital.