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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 3733-3737, June 1986 Biochemistry

Phosphorylation and dephosphorylation of dihydropyridinesensitive voltage-dependent Ca2" channel in skeletal muscle membranes by cAMP- and Ca2"-dependent processes (1,4-dihydropyridine receptor/calmodulin/calcineurin/transverse-tubule membranes)

M. MARLENE HOSEY*, MARC BORSOTTO, AND MICHEL LAZDUNSKI Centre de Biochimie, Centre National de la Recherche Scientifique, Universite de Nice, Parc Valrose, 06034 Nice, France

Communicated by Philip Siekevitz, February 5, 1986

ABSTRACT The phosphorylation and dephosphorylation of the dihydropyridine-sensitive Ca2+ channel was studied in transverse-tubule membranes isolated from rabbit skeletal muscle. Exposure of these membranes to either the cAMPdependent protein kinase or a Ca2+/calmodulin-dependent protein kinase resulted in a rapid phosphorylation of a protein with properties similar to the major component of the skeletal muscle Ca2+ channel. The molecular mass of the phosphoprotein was 140 or 160 kDa, depending on the electrophoretic conditions. The stoichiometry of the phosphorylation was calculated to be 0.4-1.0 mol of phosphate per mol of protein. Neither the rate nor the extent of phosphorylation was affected by dihydropyridines. Limited proteolytic digestion of the protein that had been phosphorylated by either or both protein kinases yielded a single phosphopeptide of 5.4 kDa. The Ca2+-dependent phosphatase calcineurin dephosphorylated the membrane-bound Ca2+ channel that had been previously phosphorylated by either protein kinase. The results suggest that the major component of the dihydropyridine-sensitive Ca2+ channel from skeletal muscle can be effectively phosphorylated and dephosphorylated in its native state by cAMP- and Ca2+-dependent processes.

Certain Ca2" channels are activated and inactivated by hormones and/or neurotransmitters that increase and decrease cAMP, respectively (3, 21). Evidence for the involvement of phosphorylation in this regulation is that microinjection of the purified cAMP-dependent protein kinase into myocytes causes activation of Ca2+ channels (22). Ca2+ channels are also regulated by Ca2+-dependent processes (2, 3, 21), which could also involve phosphorylation/dephosphorylation reactions. The biochemical events involved in the phosphorylation/dephosphorylation-dependent regulation of Ca2+ channels have not been defined. Ca2+ channels in skeletal muscle are activated by cAMP (23), but a recent study reported that the major component of the skeletal muscle Ca2+ channel is not phosphorylated in T-tubule membranes by cAMP-dependent protein kinase (24). However, by using preparations highly enriched in Ca2+ channels, we now show that a protein similar or identical to the major component of the skeletal muscle Ca2+ channel can be phosphorylated in its membrane-bound state by cAMP- and Ca2+-dependent protein kinases and dephosphorylated by a Ca2+-dependent protein phosphatase, calcineurin (25, 26).

Ca2l movement into and out of cells is controlled by several

MATERIALS AND METHODS Membrane and Protein Isolation. T-tubule membranes were isolated from rabbit skeletal muscle as described (15, 27). The concentration of Ca2+ channels in these membranes, as assessed by dihydropyridine binding assays using [3H]PN 200-110 as the ligand, varied between 40 and 90 pmol/mg of protein. The catalytic subunit of the cAMP-dependent protein kinase was purified to homogeneity from bovine heart (28). The Ca2 + /calmodulin-dependent protein kinase ("synapsin I kinase", ref. 29), purified to homogeneity from rat brain, and the calcineurin, purified to homogeneity from bovine brain (26), were generous gifts from C. B. Klee (National Institutes of Health). Protein Phosphorylation Reactions. T-tubule membranes were phosphorylated with cAMP-dependent protein kinase in reaction mixtures that contained 50 mM Hepes (Na+) (pH 7.4), 10 mM MgSO4, 2.5 mM EGTA, 0.1 mM [y-32P]ATP (300-1000 cpm/pmol), 0.1-1 ,AM catalytic subunit, and 1-2 mg of T-tubule membrane protein per ml. Ca2+/calmodulindependent phosphorylation reaction mixtures contained 50 mM Tris HCl (pH 7.4), 10 mM MgSO4, 0.1 mM [y-32P]ATP, 0.5 mM 2-mercaptoethanol, ±1 ,tM calmodulin, ±0.5 mM CaCl2, 2.5 ,g of the Ca2+/calmodulin-dependent kinase per ml, and 1-2 mg of membrane protein per ml. All incubations were for 2-3 min at ambient temperature and were terminated

different transporting processes located in cellular membranes (1). The most important of these in terms of regulating Ca2+ entry appears to be Ca2+ channels. Ca2' channels exist mainly in excitable cells (2, 3), such as muscle and nerve, but also in secretory cells, such as adrenal chromaffin cells (4), and in some nonexcitable cells, such as sperm (5) and basophils (6). Some, but not all (7), Ca2+ channels can be activated or inactivated by 1,4-dihydropyridine derivatives (e.g., nitrendipine, PN 200-110, Bay K 8644) (8-12). These drugs are important tools for biochemists because they bind to receptors located on the Ca2+ channel itself, and the radiolabeled forms can be used to specifically label Ca2+channel proteins (8-12). Comparison of data obtained in electrophysiological and biochemical studies suggests that the dihydropyridine receptors are indeed valid biochemical markers of voltage-dependent Ca2+ channels (13, 14). The transverse(T)-tubular system of skeletal muscle membranes contains the greatest density of high-affinity dihydropyridine binding sites so far described (15, 16). This high density of sites has facilitated the biochemical characterization (see refs. 10 and 12) and subsequent purification (17-19) of the Ca2+ channel from skeletal muscle. The major component of this Ca2+ channel is a 140-kDa protein (17-19) that contains receptor sites for dihydropyridines as well as for two other different types of Ca2+-channel inhibitors, diltiazem and bepridil (20).

Abbreviations: T-tubule, transverse-tubule; WGA, wheat germ agglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. *Permanent and present address: Department of Biological Chemistry and Structure, University of Health Sciences/The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064.

The publication costs of this article were defrayed in part by page charge

payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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by adding 1-2 vol of ice-cold buffer A (50 mM NaF/50 mM sodium/potassium phosphate, pH 7.4/20 mM EDTA) or the NaDodSO4-gel denaturing buffer (30). All solutions used in this study (except the phosphorylation reaction mixtures) contained 1 mM iodoacetamide, 0.1 mM phenylmethylsulfonyl fluoride, 1 gM pepstatin A, and 2 gg of leupeptin per ml. Solubilization and Partial Purification of the Phosphorylated Ca2+ Channel. T-tubule membranes were phosphorylated, washed with buffer A, and subsequently solubilized by adding 1 vol of buffer A plus 1 vol of 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 5% glycerol, 140 mM NaCi, and 20 mM Tris HCl (pH 7.5). After 30 min at 40C, the mixture was centrifuged at 160,000 x g for 30 min. The supernatant was incubated with wheat germ agglutinin (WGA)-Sepharose (5 vol of extract to 1 vol of packed gel) for 2 hr at 4°C, and the Ca2+-channel protein was eluted as described (19). The fractions were analyzed for [3H]PN 200-110 binding activity (19, 31), for protein content (32), and for peptide and phosphopeptide content by NaDodSO4 gel electrophoresis (30) and autoradiography. Dephosphorylation Reactions. Phosphorylated T-tubule membranes were washed once with buffer A and then washed twice with 5 ml of ice-cold 20 mM Tris HCl, pH 8/1 mM EDTA. All solutions contained the protease inhibitors listed above plus 10 ug of aprotinin per ml and 10 ,ug of soybean trypsin inhibitor per ml. The membranes were subsequently added to dephosphorylation reaction mixtures that contained 20 mM Tris HCl (pH 8), 0.1 M NaCl, 6 mM MgCl2, and either 2.5 mM CaCl2 or 2.5 mM EGTA, ±1 ,uM calmodulin, +0.075-0.4 ,M calcineurin, in a final volume of 0.2-0.25 ml. The reactions were performed at 25°C for the indicated times and stopped by adding aliquots to tubes containing the NaDodSO4-gel denaturing buffer. Gel Electrophoresis. Samples were denatured in 2% NaDodSO4 containing 10 mM Tris HCl (pH 6.8), 7.5% sucrose, and either 0.5 M 2-mercaptoethanol or 8 mM iodoacetamide, as indicated, and heated at 95°C for 5 min. Electrophoresis was performed on either 4-14% linear gradient gels or 5% linear gels (30). Peptide mapping of bands excised from the gels was performed according to the method of Cleveland et al. (33) as modified by Huttner and Greengard (34). The gels used to resolve the proteolytic products contained 16% acrylamide and were processed directly for

autoradiography. Materials. [3H]PN 200-110 and [_y-32P]ATP were purchased from Amersham. CHAPS and WGA-Sepharose were from Sigma. Molecular mass markers were from Bio-Rad or Bethesda Research Laboratories. Calmodulin was purchased from IBF (France) or prepared according to Yazawa et al.

(35). RESULTS Phosphorylation Catalyzed by cAMP-Dependent Protein Kinase. T-tubule membranes were phosphorylated by the cAMP-dependent protein kinase and the resulting phosphopeptides were then analyzed by NaDodSO4 gel electrophoresis in three types of preparations: (0) the intact T-tubule membranes, (ii) the CHAPS-solubilized preparations, and (iii) the partially purified Ca2+ channels obtained by WGASepharose chromatography (19). The Ca2+-channel protein had an apparent molecular mass of 140 kDa when electrophoresed in the presence of 2-mercaptoethanol (17-19) or 160 kDa when electrophoresed in the presence of iodoacetamide (Fig. 1). This can be seen in the membranes, CHAPS extract, and fractions eluted from the WGA-Sepharose (Fig. 1 Upper, lanes 1, 2, and 4). This protein was absent in the breakthrough fraction from the WGA-Sepharose column (Fig. 1 Upper, lane 3). A phosphoprotein that exhibited similar electropho-

Proc. Natl. Acad. Sci. USA 83 (1986) 2-mercaptoethanol

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FIG. 1. Phosphorylation of the major component of the skeletal muscle Ca2l channel in T-tubule membranes by cAMP-dependent protein kinase. T-tubule membranes (2 mg) were phosphorylated in a reaction volume of 3 ml. The Ca2l-channel protein was then solubilized with CHAPS and purified by WGA-Sepharose chromatography. (Upper) Silver-stained gel (4-14%) patterns of aliquots of T-tubule membranes (lanes 1), supernatant after solubilization of the T-tubule membranes with CHAPS (lanes 2), breakthrough fraction from the WGA-Sepharose column (lanes 3), and fraction eluted from the WGA-Sepharose column with 0.2 M N-acetylglucosamine (lanes 4). Samples shown on the left were electrophoresed in the presence of 2-mercaptoethanol; those on the right were run in the presence of iodoacetamide. Molecular masses are given in kDa. (Lower) Autoradiogram produced from the corresponding samples shown in Upper. The specific activity of the [y-32P]ATP was 580 cpm/pmol. The autoradiogram was produced by exposing lanes 1 and 2 for 66 hr and lanes 3 and 4 for 19 hr to Kodak XAR film.

retic behavior-i.e., molecular mass of 140 or 160 kDa when electrophoresed in the presence of 2-mercaptoethanol or iodoacetamide, respectively-was detected in the membranes and CHAPS extract (Fig. 1 Lower, lanes 1 and 2), was absent in the breakthrough fractions (Fig. 1 Lower, lane 3), and coeluted from the WGA-Sepharaose column with the fractions containing the dihydropyridine receptor (Fig. 1 Lower, lane 4). In addition to the phosphopeptide of 140 or 160 kDa, phosphopeptides of 60 and 55 kDa were observed in the fractions eluted from the WGA-Sepharose column (Fig. 1 Lower, lane 4). These peptides were not readily visualized in the gels by either Coomassie blue or silver staining. Very little phosphorylation was observed in the absence of the cAMP-dependent kinase under the conditions used for this assay (but see below). The results in Fig. 1 were obtained using 1 ,M kinase; similar results were obtained using 0.1-0.5 uM of the kinase. The time course of the phosphorylation was rapid (Fig. 2) and was identical whether the protein was analyzed as the 140- or 160-kDa peptide (Fig. 2). The stoichiometry of the phosphorylation catalyzed by the cAMP-dependent protein kinase was 0.6-1.0 mol of phosphate per mol of protein. Nitrendipine was reported to modulate the phosphorylation of a 42-kDa peptide in cardiac microsomes (36); however, neither the rate nor the extent of phosphorylation of the skeletal muscle Ca2+ channel was affected by prior exposure of the T-tubule membranes to either the dihydropyridine activator Bay K 8644 or the inhibitor (+)-PN 200-110 under the conditions tested (Fig. 2).

Biochemistry: Hosey et al. c

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Proc. Natl. Acad. Sci. USA 83 (1986)

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Phosphorylation of the Ca21 Channel by a Purified Ca2+/Calmodilin-Dependent Protein Kinase. T-tubule mem-

FIG. 2. Time course of phosphorylation of the major component of the Ca2l channel in T-tubule membranes by cAMP-dependent protein kinase in the presence and absence of dihydropyridines. T-tubule membranes (1 mg/ml) were preequilibrated with buffer only (control), 1 ,uM Bay K 8644, or 1 MiM (+)-PN 200-110 for 10 min at ambient temperature. The membranes were phosphorylated and the reactions were terminated by adding aliquots to NaDodSO4 sample buffer. The samples were electrophores~d on 5% gels and the radioactivity in the Ca2+-channel protein was quantitated by autoradiography and by direct counting of gel slices corresponding to the Ca2+-channel. (Left) The 140-kDa peptide. (Right) The 160-kDa peptide.

parent molecular masses of 5.4 kDa. The sizes of the phosphorylated proteolytic fragments generated from either the band migrating at 140 or 160 kDa were also similar (Fig. 4). Digestion of the 55- and 60-kDa phosphopeptides with the S. aureus V8 protease also yielded phosphopeptides of 5.4 kDa (Fig. 4). The results suggest that the 55- and 60-kDa phosphopeptides may be related to, or derived from, the 140/160-kDa peptide. Dephosphorylation of the Phosphorylated Ca2+ Channel by Calcineurin. T-tubule membranes were phosphorylated with either the cAMP- or Ca2+/calmodulin-dependent protein kinase, extensively washed, and then exposed to dephosphorylation conditions in the presence or absence of the phosphoprotein phosphatase calcineurin (25, 26). All reactions were evaluated by analyzing the phosphopeptide content of T-tubule membranes electrophoresed in the presence of iodoacetamide. Calcineurin catalyzed a time-dependent dephosphorylation of the 160-kDa protein previously phosphorylated by either protein kinase (Fig. 5). The results shown in Fig. S were obtained with 0.4,uM calcineurin. When the concentration of the phosphatase was lowered to 0.07 MtM, the phosphatase showed greater specificity for the Ca2+ channel than for other T-tubule proteins (not shown). The calcineurin-catalyzed dephosphorylation of the Ca2+-channel protein was stimulated 2- to 3-fold by Ca2 /calmodulin (data not shown). Ca2+/calmodulin had no effect on the dephosphorylation observed in the absence of calcineurin. Loss of 32p from a phosphopeptide could be due to either dephosphorylation or limited proteolysis of a protein. To check against the latter possibility we determined if the Ca2+ channel that had been phosphorylated and subsequently dephosphorylated by calcineurin could then be rephosphorylated. The results showed that the Ca2+ channel could be fully rephosphorylated after the calcineurin treatment (data

branes were phosphorylated under various conditions with and without the Ca2+/calmodulin-dependent protein kinase. The Ca2" channel was subsequently solubilized, purified by WGA-Sepharose chromatography, and analyzed by electrophoresis. The results showed that the 140/160-kDa protein was phosphorylated in the presence of the Ca2+/calmodulindependent protein kinase in a manner that was completely dependent on the presence of Ca2' plus calmodulin (Fig. 3). The T-tubule membranes themselves possessed an endogenous protein kinase activity that phosphorylated the 140/160kDa protein in reactions that did not contain EGTA. The activity was not increased by addition of Ca2+/calmodulin but was suppressed by the exogenous protein kinase, which carried 10 ,M EGTA into the reactions. The stoichiometry of incorporation catalyzed in the presence of the calmodulindependent protein kinase was 0.2-0.4 mol of phosphate per mol of protein. (Because of the limited availability and stability of the kinase, it was not determined if a higher stoichiometry could be achieved under other conditions.) A peptide of 55 kDa was also phosphorylated in the presence of the calmodulin-dependent kinase and copurified with the 140/160-kDa peptide on the WGA-Sepharose column (Fig. 3). This protein was not a subunit of the synapsin I kinase because it was also observed in the T-tubule membranes phosphorylated in the absence of the kinase (Fig. 3), and the kinase should not absorb to the WGA-Sepharose column. Identification of the Site(s) Phosphorylated by the cAMPand Ca2+/Calmnodulin-Dependent Protgin Kinases. Limited proteolytic digestion with Staphylococcus aureus V8 protease (33, 34) of the 140/160-kDa channel protein that had been phosphorylated in the presence of either or both of the protein kinases generated similar phosphopeptides with ap2-mercaptoethanol

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FIG. 3. Phosphorylation of the major component of the skeletal muscle Ca2l channel in T-tubule *- 68 membranes by a purified Ca2+-calmodulin-dependent protein kinase. T-tubule membranes (0.75 mg * * per reaction) were phosphorylated in the presence and absence of Ca2+/calmodulin and/or the Ca2+/calmodulin-dependent protein kinase as indicated. The Ca2+-channel protein was solubilized - 31 with CHAPS, purified by WGA-Sepharose chro_ matography; gnd electrophoresed on a 4-14% gel. do The autoradiogram of the purified fractions is shown. The specific activity of the [y32P]ATP was - + - 4 400 cpm/pmol. The autoradiogram was prepared after a 24-hr exposure. Molecular masses are given 97

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Biochemistry: Hosey et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

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DISCUSSION The relevant findings of this study are that a protein similar or identical to the major component of the dihydropyridinesensitive Ca2" channel in skeletal muscle membranes can be (i) efficiently phosphorylated in its membrane-bound state in the presence of either the cAMP-dependent and/or a Ca2+/calmodulin-dependent protein kinase and (ii) dephosphorylated by the Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin. These findings have potential physiological relevance since dihydropyridine-sensitive voltage-dependent Ca2+ channels are regulated in situ by cAMP-dependent and Ca2+-dependent events (2, 3, 21). The coidentity of the 140/160-kDa phosphoprotein and the major component of the Ca2" channel was suggested by several criteria. (i) The phosphoprotein and stained protein copurified on WGA-Sepharose under conditions previously established to purify the dihydropyridine receptor (17, 19). (it) The stained protein and phosphoprotein migrated on 4-14% gradient gels with an apparent molecular mass of 140 kDa when analyzed in the presence of 2-mercaptoethanol or 160 kDa when analyzed in the presence of iodoacetamide. (iii) The phosphoprotein and the stained protein comigrated on NaDodSO4 gels of different acrylamide composition (not shown). Since glycoproteins migrate anomalously on NaDodSO4 gels (37), different glycoproteins would not be expected to migrate identically on different gels. (iv) The stained protein and phosphoprotein were immunoprecipitated by specific monoclonal antibodies raised against the purified dihydropyridine receptor (S. Vandaele, M. Fosset, M.M.H., and M.L., unpublished data). Consequently, we conclude that the 140/160-kDa protein phosphorylated in the T-tubule membranes is identical to or closely associated with the major component of the dihydropyridine-sensitive Ca2" channel of skeletal muscle.

+

FIG. 4. Phosphopeptides generated by limited proteolytic digestion of the Ca2l-channel protein phosphorylated by the cAMP-dependent and/or the Ca2+/calmodulin-dependent protein kinase. Ttubule membranes (1 mg per reaction) were phosphorylated with either the cAMP-dependent protein kinase (indicated by "cAMP") or the Ca2+/calmodulin-dependent kinase (indicated by "Ca2+/calmodulin") or by both kinases simultaneously in a reaction volume of 1 ml. The phosphorylated Ca2l channel was solubilized and purified by WGA-Sepharose. (Upper) Autoradiograms prepared from 4-14% gels in which the purified protein was electrophoresed in either the presence of 2-mercaptoethanol (A) or iodoacetamide (B). The specific activity of the [y-32P]ATP was 1300 cpm/pmol. The autoradiogram was prepared after a 24-hr exposure. The bands corresponding to the 140-kDa phosphopeptides in A, the 160-kDa phosphopeptides in B, and the 60- and 55-kDa peptides in B (lane 3) were excised and subjected to digestion with S. aureus V8 protease (0.02 mg) in a second gel as described (33). (Lower) Resulting autoradiogram ("Proteolytic Phosphopeptides"). The origin of each proteolytic fragment is given in the legend. Molecular masses are given in kDa. The apparent molecular mass of the proteolytic fragment in each case was 5.4 kDa.

The phosphorylation of the 140/160-kDa Ca2l-channel protein by cAMP-dependent kinase described here contrasts to a report by Curtis and Catterall (24) in which it was concluded that this protein was not phosphorylated in its membrane-bound state. Their result was based on detection of the phosphorylated Ca2+ channel in isoelectric focusing gels. However, it is not clear that the Ca2+ channel was focused under the conditions used (24). In addition, the membranes used by Curtis and Catterall contained significantly fewer dihydropyridine receptors (6 pmol/mg of protein, ref. 18) than the membranes used in the present study (40-90 pmol/mg of protein, refs. 15 and 27). We have found in our own studies with microsomal membranes, which have 4-10 pmol of dihydropyridine receptors per mg of protein, that it is more difficult to detect the phosphorylated Ca2+ channel (data not shown). This is because the cruder membranes contain (i) fewer Ca2+ channels, (ii) other phosphoproteins of 140-150 kDa, which are not related to the Ca2+ channel (24), and (iiM) phosphatases that dephosphorylate the Ca2' channel. In spite of these difficulties, phosphorylation of the Ca2+ channel in the cruder membranes can be shown by purifying the phosphorylated Ca2+ channel in the presence of phosphatase inhibitors and by analyzing the channel under conditions in which it is resolved from the non-Ca2'-channel proteins (C. Cooper, C. O'Callahan, and M.M.H., unpublished data). It is conceivable that phosphorylation/dephosphorylation reactions similar to the in vitro reactions described in this study may also occur in skeletal muscle in vivo. The skeletal muscle Ca2+ channel is activated in intact cells by cAMP (23). The concentrations of ATP and cAMP-dependent protein kinase used in the in vitro phosphorylation reactions were less than or equal to those found in normal muscle cells (38). Calmodulin (39) and a Ca2+/calmodulin-dependent kinase (40) similar to the kinase used in this study (41) also exist in skeletal muscle cells and thus could cause phosphorylation of the Ca2' channel in the intact cell. It has been previously suggested that the Ca2+ channel may be inactivated in vivo by dephosphorylation (3, 21). We showed herein that the Ca2+-

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Biochemistry: Hosey et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

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1. Carafoli, E. (1984) Adv. Cyclic Nucleotide Protein Phosphorylation Res. 17, 543-549. 2. Tsein, R. W. (1983) Annu. Rev. Physiol. 45, 341-358. 3. Reuter, H. (1983) Nature (London) 301, 569-574. 4. Baker, P. F., Knight, F. R. S. & Knight, D. E. (1981) Philos. Trans. R. Soc. London Ser. B 296, 83-103. 5. Kazazoglou, T., Schackmann, R. W., Fosset, M. & Shapiro, B. M. (1985) Proc. Natl. Acad. Sci. USA 82, 1460-1464. 6. Mazurek, N., Schindler, H., Schurholz, T. L. & Pecht, I. (1984) Proc. Natl. Acad. Sci. USA 81, 6841-6845. 7. Hess, P., Lansman, J. B. & Tsien, R. W. (1985) Nature (London) 316, 443-446. 8. Fleckenstein, A. (1983) Circ. Res. 53, Suppl. I, 3-16. 9. Janis, R. A. & Triggle, D. J. (1984) Drug Dev. Res. 4, 257-274. 10. Glossman, H. & Ferry, D. R. (1985) Methods Enzymol. 109, 513-550. 11. Miller, R. J. & Freedman, S. B. (1984) Life Sci. 34, 1205-1221. 12. Fosset, M. & Lazdunski, M. (1986) Recept. Biochem.

+

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+

+

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0

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45

Dephosphorylation of the major component of the skelby calcineurin. T-tubule membranes were phosphorylated by either the cAMP-dependent protein kinase (Upper) or the Ca2'/calmodulin-dependent protein kinase (Lower), washed three times, and then subjected to dephosphorylation conFIG. 5.

etal muscle Ca2+ channel

ditions. Ca2+/calmodulin

was

present in all reactions shown and

specific activities of the cpm/pmol (Upper) and 760 cpm/pmol (Lower).- The autoradiograms were prepared after a 2-day exposure (Upper) and a 5-day exposure (Lower). Molecular mass is given in

calcineurin

[y-32p]ATP

was

present

used

were

as

indicated. The

360

kDa.

dephosphorylated by calcineurin, a phosphatase. Although calcineurin is found primarily in neuronal cells (26), a calcineurinlike phosphatase (phosphatase 2B) is known to be present in relatively high levels in skeletal muscle (25, 42). In view of the

channel

protein

was

Ca2+/calmodulin-dependent

present results, it

seems

reasonable to suggest that the in vivo

regulation of the Ca2+ channel by phosphorylation/dephosphorylation reactions may be mediated at the level of the major component of this channel. Future studies will determine if such reactions

occur

in intact cells and if the electrical

activity of the skeletal muscle Ca2+ channel can be regulated by the cAMP- and Ca2+-dependent protein kinases and by

calcineurin. We

thank Dr. C. B. Klee

helpful

advice,

membranes, work

and

Dr.

C.

M.

for

Fosset

Johnson

Caj2-dependent

and

colleagues for

enzymes the

Centre

National de la Recherche

des Myopathes de France, and Health (Grant HL 23306). M.M.H. is

National Institutes of Established Investigator

and

T-tubule

for expert secretarial assistance. This

supported by the Scientifique, the Association was

the

of the American Heart

3737

Association.

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