Effect of thiol group modification on ion flux and ligand binding properties of the GABAA-benzodiazepine receptor chloride channel complex.

Agents that modify thiol groups have been shown to alter ligand binding at a variety of receptor sites. In addition, alkylation of sulfhydryls has been shown to block ion channel conductance. We studied the effects of thiol reagents on gamma-aminobutyric acid (GABA)-activated chloride flux (36Cl-) and [3H]-diazepam binding in mouse brain membrane preparation (microsacs). Incubation of microsacs in the presence of: mercuric chloride (HgCl2), p-chloromercuriphenylsulfonic acid (pCMBS), hydroxymercuribenzoate (HMB), n-ethylmaleimide (NEM), or iodoacetic acid (IAA) attenuated GABA-stimulated Cl- uptake. The thiol reagents reduced both maximal stimulation and the potency of GABA to induce Cl- uptake. Thiol reagent treatment decreased the affinity of high-affinity [3H]-muscimol equilibrium binding. Supernatant prepared from microsacs treated with pCMBS stimulated Cl- uptake in the absence of GABA agonist in microsacs unexposed to thiol reagents. The supernatant taken from pCMBS-treated microsacs also stimulated [3H]-diazepam binding. This effect was blocked by the addition of the GABA receptor antagonist bicuculline. The concentration of endogenous GABA in supernatant from pCMBS-treated microsacs was sixfold greater than that in supernatant from control microsacs. This increase in levels of endogenous GABA by thiol reagents was due to both an increase in GABA release and a decrease in high-affinity GABA uptake.