PHOSPHORYLATION-INDEPENDENT EFFECTS OF 2ND MESSENGER SYSTEM MODULATORS ON GAMMA-AMINOBUTYRIC ACIDA RECEPTOR COMPLEX FUNCTION

Recent studies investigating the functional significance of gamma-aminobutyric acid(A) (GABA(A)) receptor complex phosphorylation have employed membrane-permeant compounds to manipulate second messenger systems. Although these compounds affect GABA(A) receptor function, the dependence of these effects on phosphorylation has not been established. Here we report that several second messenger system modulations can decrease GABA(A) receptor function independently of their effects on protein phosphorylation. Brain membrane vesicles were lysed and resealed in the presence of EDTA to chelate internal Mg2+. Under these conditions, phosphorylation of vesicle proteins was almost completely inhibited, as determined by incorporation of P-32 into phosphoproteins. In these lysed/resealed vesicles, an inhibition of muscimol-stimulated Cl-36- uptake was observed with the cAMP analogs 8-(4-chlorophenylthio)-cAMP, N6,O2'-dibutyryl-cAMP, and 8-bromo-cAMP, the protein kinase inhibitor H7, and the adenylate cyclase activator forskolin. In both intact and EDTA-treated lysed/resealed microsacs, cAMP analogs and H7 inhibited binding of the GABA(A) receptor ligand [H-3]SR 95531 at concentrations shown to inhibit muscimol-stimulated Cl-36- uptake. Forskolin was observed to inhibit the binding of t-butylbicyclophosphoro-[S-35]thionate, a ligand that binds to a site on the chloride channel. These results demonstrate that compounds commonnly used to alter second messenger systems affect the receptor sites and function of the GABA(A) receptor chloride channel by mechanisms that do not involve protein phosphorylation. In light of these findings, results obtained with these compounds should be interpreted with caution.