Abstract
Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. We have identified soluble rat brain phosphatases that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity were observed. Three 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 monoclonal antibody directed against the catalytic subunit of phosphatase 2A. Immunoblots performed using antibodies specific for isoforms of the B subunit of phosphatase 2A indicate that the two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and calmodulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcinerurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A or cyclosporin A to inhibit calcineurin increased apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites, as assayed by back phosphorylation. These results indicate that phosphatase 2A and calcineurin dephosphorylate sodium channels in brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity.
Highlights
From the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721 and :\:Departmentof Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153
Three forms are identical or related to phosphatase 2A, since they were 85-100% inhibited by 10 DM okadaic acid and contained a 36-kDa polypeptide recognized by a monoclonal antibody directed against the catalytic subunit of phosphatase 2A
Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and cal. modulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcineurin
Summary
Materials-Materials were purchased from the following sources: chromatography media, Pharmacia Biotech Inc.; [y"2P1ATP (3000 Ci/ mmol), DuPont NEN; [3H1Saxitoxin (20-40 Ci/mmol), Amersham Corp.;Immobilonpolyvinylidene difluoride,Millipore; forskolin,Calbiochern; okadaic acid, Biomol Research Laboratories, Inc. The resulting supernatant (soluble fraction) was assayed for sodium channel phosphatase activity as described below. Phosphatase Assay-In most assays, samples were mixed with 32p_ labeled sodium channels (300-500 fmol) in 20 IDM Tris-HCl, pH 7.6, 1 IDM CaCI 2, 50 nM calmodulin, 10 mM MgCI2, 50 mM NaCl, 0.2% Triton X-100, 0.3 mM EDTA, 0.1% l3-mercaptoethanol, and 1 mg/ml bovine serum albumin in a final volume of 30 fll. After drug treatment, synaptosomes were solubilized in (50 mM KP04, 50 mM NaP04 50 mM NaF, 10 mM EDTA, 5 mM l3-g1ycerophosphate, 3% Triton X-lOO, 0.1 mM phenylmethylsulfonyl fluoride, 1 IJ-M pepstatin A, 5 IJ-g/ml leupeptin, pH 6.8, at 0 °C), and incubated at 4°C for 2 h with monoclonal antibody 1Gll directed against the sodium channel a subunit.
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