Pharmacological and biophysical characteristics of picrotoxin-resistant, δsubunit-containing GABAA receptors

GAB

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 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 GABA(A) receptor activation and desensitization

This study examines how site-specific binding to three identified neurosteroid-binding sites in the

Steroid Interaction with a Single Potentiating Site Is Sufficient to Modulate GABA-A Receptor FunctionS⃞

Neuroactive steroids are efficacious potentiators of GABA-A receptors. Recent work has identified a site in the α1 subunit of the GABA-A receptor, that is essential for potentiation by steroids. However, each receptor contains two copies of the α1 subunit. We generated concatemers of subunits so that the α1 subunits could be mutated separately and examined the consequences of mutations that remove potentiation by most neurosteroids (α1 Q241L, α1 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 α1 Q241L mutation is present in α1 subunits the EC50 for activation by GABA is shifted to higher concentration and potentiation by neurosteroids is diminished. When the α1 Q241W mutation is expressed, the EC50 for GABA is shifted to lower concentration, the relative efficacy of P4S is increased, and potentiation by neurosteroids is diminished. Mutation of only one α1 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