©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Soluble Protein Phosphatases That Dephosphorylate Voltage-sensitive Sodium Channels in Rat Brain (*)

(Received for publication, July 22, 1994; and in revised form, December 27, 1994)

Tzu-chin Chen Brian Law (1) Tamara Kondratyuk (1) Sandra Rossie (1)(§)

From the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721 and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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 Balpha. 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 CaCl(2) and calmodulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcineurin. 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.


INTRODUCTION

Voltage-sensitive sodium channels mediate sodium influx during the initial phase of the action potential in excitable cells. Sodium channels from rat brain contain three glycosylated subunits: alpha (260 kDa), which contains both the ion pore and voltage sensor; beta1 (36 kDa), which is associated with alpha by ionic interactions and modulates channel inactivation; beta2 (33 kDa), which is covalently attached to alpha by disulfide bonds (reviewed in (1) ). The alpha subunit contains four homologous domains, termed I-IV, each of which has multiple membrane spanning segments. The electrophysiological responses of brain sodium channels are modified by cAMP-dependent protein kinase (PKA) (^1)and protein kinase C. Cyclic AMP-dependent phosphorylation decreases sodium current during depolarization(2, 3) . Protein kinase C-dependent phosphorylation slows channel inactivation (4) and also decreases sodium current during depolarization(4, 5, 6) . The alpha subunit of brain sodium channels is phosphorylated in synaptosomes and in cultured cells by cAMP-dependent protein kinase on Ser-573, Ser-610, Ser-623, and Ser-687, which are clustered in a single intracellular loop of the alpha subunit between homologous domains I and II of the alpha subunit(7, 8, 9, 10, 11) . Protein kinase C phosphorylates the alpha subunit of purified reconstituted sodium channels on two sites, Ser-610, which is also phosphorylated by PKA, and Ser-1506 which is not phosphorylated by PKA and is located within an intracellular loop connecting homologous domains III and IV of the alpha subunit(12) . Phosphorylation of Ser-1506 mediates the effect of protein kinase C on channel inactivation (13) and enhances the effect of cAMP on channel function(14) .

Purified soluble sodium channels were dephosphorylated by purified calcineurin, a calcium- and calmodulin-dependent protein phosphatase, and the catalytic subunit of phosphatase 2A, but not the catalytic subunit of phosphatase 1(11) . A mixture of the purified catalytic subunits of phosphatases 1 and 2A reversed the electrophysiological response of sodium channels to cAMP in Chinese hamster ovary cells tranfected with the brain sodium channel alpha subunit(3) . When brain sodium channels were expressed in Xenopus oocytes, the catalytic subunit of phosphatase 2A, but not that of phosphatase 1, could reverse the effect of PKA on sodium current(2) . It is not known which of these serine/threonine phosphatases dephosphorylate sodium channels in vivo. To fully understand how neuronal sodium channels are regulated by reversible phosphorylation, it is necessary to identify the phosphatases that dephosphorylate sodium channels in brain and to learn how their activity is controlled. In this study we demonstrate that calcineurin and phosphatase 2A in rat brain extracts dephosphorylate sodium channels that have been phosphorylated at cAMP-dependent phosphorylation sites. We also provide evidence that both of these phosphatases dephosphorylate sodium channels in synaptosomes. These observations establish that calcineurin and phosphatase 2A participate in controlling the phosphorylation state of sodium channels in brain.


EXPERIMENTAL PROCEDURES

Materials

Materials were purchased from the following sources: chromatography media, Pharmacia Biotech Inc.; [P]ATP (3000 Ci/mmol), DuPont NEN; [^3H]Saxitoxin (20-40 Ci/mmol), Amersham Corp.; Immobilon polyvinylidene difluoride, Millipore; forskolin, Calbiochem; okadaic acid, Biomol Research Laboratories, Inc. Cyclosporin A was a gift from Sandoz, Inc. All other reagents were purchased from Sigma.

Purified Proteins and Antibodies

Sodium channels were purified from rat brain through the wheat germ agglutinin chromatography step(15) . The catalytic subunit of PKA was purified from bovine heart(16) , and the catalytic subunit of phosphatase 2A was purified from rabbit skeletal muscle according to Cohen et al.(17) . Mouse monoclonal antibody 1G11 raised against purified brain sodium channels was kindly provided by Dr. William A. Catterall, Department of Pharmacology, University of Washington, Seattle. Antibody 1G11 specifically recognizes the alpha subunit of rat brain sodium channels (18) and does not interfere with phosphorylation by PKA. (^2)A mouse monoclonal antibody raised against the catalytic subunit of phosphatase 2A from bovine cardiac muscle (19) and rabbit antisera raised against phosphatase 2A B`and Balpha (20, 21) were kindly provided by Dr. Marc Mumby, Department of Pharmacology, University of Texas Health Science Center, Dallas. Purified calcineurin and a mouse monoclonal antibody raised against the B subunit of calcineurin was purchased from Upstate Biotechnology, Inc. Biotinylated anti-mouse or anti-rabbit IgG antibodies and streptavidin-alkaline phosphatase immunostaining kits were purchased from Zymed Laboratories, Inc. Phosphorylase b and phosphorylase kinase were purchased from Sigma. Gel filtration molecular weight standards were purchased from Pharmacia. Prestained molecular weight markers were purchased from Life Technologies, Inc. Biotinylated molecular weight markers were purchased from Bio-Rad.

Preparation of P-Labeled Substrates

Purified sodium channels were phosphorylated to 3-4-mol of phosphate/mol of channel with 50-100 µM [P]ATP (3-5 Ci/mmol) and the catalytic subunit of PKA as described previously(11) . P-labeled phosphorylase a was prepared as described by Cohen et al.(17) .

Separation and Characterization of Sodium Channel Phosphatases

All procedures were performed at 4 °C. For initial studies, soluble and particulate fractions were prepared by homogenizing a freshly dissected rat brain in 12 ml of homogenization buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1% beta-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 1 µg/ml leupeptin, and 10 µg/ml trypsin inhibitor). The homogenate was centrifuged at 1000 times g for 10 min, and the supernatant was retained. The pellet was re-extracted in 6 ml of buffer, and the combined supernatants were then centrifuged at 100,000 times g for 1 h. The resulting supernatant (soluble fraction) was assayed for sodium channel phosphatase activity as described below. The pellet (particulate fraction) was resuspended in homogenization buffer and assayed for phosphatase activity.

For all other studies, one to three fresh rat brains were homogenized in homogenization buffer, 23 ml/brain, and centrifuged at 100,000 times g for 1 h. The resulting supernatant was applied to a DEAE-Sephadex ion exchange column (2.4 times 20 cm) equilibrated in the same buffer. The column was washed until the effluent absorbance at 280 nm was less than 0.1. Bound material was then eluted at 1 ml/min with a 600-ml gradient of 0-1 M NaCl. Three-ml fractions were collected and assayed for sodium channel phosphatase activity.

DEAE fractions containing sodium channel phosphatase activity were pooled, and saturated (NH(4))(2)SO(4) (pH 7.4) was added to a final concentration of 55% at 4 °C. The mixture was stirred for 30 min, then centrifuged at 10,000 times g for 30 min. The pellet was resuspended in 1 ml of homogenization buffer, applied to a Sephacryl S-300 gel filtration column (1.7 times 96 cm) equilibrated in homogenization buffer containing 0.1 M NaCl, and eluted at 0.2 ml/min. One-ml fractions were collected and assayed for phosphatase activity.

Phosphatase Assay

In most assays, samples were mixed with P-labeled sodium channels (300-500 fmol) in 20 mM Tris-HCl, pH 7.6, 1 mM CaCl(2), 50 nM calmodulin, 10 mM MgCl(2), 50 mM NaCl, 0.2% Triton X-100, 0.3 mM EDTA, 0.1% beta-mercaptoethanol, and 1 mg/ml bovine serum albumin in a final volume of 30 µl. The reaction was initiated by the addition of extract, incubated at 30 °C for 10 min, then terminated with 100 µl of ice-cold 10% trichloroacetic acid. Samples were left at room temperature for 2 min, then centrifuged 12,000 times g for 5 min, and acid-soluble [P]P(i) was quantified by liquid scintillation counting. One unit of phosphatase activity was defined as 1 pmol of phosphate released/min at 30 °C. To ensure that acid-soluble [P]P(i) represented released inorganic phosphate, phosphate release from P-labeled sodium channels by soluble extract was also measured by extraction of inorganic phosphate(22) . Results obtained were comparable to those obtained by the acid precipitation method. The simpler acid precipitation assay was therefore used routinely. Exceptions to these standard assay conditions are outlined below.

When P-labeled phosphorylase a was used as substrate, it was present at a final concentration of 10 µM and 5 mM caffeine was included in the assay mixture. Sodium channel phosphatase activity was not affected by caffeine, and phosphorylase a phosphatase activity was not affected by the presence of 0.2% Triton X-100. For samples treated with okadaic acid, a concentrated stock (124 µM) of okadaic acid in N,N-dimethylformamide was diluted to a final concentration of 10 nM or 500 nM in assay samples. Control samples received an equal amount of N,N-dimethylformamide, which did not alter phosphatase activity. When testing the sensitivity of phosphatase activity to CaCl(2) and calmodulin, samples were assayed in the presence of 2 mM EGTA and the absence of CaCl(2) and calmodulin.

SDS-PAGE

SDS-PAGE was performed according to the method of Laemmli as described by Maizel(23) , using a 3% stacking gel and a 12% running gel for samples containing phosphatase 2A, a 15% running gel for samples containing calcineurin, or a 6% running gel for sodium channel samples.

Immunoblot Analysis

For immunoblots using antibodies specific for either the catalytic subunit of phosphatase 2A or the B subunit of calcineurin, after SDS-PAGE, proteins were electrophoretically transferred to Immobilon polyvinylidene difluoride membranes at 100 V for 30 min in (10 mM 3-[cyclohexyl]-1-propanesulfonic acid, pH 10.5, 10% methanol). For immunoblots using antibodies toward the B subunits of phosphatase 2A, after SDS-PAGE, proteins were transferred to nitrocellulose at 100 V for 30 min in (25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3). All blots were then incubated overnight at 4 °C or 1 h at room temperature in (50 mM Tris-HCl, pH 8.0, 80 mM NaCl, 2 mM CaCl(2), 0.2% Tween 20 with 10% bovine serum albumin), then probed with antibody. Blots were washed twice in (50 mM Tris-HCl pH 8.0, 80 mM NaCl, 2 mM CaCl(2), 1% bovine serum albumin, 1% Tween 20, and 0.2% SDS), then twice in (50 mM Tris-HCl, pH 8.0, 80 mM NaCl, 2 mM CaCl(2), 0.05% Tween 20). Antibody binding was detected using biotinylated secondary antibodies to mouse or rabbit IgG and a streptavidin-alkaline phosphatase immunostaining kit according to instructions.

Synaptosome Preparation

Synaptosomes were prepared as described by Gray and Whitaker(24) , with the indicated modifications (25) . Until the final resuspension, all procedures were performed at 4 °C. Briefly, a freshly dissected rat brain was homogenized in 12.5 ml (0.32 M sucrose, 5 mM HEPES, pH 7.4, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µM pepstatin A), centrifuged at 1000 times g for 6 min, and the supernatant was saved. The pellet was re-extracted in 6 ml of buffer, and the combined supernatants were centrifuged at 17,300 times g for 12 min. The resulting pellet was resuspended in 9 ml of buffer, and 3-ml aliquots were loaded onto discontinuous gradients containing 3.5 ml each of 7.5 and 13% Ficoll in homogenization buffer, then centrifuged in a swinging bucket rotor at 72,000 times g for 60 min. The fraction at the 7.5%/13% interphase, which contained the majority of sodium channels as measured by [^3H]saxitoxin binding (26) , was collected and diluted with 10 ml of homogenization buffer, then recentrifuged at 17,300 times g for 10 min. The synaptosome pellet was finally resuspended in buffer (20 mM HEPES, pH 7.4, 10 mM glucose, 132 mM NaCl, 4.8 mM KCl, 2.4 mM MgSO(4), 0.1 mM EGTA, 1.1 mM CaCl(2)) to a protein concentration of approximately 1.5 mg/ml. Resuspended synaptosomes were incubated for 5 min at 37 °C before addition of drug.

Isolation and Back Phosphorylation of Sodium Channels

Solubilization, immunoprecipitation, and back phosphorylation of sodium channels were performed as previously reported (27) with minor changes. Briefly, after drug treatment, synaptosomes were solubilized in (50 mM KPO(4), 50 mM NaPO(4) 50 mM NaF, 10 mM EDTA, 5 mM beta-glycerophosphate, 3% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 5 µg/ml leupeptin, pH 6.8, at 0 °C), and incubated at 4 °C for 2 h with monoclonal antibody 1G11 directed against the sodium channel alpha subunit. Protein A-Sepharose was then added, and the sample was mixed for an additional 45 min. Immunoprecipitates were sedimented briefly in a microcentrifuge, washed twice with the same buffer, then five times with 25 mM HEPES/Tris, pH 7.4, 5 mM MgCl(2), 5 mM EGTA, 0.1% Triton X-100 and stored at -80 °C until analyzed. To determine the extent of endogenous channel phosphorylation, immunoprecipitates were incubated with [P]ATP (3000 Ci/mmol) and the catalytic subunit of PKA in 25 mM HEPES/Tris, pH 7.4, 5 mM MgCl(2), 5 mM EGTA, 0.1% Triton X-100 in a final volume of 50 µl for 30 s at 30 °C. Phosphorylation was terminated with 50 µl of concentrated SDS gel loading buffer (6% SDS, 60 mM Tris, pH 6.8, 25 mM EDTA, 40% glycerol, 0.3% beta-mercaptoethanol, 0.2 mg/ml bromphenol blue). Samples were boiled for 2 min and briefly centrifuged, and the supernatants were subjected to SDS-PAGE. Gels were fixed, dried, and subjected to autoradiography. Gel bands corresponding to P-labeled sodium channel alpha subunits were excised and P was quantified by liquid scintillation counting.

Two-dimensional Phosphopeptide Maps

Two-dimensional phosphopeptide maps were prepared using the method of Beemon and Hunter (28) with minor modifications(29) , as described in detail(27) .


RESULTS

Sodium Channel Phosphatase Activity in Crude Rat Brain Extract

Soluble and particulate fractions prepared from rat brain were assayed for sodium channel phosphatase activity. Since the four major serine/threonine phosphatases, phosphatases 1, 2A, 2B (also known as calcineurin), and 2C all are present in brain (reviewed in (30) ), assays were performed under conditions that would support activity of all four enzymes. One millimolar CaCl(2) and 50 nM calmodulin were included in order to promote calcineurin activity, and 10 mM MgCl(2) to support the activity of phosphatase 2C. The soluble fraction from one rat brain contained roughly twice as much sodium channel phosphatase activity (530 units) as did the particulate fraction (235 units).

Okadaic acid inhibits serine/threonine protein phosphatases to varying degrees(31) . The effect of okadaic acid or CaCl(2) and calmodulin on phosphatase activity in the soluble extract was determined using two substrates, sodium channel and phosphorylase a, a substrate for phosphatases 1 and 2A (Fig. 1). Two concentrations of okadaic acid were tested: 10 nM to preferentially inhibit phosphatase 2A, and 500 nM to inhibit both phosphatases 2A and 1. Phosphorylase a phosphatase activity was 40% inhibited by 10 nM okadaic acid and completely inhibited by 500 nM okadaic acid, consistent with the presence of both phosphatase 1 and 2A. Sodium channel dephosphorylation was 75% inhibited by 10 nM okadaic acid, however little or no further inhibition was achieved by 500 nM okadaic acid. This suggests that phosphatase 2A or a phosphatase with similar sensitivity to okadaic acid in the brain extract dephosphorylates sodium channels. In addition, those form(s) of phosphatase 1 present in the soluble extract and active toward phosphorylase a do not dephosphorylate sodium channels. Removal of CaCl(2) and calmodulin further reduced sodium channel dephosphorylation to 6% of maximal activity, suggesting that calcineurin in the crude soluble extract may also dephosphorylate sodium channels.


Figure 1: Dephosphorylation of sodium channels or phosphorylase a by soluble rat brain extract. Dephosphorylation of P-labeled sodium channels () or P-labeled phosphorylase a (&cjs2112;) by soluble rat brain extract was measured as described under ``Experimental Procedures.'' Dephosphorylation in the presence of 10 nM or 500 nM okadaic acid, or in the presence of 10 nM okadaic acid and 2 mM EGTA is expressed as a percent of control dephosphorylation (100%) in the presence of 1 mM CaCl(2) and 50 nM calmodulin. Average values ± S.E. from three separate experiments are shown. (O.A., okadaic acid; *, no detectable dephosphorylation; N.D., not determined).



Separation and Characterization of Sodium Channel Phosphatases

DEAE ion exchange chromatography of soluble brain extract yielded three major peaks of sodium channel phosphatase activity, A, B, and C (Fig. 2). Each peak was pooled, concentrated by precipitation with 55% ammonium sulfate, and subjected to gel filtration. Gel filtration resolved DEAE peaks A and B each into two peaks of sodium channel phosphatase activity, termed A1 and A2, or B1 and B2 (Fig. 3, A and B). Peak C from DEAE chromatography yielded a single peak of activity upon gel filtration (Fig. 3C). Apparent molecular weights for each peak, based on comparison with the elution of protein standards, were estimated to be: A1, 234,000; A2, 99,000; B1, 234,000; B2, 102,000; C, 192,000.


Figure 2: DEAE chromatogram of soluble rat brain sodium channel phosphatase activity. Soluble rat brain extract was subjected to DEAE ion exchange chromatography as described under ``Experimental Procedures.'' Phosphatase activity was measured by the release of phosphate from P-labeled sodium channels. Results from a single experiment are shown and are representative of five separate studies.




Figure 3: Gel filtration chromatograms of DEAE peaks A, B, and C. DEAE peaks A, B, and C were each pooled, concentrated, and subjected to gel filtration through Sephacryl S-300. Sodium channel phosphatase activity was measured as described under ``Experimental Procedures.'' The elution of gel filtration standards are indicated: a, ferritin, 440,000; b, catalase, 232,000; c, aldolase, 158,000; d, bovine serum albumin, 66,000; e, carbonic anhydrase, 24,000; f, cytochrome c, 12,000. Results from a single experiment are shown and are representative of four separate studies.



Fractions A1, A2, B1, and B2 obtained from gel filtration and pooled concentrated fractions from DEAE peak C were assayed for sensitivity to calcium and calmodulin or okadaic acid (Fig. 4). A1, B1, and C were unaffected by the removal of CaCl(2) and calmodulin, but were 80-100% inhibited by 10 nM okadaic acid. In contrast, A2 and B2 were insensitive to 10 nM okadaic acid, but were reduced in activity by 85-100% in the absence of CaCl(2) and calmodulin. These results suggest that A1, B1, and C are similar to phosphatase 2A with regard to okadaic acid sensitivity, whereas A2 and B2 may represent calcineurin.


Figure 4: Sensitivity of sodium channel phosphatases to okadaic acid or CaCl(2) and calmodulin. Gel filtration peaks A1, A2, B1, B2, and DEAE peak C were assayed for sodium channel phosphatase activity in: control buffer containing 1 mM CaCl(2) and 50 nM calmodulin (&cjs2108;); buffer containing 2 mM EGTA with no added CaCl(2) or calmodulin (); buffer containing 10 nM okadaic acid (&cjs2113;). Dephosphorylation is expressed as a percent of control dephosphorylation (100%) in the presence of CaCl(2) and calmodulin. Average values ± S.E. from three separate experiments are shown, except for B2, which was assayed once in triplicate. (*, no detectable dephosphorylation).



To test whether the okadaic acid-sensitive sodium channel phosphatase fractions contained phosphatase 2A, immunoblots were performed using antibodies specific for the catalytic (C) subunit of phosphatase 2A. Fractions of A1 and B1 from gel filtration chromatography and C from DEAE chromatography were immunoreactive with a monoclonal antibody that specifically recognizes the catalytic subunit of phosphatase 2A (Fig. 5A). Mobility of the immunoreactive species in these samples coincided with that of the 36-kDa catalytic subunit of phosphatase 2A purified from rabbit skeletal muscle. In contrast, A2, which was active only in the presence of calcium and calmodulin, did not contain an immunoreactive polypeptide of 36 kDa. Recognition of the 36-kDa polypeptide in soluble brain extract was blocked by preincubation of antibody with the purified catalytic subunit of phosphatase 2A (data not shown), indicating that recognition was specific. Western (21, 55) and Northern (38, 39, 55) blot analyses indicate that as many as five forms of the phosphatase 2A B subunits may be present in brain. Immunoblots using antibodies specific for two isoforms of the B subunit, Balpha and B`, demonstrated that A1 was enriched in B` (Fig. 5B), whereas B1 was enriched in Balpha (Fig. 5C). Peak C stained weakly with antisera for both types of B subunits. The doublet visualized with the B` subunit antiserum has also been reported in cardiac tissue(20) , where the B`-containing isoform is the major species of phosphatase 2A. These data indicate that A1 contains phosphatase 2A(0), and B1 contains phosphatase 2A(1). Fraction C may contain proteolyzed forms of both phosphatase 2A(0) and 2A(1), as has been observed by others(17) .


Figure 5: Immunoblot analysis of sodium channel phosphatases with antibodies to the catalytic or B subunit of phosphatase 2A. Peak fractions from DEAE or gel filtration chromatography were subjected to SDS-PAGE, then transferred to membrane and probed as described under ``Experimental Procedures'' with antibodies to either the catalytic subunit (A) or the B` (B) or Balpha (C) subunits of phosphatase 2A (B). A gel filtration peak fractions A1, A2, B1, and DEAE peak C, and the catalytic subunit of phosphatase 2A purified from rabbit skeletal muscle (PP-2Ac). B and C, gel filtration peak fractions A1, B1, and C. The migration of prestained standards (bovine serum albumin (76 kDa), carbonic anhydrase (28 kDa), biotinylated standards (phosphorylase b` (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase B (31 kDa), and the catalytic subunit (PP2A-C), the B` subunit (PP2A-B`) or the Balpha subunit (PP2A-Balpha) of phosphatase 2A are indicated.



Immunoblots were also performed to determine whether the calcium and calmodulin-sensitive fractions contained calcineurin. A polypeptide of approximately 19 kDa in peaks A2 and B2 was detected using an antibody directed against the B subunit of calcineurin (Fig. 6). The migration of this polypeptide coincided with that of the B subunit of purified calcineurin. A1, which was insensitive to calcium and calmodulin, was not recognized by this antibody. Recognition of the 19-kDa polypeptide in soluble brain extract was blocked by preincubation of antibody with the purified calcineurin (data not shown), again indicating that recognition was specific.


Figure 6: Immunoblot analysis of sodium channel phosphatases with an antibody to the B subunit of calcineurin. Peak fractions A1, A2, and B2 from DEAE chromatography and purified calcineurin (CN) were subjected to SDS-PAGE, transferred, and probed as described under ``Experimental Procedures.'' The migration of prestained standards (carbonic anhydrase (28 kDa), and lysozyme (16 kDa)) and the B subunit of calcineurin (CNB) are indicated.



To determine whether A2 and B2 represent two separate peaks of CaCl(2)/calmodulin-sensitive phosphatase activity, soluble brain extract was subjected to DEAE chromatography as before. Fractions were then assayed in the presence or absence of 10 nM okadaic acid (Fig. 7). In the presence of okadaic acid, two distinct peaks of activity were observed. In the presence of 10 nM okadaic acid and the absence of CaCl(2) and calmodulin, all sodium channel phosphatase activity was lost. This indicates that A2 and B2 are separate peaks containing calcineurin, but it is unclear whether they represent distinct isoforms of calcineurin, or if one is derived from the other via proteolysis.


Figure 7: Two separable peaks of CaCl(2)/calmodulin-sensitive sodium channel phosphatase activity. DEAE ion exchange chromatography was performed as in Fig. 2, with phosphatase activity measured in the presence of CaCl(2) and calmodulin (bullet) as usual, in the presence of CaCl(2), calmodulin and 10 nM okadaic (circle), or in the presence of 10 nM okadaic acid and 2 mM EGTA with no added CaCl(2) or calmodulin (times).



Sodium Channel Dephosphorylation in Synaptosomes

To determine if phosphatase 2A and calcineurin dephosphorylate sodium channels under physiological conditions, back phosphorylation was used to measure changes in sodium channel phosphorylation in synaptosomes. In this technique, the substrate of interest is isolated from cells or tissue under conditions which preserve endogenous phosphorylation, then phosphorylated with [P]ATP in vitro to radiolabel phosphorylation sites that were unmodified in situ(32) . Back phosphorylation has been successfully used to measure changes in phosphorylation of sodium channels in synaptosomes (7) and cultured neurons(27) . When synaptosomes were treated with forskolin to increase cAMP levels and trigger cAMP-dependent phosphorylation, back phosphorylation of sodium channels was decreased by 40%, indicating that endogenous phosphorylation had been increased (Fig. 8A). When synaptosomes were treated with cyclosporin A to inhibit calcineurin (33, 34) or with okadaic acid, back phosphorylation was also reduced by 40-60%. Under these same conditions, okadaic acid and cyclosporin A had no effect on the activity of PKA, measured as described by Reimann et al.(35) (data not shown). The influence of okadaic acid on sodium channel back phosphorylation was half-maximal at 1-2 nM (Fig. 8B), consistent with inhibition of phosphatase 2A or a phosphatase with similar sensitivity to okadaic acid. Thus, by inhibiting dephosphorylation, okadaic acid or cyclosporin A each caused a net increase in phosphorylation of sodium channel cAMP-dependent phosphorylation sites.


Figure 8: Back phosphorylation of sodium channels from synaptosomes treated with okadaic acid or cyclosporin A. A, synaptosomes were incubated at 37 °C with 10 µM forskolin for 10 min, 300 nM okadaic acid for 15 min, or 1 µM cyclosporin A for 30 min, and then sodium channels were isolated and back phosphorylated with the catalytic subunit of PKA and []P-labeled ATP as described under ``Experimental Procedures.'' B, dose dependence of the effect of okadaic acid on synaptosomes. Results are expressed as the percent decrease in back phosphorylation compared to control in the absence of drug, with appropriate vehicle controls. Results are the average ± S.E. of three or more experiments.



Previous studies have shown that PKA phosphorylates four serine residues in sodium channel alpha subunits in vitro and in situ(11) . These sites incorporated different levels of P during back phosphorylation or metabolic labeling, suggesting that they may be differentially phosphorylated by PKA. In back phosphorylation studies, Ser-623 incorporated nearly twice as much P as other residues, whereas in metabolic labeling studies, Ser-687 incorporated twice as much P as any of the other sites. To determine whether calcineurin or phosphatase 2A selectively dephosphorylated any of the four identified PKA phosphorylation sites, phosphopeptide maps were generated from back phosphorylated sodium channels isolated from synaptosomes treated with phosphatase inhibitors or forskolin, and phosphate incorporated into each phosphorylation site quantified separately as described previously (11) (Fig. 9). This allows changes in phosphorylation of each site to be examined, regardless of the total amount of P incorporated. Treatment of synaptosomes with forskolin, okadaic acid or cyclosporin A led to a moderate decrease in back phosphorylation of each Ser residue phosphorylated by PKA. Ser-573 was less affected by treatment with forskolin or okadaic acid than were the other phosphorylation sites, although the effect of treatments on back phosphorylation of this site was variable. Since Ser-573 accounts for 20-25% of the total phosphate incorporated into all four sites (11) and is the least affected of any site by all treatments, differences in back phosphorylation at this site contribute modestly to the overall changes in back phosphorylation seen when all four sites are considered together (Fig. 8). Ser-687 was more sensitive to treatment with okadaic acid than forskolin or cyclosporin A, indicating that phosphatase 2A exerted greater influence on the phosphorylation status of this site than did PKA or calcineurin.


Figure 9: Phosphopeptide analysis of sodium channels after treatment of synaptosomes with okadaic acid or cyclosporin A. Synaptosomes were treated as in Fig. 7with A, forskolin; B, okadaic acid; or C, cyclosporin A, then sodium channels were isolated, back phosphorylated, and phosphopeptide maps generated as described under ``Experimental Procedures.'' Phosphopeptides corresponding to each serine residue were scraped and counted. Results are expressed as the percent decrease in back phosphorylation compared to control in the absence of drug, with appropriate vehicle controls. Results are the average ± S.E. of three or more experiments.




DISCUSSION

At least two different types of serine/threonine phosphatases with properties similar to those of phosphatase 2A and calcineurin dephosphorylate sodium channels in brain. These two phosphatases appear to account for all the sodium channel phosphatase activity detected in a soluble rat brain extract.

Activity present in gel filtration peaks A1 and B1, and in DEAE peak C resembles phosphatase 2A in its sensitivity to low concentrations of okadaic acid, and each peak contains a 36-kDa polypeptide recognized by an antibody directed against the catalytic subunit of phosphatase 2A. The best characterized forms of phosphatase 2A contain a catalytic subunit complexed with one or more regulatory subunits, A (61 kDa) and B (54-74 kDa), which can influence the activity and substrate specificity of the catalytic subunit (summarized in (36) ). Distinct B subunits have been observed in brain(21, 55) , and multiple forms of all three subunits in brain are predicted from cloning studies(37, 38, 39, 55) . Immunoblots indicate that peaks A1 and B1 contain phosphatase 2A(0) (AC-B`), and phosphatase 2A(1)(AC-Balpha), respectively, suggesting that both of these isoforms of phosphatase 2A may be active toward sodium channels. However, further work will be required to determine the contribution of these and other brain forms of phosphatase 2A to sodium channel dephosphorylation. The activity and substrate specificity of phosphatase 2A is modulated by the type of B subunit present(21) . Recent studies suggest that phosphatase 2A may also be regulated by phosphorylation(40, 41, 42, 43) , the binding of ceramide (44, 45) , and possibly by carboxymethylation(54) , but it is not yet clear how these events are controlled in brain. It will be important to determine whether post-translational modifications or B subunit isoforms modulate the activity of phosphatase 2A toward sodium channels. In addition, it will be interesting to determine whether any of the brain forms of phosphatase 2A are colocalized with sodium channels in neurons.

Two separable calcineurin-like phosphatase activities, A2 and B2, were also detected using sodium channel as substrate. Each was dependent on calcium and calmodulin for activity, was insensitive to 10 nM okadaic acid, and contained a 19-kDa polypeptide recognized by antibodies directed against the B subunit of bovine brain calcineurin. Calcineurin is a heterodimer containing a catalytic subunit, termed A (60 kDa) and a regulatory B subunit (19 kDa). Molecular biological, immunological, and biochemical studies indicate that multiple isoforms of the A subunit are expressed in brain (46, 47, 48, 49, 50, 51, 52, 53) . Further studies will be required to determine whether the two peaks of calcineurin-like activity are distinct forms of the enzyme or if the minor peak, B2, is derived by proteolysis. If A2 and B2 represent distinct forms of calcineurin, it will be of interest to determine whether they play distinct roles in sodium channel dephosphorylation.

The observation that phosphatase 2A and calcineurin can dephosphorylate sodium channels in brain extracts and in synaptosomes suggests a role for these phosphatases in controlling the state of sodium channel phosphorylation. This conclusion is consistent with previous in vitro studies showing that purified calcineurin and the catalytic subunit of phosphatase 2A, but not that of phosphatase 1, can dephosphorylate sodium channels(11) , and with electrophysiological studies showing that the catalytic subunit of phosphatase 2A, but not phosphatase 1, can reverse the effects of PKA on sodium channel function(2) . Despite these observations, it remains possible that a latent form of phosphatase 1 may dephosphorylate sodium channels when appropriately activated.

Inhibition of phosphatase 2A or calcineurin in synaptosomes led to an apparent increase in phosphorylation of all four cAMP-dependent phosphorylation sites of the sodium channel alpha subunit. This result differs from earlier findings that purified calcineurin or the catalytic subunit of phosphatase 2A selectively dephosphorylated Ser-623 or Ser-610 in purified sodium channels, respectively(11) . These differences may arise from the presence of additional subunits or post-translational modifications of enzymes or substrate, or may reflect the competing basal activity of phosphatases and PKA within synaptosomes.

The identification of phosphatase 2A and calcineurin as enzymes that dephosphorylate sodium channels in brain is an important step in understanding how the state of sodium channel phosphorylation is regulated. The physiological circumstances controlling the activity of calcineurin and phosphatase 2A toward sodium channels remain to be determined. Since the sodium channel contains multiple phosphorylation sites and is a target for multiple protein kinases and phosphatases, control of its state of phosphorylation is expected to be complex.


FOOTNOTES

*
This work was supported by National Multiple Sclerosis Society Grant RG2060-A-1 and National Institutes of Health Grant NS31221 (to S. R.). This is Journal Paper No. 14322 from the Purdue University Agriculture Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 317-494-3112; Fax: 317-494-7897.

(^1)
The abbreviations used are: PKA, cAMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis.

(^2)
S. Rossie and W. A. Catterall, unpublished observation.


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