Endogenous phosphorylation of the GABA(A) receptor protein is counteractedby a membrane-associated phosphatase

Incubation of bovine brain membranes with [gamma-P-33]ATP phosphorylated ma inly a 51-kDa band. Electrophoretic co-migration was observed for P-33- and [H-3]flunitrazepam-labeled bands in both membrane fractions and in affinit y-purified GABA(A) receptor (GABA(A)-R) preparations. An a-subunit monoclon al antibody adsorbed most of the radiolabeled-band, suggesting that the lab eled-membrane polypeptide corresponds to the GABA(A)-R alpha(1)-subunit, wh ich is the only GABA(A)-R subunit with a molecular weight of 51 kDa. The ph osphorylation rate was much faster in membranes than in purified receptor. Dephosphorylation was detected in membranes only. The membrane-bound phosph atase was potently inhibited by vanadate and Zn2+>>Mn2+, but was insensitiv e to okadaic acid (a phosphatase 1, 2 and 2B inhibitor), cyclosporin (speci fic calcineurin inhibitor) and phosphatase-l inhibitor. Endogenous kinase w as activated by divalent cations including calcium (Mg2+ > Mn2+ > Ca2+), wh ilst dephosphorylation did not require the presence of Ca2+ ions. This sugg ests that at least one membrane-bound phosphatase counteracts the endogenou s phosphorylation of the GABA(A)-R: the lack of dephosphorylation in the pu rified receptor preparation indicates that. in contrast to the endogenous k inase, no phosphatase is closely associated with the receptor protein compl ex. (C) 2000 Elsevier Science Ltd. All rights reserved.