Site-Specific Fluorescence Reveals Distinct Structural Changes Induced in the Human ρ1 GABA Receptor by Inhibitory Neurosteroids

Abstract

The ρ1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (Li et al., 2007, JPET 323:236-247) focusing on the electrophysiological effects of inhibitory steroids on the ρ1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We have selected representatives of the three groups (β-estradiol, pregnanolone and pregnanolone sulfate), and probed how these steroids modify fluorescence changes from the Alexa 546 C5 maleimide fluorophore attached to residues in the extracellular region of the receptor. The results indicate that the steroids have distinct effects on fluorescence changes. Pregnanolone sulfate diminished the fluorescence change produced by GABA at the K217C and S66C positions. The application of β-estradiol reduced the fluorescence change at the L166C and S66C positions. Introduction of the T298F mutation abolished the β-estradiol-induced reduction in fluorescence change at the L166C and S66C sites, and also abolished the functional inhibition produced by the steroid. Pregnanolone did not affect fluorescence changes at any of the sites examined. The findings are consistent with the steroids acting as allosteric inhibitors of the ρ1 GABA receptor, and support the hypothesis that divergent mechanisms underlie the action of inhibitory steroids on the ρ1 GABA receptor.