Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor.

ABSTRACT: Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as

SUBMITTER: Chen ZW 

PROVIDER: S-EPMC3422701 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature