Identification of Neurosteroid Binding Sites on GABAA Receptors using Photolabeling with Mass Spectrometry

Abstract

Neurosteroids are endogenous modulators for neuronal excitability and nervous system development and are being therapeutically applied as anesthetic agents and treatments for psychiatric disease. While GABAA receptors are the prime molecular targets of neurosteroid action, the molecular details of neurosteroid binding to these proteins are not defined. Accumulated evidence suggests that neurosteroids may modulate GABAA receptor via multiple binding sites. In this study, we synthesized neurosteroid analogue photolabeling reagents in which diazirine or trifluromethylphenyldiazirine photolabeling groups were placed at the 3, 6, 17 or 20-positions of the neurosteroid, allowing us to identify binding sites and orientations of neurosteroids in these sites. A stable HEK cell line expressing α1FLAG-HISβ3 GABAA receptors was generated and membranes from these cells (expressing ∼25 pmol of receptor/mg protein), were used for photolabeling. The photolabeled proteins were detergent-solubilized and affinity purified on anti-FLAG beads. The purified receptors were digested with trypsin and the resulting transmembrane domain (TMD) peptides were analyzed using middle-down mass spectrometry. Three clusters of photolabeled residues were identified consistent with three distinct neurosteroid binding sites. One site was located in the interface between β3-TM3 and α1-TM1, in the intracellular end of the TMDs; this inter-subunit site is consistent with our previous identification of F301 as a neurosteroid binding site in β3 homomeric GABAA receptors. Intra-subunit sites were located in both the α1 and β3 subunits, with labeling at the extracellular end of TM1 and TM4. Docking studies performed using AutoDock and a structural model based on the X-ray structure of the β3 homomer were consistent with neurosteroid binding at the sites identified by photolabeling. Further mutagenesis and computational simulation studies will be pursued to confirm these two photolabeling sites.