Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Photoaffinity labeling of γ-aminobutyric acid type A (GABAA)-receptors (GABAAR) with an etomidate analog and mutational analyses of direct activation of GABAAR by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABAARs in the transmembrane domain at the interface between the β and α subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate photolabeling of GABAAR α1Met-236 and βMet-286 (in αM1 and βM3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of β and α subunits (two copies per pentamer) is consistent with a GABAAR homology model based upon the structure of the nicotinic acetylcholine receptor and with recent αM1 to βM3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABAAR subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABAAR-modulating neurosteroids do not inhibit photolabeling of GABAAR α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486–489) are located at the subunit interface defined by our etomidate site model.