Sorcin Associates with the Pore-forming Subunit of Voltage-dependent L-type Ca2+ Channels*

Marian B. MeyersDagger §, Tipu S. Puri, Andy J. Chien, Tianyan Gao, Pei-Hong HsuDagger , M. Marlene Hosey, and Glenn I. FishmanDagger parallel

From the Dagger  Department of Medicine, Cardiovascular Institute, Mount Sinai School of Medicine, New York, New York 10029 and the  Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intracellular Ca2+ release in muscle is governed by functional communication between the voltage-dependent L-type Ca2+ channel and the intracellular Ca2+ release channel by processes that are incompletely understood. We previously showed that sorcin binds to cardiac Ca2+ release channel/ryanodine receptors and decreases channel open probability in planar lipid bilayers. In addition, we showed that sorcin antibody immunoprecipitates ryanodine receptors from metabolically labeled cardiac myocytes along with a second protein having a molecular weight similar to that of the alpha 1 subunit of cardiac L-type Ca2+ channels. We now demonstrate that sorcin biochemically associates with cardiac and skeletal muscle L-type Ca2+ channels specifically within the cytoplasmically oriented C-terminal region of the alpha 1 subunits, providing evidence that the second protein recovered by sorcin antibody from cardiac myocytes was the 240-kDa L-type Ca2+ channel alpha 1 subunit. Anti-sorcin antibody immunoprecipitated full-length alpha 1 subunits from cardiac myocytes, C2C12 myotubes, and transfected non-muscle cells expressing alpha 1 subunits. In contrast, the anti-sorcin antibody did not immunoprecipitate C-terminal truncated forms of alpha 1 subunits that were detected in myotubes. Recombinant sorcin bound to cardiac and skeletal HIS6-tagged alpha 1 C termini immobilized on Ni2+ resin. Additionally, anti-sorcin antibody immunoprecipitated C-terminal fragments of the cardiac alpha 1 subunit exogenously expressed in mammalian cells. The results identified a putative sorcin binding domain within the C terminus of the alpha 1 subunit. These observations, along with the demonstration that sorcin accumulated substantially during physiological maturation of the excitation-contraction coupling apparatus in developing postnatal rat heart and differentiating C2C12 muscle cells, suggest that sorcin may mediate interchannel communication during excitation-contraction coupling in heart and skeletal muscle.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The release of Ca2+ from muscle sarcoplasmic reticulum (SR)1 is the principal link between electrical excitation of the sarcolemma and mechanical activation of the myofilaments, a process known as excitation-contraction (E-C) coupling. Ca2+ release from stores in the cardiac SR occurs via Ca2+ release channels that are referred to as ryanodine receptors (RyRs). In the heart, Ca2+ release from the SR is largely triggered by Ca2+ influx at the plasma membrane via voltage-dependent L-type Ca2+ channels that are also dihydropyridine receptors (DHPRs) (1-7). In skeletal muscle, DHPRs serve as voltage sensors to detect depolarization of the sarcolemmal transverse tubules (T-tubules) and provide the physical impetus for opening of the SR Ca2+ release channels (1, 8-11). In both tissues, the geometry and close spatial relationship between sarcolemmal L-type channels and SR RyRs is critical in determining the time course of Ca2+ release and E-C coupling; however, the molecular mechanisms that mediate interchannel communication are incompletely understood (1, 12-14). The possibility that additional proteins or factors might be interposed to facilitate cross-talk has often been suggested (1, 8, 12).

Sorcin, a 22-kDa Ca2+-binding protein first identified in multidrug-resistant cells, is widely distributed among mammalian tissues, including heart and skeletal muscle (15-19). At the subcellular level, sorcin localizes to T-tubule junctions of cardiac SR (19) and co-localizes with brain RyR in rat brain caudate-putamen nucleus (20, 21). We previously demonstrated that introduction of sorcin into nonmuscle cells confers the property of caffeine-activated intracellular Ca2+ release, suggesting a role for sorcin in modulating RyR function (19). This hypothesis was further strengthened by the demonstration that sorcin completely inhibits ryanodine binding to cardiac RyRs and substantially decreases the open probability of the Ca2+ release channels reconstituted in lipid bilayers (22). Sorcin is, therefore, one of a group of modulators of RyR gating that includes calmodulin and FK506-binding protein, ligands that bind to a RyR domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor (23-27).

Anti-sorcin antibody immunoprecipitates two proteins from metabolically labeled cardiac myocytes (19), one of which we previously identified as the 565-kDa cardiac RyR (19), and the other (~220 kDa) displayed an electrophoretic mobility similar to that of the major, pore-forming alpha 1 subunit of the cardiac L-type Ca2+ channel (alpha 1C) (28, 29). Here we directly test the possibility that the unidentified protein is alpha 1C by analyzing the interaction of sorcin and the alpha 1 subunits of cardiac and skeletal muscle Ca2+ channels. The ability of sorcin to interact with sarcolemmal L-type channels, in concert with its modulatory effect on RyR gating, positions sorcin as a candidate regulator of interchannel cross-talk.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Lines and Cultured Cardiac Myocytes-- COS-1 cells were obtained from ATCC (Rockville, MD) and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Culture conditions for human embryonic kidney (HEK) 293 and Sf9 insect cells have been described (29-31). Mouse C2C12 myoblasts (32-34) were maintained in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and were transferred to Dulbecco's modified Eagle's medium containing 2% horse serum to initiate differentiation, a process that serves as a model of normal myogenesis. Formation of multinucleated myotubes was observed within 48 h after medium transfer. Preparation of rat cardiac myocytes was carried out according to published procedures (19, 35).

Membrane Preparations-- Sf9 and HEK 293 cell membranes were prepared as described previously (29, 30). Fractionation of rat heart tissue for soluble and crude membrane components was carried out by differential centrifugation according to our published procedures (29). In brief, rat heart tissue was homogenized in buffer containing 0.25 M sucrose, 0.25 M KCl, 10 mM imidazole (pH 7.4), 5 mM MgCl2, 10 mM EDTA, and protease inhibitors. After centrifugation at 5000 × g for 10 min, supernatants were removed and frozen, and pellets were quickly washed with lysis buffer containing 0.6 M KCl to extract myosin, which obscures alpha 1c on gels. Washed pellets were solubilized and analyzed for alpha 1c content, and supernatants were analyzed for sorcin by Western blot as described below. In some experiments, heart tissue was lysed by sonication in 50 mM Tris (pH 7.4) containing 1% Nonidet P-40, 150 mM NaCl, 20 mM NaF, 10 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride (buffer A).

Antibodies and Expression Vectors-- Preparation and characterization of sorcin antibodies (19); of SKN, SKC, Card I, and Card C antibodies; and of alpha 1C and alpha 1S expression vectors has been described previously (30, 36). Briefly, the SKN and SKC antibodies recognize N-terminal and C-terminal domains on the alpha 1S subunit (36), while the Card I and Card C antibodies recognize internal and C-terminal domains of the alpha 1C subunit (30). The RyR antibody was a generous gift of Dr. Gerhard Meissner (University of North Carolina) (37). The anti-myosin heavy chain (MHC) antibody F59 has been described (34). Anti-polyhistidine was purchased from Sigma, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used according to the manufacturer's directions.

Western Blot Analysis and Immunoprecipitation-- Soluble fractions of cultured cells and tissues, as well as solubilized membrane fractions (described above) were prepared for analysis in buffer A. Samples containing 60 µg of protein (38) were separated by gel electrophoresis on 6% (for analysis of alpha 1 subunits, RyR, and MHC) or 11% (for sorcin and HIS6-tagged proteins) polyacrylamide gels under denaturing conditions (39). Proteins were transferred to nitrocellulose, and the membranes were processed for Western blotting with detection by chemiluminescence (Amersham Pharmacia Biotech) as described previously (19). Additional details are given in the figure legends. For immunoprecipitation assays, aliquots of protein samples in buffer A containing 100 or 200 µg of protein in a total of 0.25 ml of buffer A were incubated with 1 µg of antibody raised against peptides from either the N or C terminus of sorcin (19) for 1 h at 4 °C. Antigen-antibody complexes were precipitated with protein G (Sigma), washed with buffer A, and solubilized in Laemmli (39) buffer at room temperature for 15 min. Immunoprecipitated proteins were analyzed by Western blot as described above and in the figure legends. Molecular weight markers were purchased from Amersham Pharmacia Biotech or Bio-Rad.

