Interaction of non-competitive blockers within the γ-aminobutyric acid type A chloride channel using chemically reactive probes as chemical sensors for cysteine mutants

Abstract

Selected channel-lining cysteine mutants from the M2 segment of rat α1 γ-aminobutyric acid (GABA) type A receptor subunit, at positions 257, 261, 264, and 272 were co-expressed with β1 and γ2 subunits in Xenopus oocytes. They generated functional receptors displaying conductance and response to both GABA and picrotoxinin similar to the wild type α1β1γ2 receptor. Three chemically reactive affinity probes derived from noncompetitive blockers were synthesized to react with the engineered cysteines: 1) dithiane bis-sulfone derivative modified by an isothiocyanate function (probe A); 2) fiprole derivatives modified by an α-chloroketone (probe B) and α-bromoketone (probe C) moiety. These probes blocked the GABA-induced currents on all receptors. This blockade could be fully reversed by a washing procedure on the wild type, the α1T261Cβ1γ2 and α1L264Cβ1γ2 mutant receptors. In contrast, an irreversible effect was observed for all three probes on both α1V257Cβ1γ2 and α1S272Cβ1γ2 mutant receptors. This effect was probe concentration-dependent and could be abolished by picrotoxinin and/or t-butyl bicyclophosphorothionate. These data indicate a major interaction of non- competitive blockers at position 257 of the presumed M2 segment of rat α1 subunit but also suggest an interaction at the more extracellular position 272.