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 ?1?3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the ?1 (labeled residues ?1-N408, Y415) and ?3 (labeled residue ?3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues ?3-L294 and G308) in the interface between the ?3(+) and ?1(-) 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 (?1-V227W, N408A/Y411F, and Q242L) indicate that both the ?1 intrasubunit and ?3-?1 intersubunit sites are critical for neurosteroid action.

SUBMITTER: Chen ZW 

PROVIDER: S-EPMC6424464 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature