IDENTIFICATION OF SOLUBLE-PROTEIN PHOSPHATASES THAT DEPHOSPHORYLATE VOLTAGE-SENSITIVE SODIUM-CHANNELS IN RAT-BRAIN

Rat brain sodium channels are phosphorylated at multiple serine residu es by cAMP-dependent protein kinase, We have identified soluble rat br ain phosphatases that dephosphorylate purified sodium channels, Five s eparable forms of sodium channel phosphatase activity were observed, T hree forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A, since they were 85-100% inhibited by 10 nM okadaic acid and contained a 36-kDa polypeptide recognized by a mo noclonal antibody directed against the catalytic subunit of phosphatas e 2A, Immunoblots performed using antibodies specific for isoforms of the B subunit of phosphatase 2A indicate that the two major peaks of p hosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) prob ably represent calcineurin, Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and calmodulin, and co ntained a 19-kDa polypeptide recognized by a monoclonal antibody raise d against the B subunit of calcineurin, Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A or cyclosporin A to inhibit ca lcineurin increased apparent phosphorylation of sodium channels at cAM P-dependent phosphorylation sites, as assayed by back phosphorylation, These results indicate that phosphatase 2A and calcineurin dephosphor ylate sodium channels in brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity.