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 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. [ABSTRACT FROM AUTHOR]