Endogenous phosphorylation of the GABA A receptor protein is counteracted by a membrane-associated phosphatase

• Published in: Neurochemistry international 2000-05, Vol.36 (6), p.499-506
• Main Authors: Minier, Frédéric, Laschet, Jacques J, Evrard, Bertrand, Bureau, Michel H
• Format: Article
• Language: English
• Subjects: Animals; Brain - enzymology; Brain - metabolism; Cattle; Female; Membrane Proteins - metabolism; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Precipitin Tests; Protein Phosphatase 1; Radioligand Assay; Receptors, GABA-A - metabolism
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/S0197-0186(99)00158-8

Incubation of bovine brain membranes with [γ- 33P]ATP phosphorylated mainly a 51-kDa band. Electrophoretic co-migration was observed for 33P- and [ 3H]flunitrazepam-labeled bands in both membrane fractions and in affinity-purified GABA A receptor (GABA A-R) preparations. An α-subunit monoclonal antibody adsorbed most of the radiolabeled-band, suggesting that the labeled-membrane polypeptide corresponds to the GABA A-R α1-subunit, which is the only GABA A-R subunit with a molecular weight of 51 kDa. The phosphorylation rate was much faster in membranes than in purified receptor. Dephosphorylation was detected in membranes only. The membrane-bound phosphatase was potently inhibited by vanadate and Zn 2+≫Mn 2+, but was insensitive to okadaic acid (a phosphatase 1, 2 and 2B inhibitor), cyclosporin (specific calcineurin inhibitor) and phosphatase-1 inhibitor. Endogenous kinase was activated by divalent cations including calcium (Mg 2+>Mn 2+>Ca 2+), whilst dephosphorylation did not require the presence of Ca 2+ ions. This suggests that at least one membrane-bound phosphatase counteracts the endogenous phosphorylation of the GABA A-R: the lack of dephosphorylation in the purified receptor preparation indicates that, in contrast to the endogenous kinase, no phosphatase is closely associated with the receptor protein complex.