Multiple functional neurosteroid binding sites on GABAA receptors

Abstract

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 α1β3 GABAA 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 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 β3-α1 intersubunit sites are critical for neurosteroid action.

Site-specific effects of neurosteroids on GABAAreceptor activation and desensitization

“…We did not test the effects of mutations on KK150 action because it minimally enhances [ 3 H]muscimol 3 binding. However, KK150 binds to all three of the identified NS binding sites, and may thus be a weak through all three sites but predominantly through the intersubunit and α1 intrasubunit sites, which we have 12 previously shown mediate PAM-NS potentiation (11). In contrast, 3β5αP and KK148 enhance…”

“…We have also shown that KK123 labeling of the α1 intrasubunit (α1Y415) and β3 intrasubunit (β3Y442) 3 sites ( Figure 5A) can be prevented by a ten-fold excess of 3α5αP (11). We thus examined whether 3β5αP 4 (30 μM) inhibited photolabeling by KK123 (3 μM).…”

“…23 To determine whether KK148 and 3β5αP stabilize the desensitized conformation of the GABAAR by 24 selectively binding to one or more of the identified NS binding sites on the GABAAR (11), we first 25 determined which of the identified NS sites they bind. We have previously shown that the 3α5αP-analogue photolabeling reagent, KK200 labels the β3(+)-α1(-) intersubunit (β3G308) and α1 intrasubunit (α1N408) 1 sites on α1β3 GABAARs ( Figure 4A), and that photolabeling can be prevented by a ten-fold excess of 3α5αP 2 (11). As a first step to determine the binding sites for 3β5αP, KK148 or KK150, we examined whether a 3 ten-fold excess of these compounds (30 μM) prevented KK200 (3 μM) photolabeling of either binding site.…”

“…The KK148-and KK150-photolabeled samples were also analyzed to directly identify the sites of supplement 2); Y415 is the same residue labeled by KK123 at the α1 intrasubunit site (11). The KK148 and 20 KK150 labeled peptides in TM4 of the β3 subunit and corresponding unlabeled peptide were identified by 21 fragmentation spectra as β3TM4 I426-N445.…”

“…To determine which of the previously identified binding sites contributes to NS enhancement of 11 [ 3 H]muscimol binding, we performed site-directed mutagenesis of the NS binding sites previously 12 determined by photolabeling ( Figure 6A) (11). Specifically, α1(Q242L)β3 targets the β3(+)-α1(-) 13 intersubunit site, α1(N408A/Y411F)β3 and α1(V227W)β3 the α1 intrasubunit site, and α1β3(Y284F) the β3 14 intrasubunit site.…”