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 (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 α 1 β 3 GABA A receptor.Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α 1 (labeled residues α 1 -N 408 , Y 415 ) and β 3 (labeled residue β 3 -Y 442 ) subunits, adjacent to the extracellular domains (ECDs).An intersubunit site (labeled residues β 3 -L 294 and G 308 ) in the interface between the β 3 (+) and α 1 (-) 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 (α 1 -V 227 W, N 408 A/Y 411 F, and Q 242 L) indicate that both the α 1 intrasubunit and β 3 -α 1 intersubunit sites are critical for neurosteroid action. Author summaryNeurosteroids 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