In Vitro Binding Assays-- Fusion proteins of the L-type Ca2+ channel alpha 1C and alpha 1S C-terminal domains were expressed in bacteria with vectors constructed by ligation of a genomic BglII-BamHI fragment of alpha 1C (GenBankTM accession number X15539) or alpha 1S (GenBankTM accession number X05921) into the BamHI site of pQE (QIAGEN, Valencia, CA). The proteins were expressed with an N-terminal HIS6 tag fused to amino acids 1622-2171 (40) of the alpha 1C C terminus or to amino acids 1497-1873 (40) of the alpha 1S C terminus. For immobilization of fusion peptides on Probond Ni2+ resin (Invitrogen Corp., San Diego, CA), bacterial lysates containing the fusion proteins were prepared according to the manufacturer's directions by sonication and freeze-thaw in phosphate-buffered saline (PBS) at pH 7.8 (binding buffer). Cleared supernatants were applied to washed resin beds by batch absorption, and treated resin samples were washed three times with binding buffer, twice with PBS at pH 6.0, and three times with PBS at pH 7.4. Aliquots of 10 µg of recombinant sorcin (41) in PBS (pH 7.4) were then combined with the protein-containing resins for 30 min at 4 °C. Resins were washed three times with PBS (pH 7.0), and bound proteins were eluted in 0.5 M imidazole. HIS6-tagged p27, a cyclin-dependent kinase inhibitor (42), was used as a negative control. Eluted proteins were analyzed by Western blot after electrophoresis on 11% acrylamide. Nitrocellulose membranes containing transferred proteins were sequentially probed with mouse monoclonal anti-polyhistidine antibody and then with rabbit polyclonal antibody to a peptide from the C terminus of sorcin (19).

Preparation and Transfection of HIS6-tagged C-terminal Fragments of alpha 1C-- A fragment (designated fragment A) of the C-terminal cytoplasmic domain of alpha 1C (amino acids 1622-2171) (41) was removed from the alpha 1C expression vector (30) and cloned in frame into the pHISA expression vector (Invitrogen), which added 30 amino acids, including the His6 epitope, at the N terminus. Fragments B (amino acids 1622-1872), C (amino acids 1749-1978), D (amino acids 1622-1772), and E (amino acids 1622-1748) from the alpha 1C C terminus were prepared by polymerase chain reaction and cloned into pHISA. Polymerase chain reaction primers included restriction sites to facilitate subsequent cloning. The following primer pairs were used: fragment B, sense GGAAACCTGGAACAAGCCAAT (also used for fragments D and E) and antisense GCTCTAGATCCTCACGTCGTAGTTGTC; fragment C, sense GCGGATCCTCCCCCAGACCTTCACTACG and antisense GCTCTAGAGCGAGGTGGGGGAATGGCT; fragment D, antisense GCTCTAGACTAGGAGTCCACCAGCTTCTCGTG; and fragment E, antisense GCTCTAGATCCTCACGTCGTAGTTGTC. Plasmids encoding the fragments were transiently co-transfected with the full-length sorcin vector pFRCMVSOR (19) in COS-1 cells or alone in HEK cells with the use of lipofectamine (Life Technologies, Inc.). Association of sorcin and fragments was studied by immunoprecipitation with sorcin antibody and Western blot analysis.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Sorcin Antibody Immunoprecipitates alpha 1C-- We previously showed that sorcin antibody recovered two proteins from rat cardiac myocytes metabolically labeled with [35S]methionine, a 565-kDa protein shown to be the RyR and an unidentified ~220-kDa protein (19). Here we demonstrate that a single protein recognized by Card I, an alpha 1C-specific antibody shown to recognize the 240-kDa alpha 1C (29, 30), was immunoprecipitated from rat heart tissue by sorcin antibody (Fig. 1A, lane 1). Card C, an antibody directed against the C terminus of alpha 1C (30), also recognized this protein (see Fig. 4). Results of immunoprecipitations carried out in the absence of Ca2+ (not shown) were indistinguishable from those shown in Fig. 1A. Rat heart contained the 22-kDa sorcin, which co-migrated with recombinant sorcin (41) (Fig. 1B, lane 1), and an 18-kDa species (Fig. 1B, lane 1). Both sorcin forms were detected in most rat (and mouse) heart samples; tissues from some animals contained only the 22-kDa form (19).


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Fig. 1.   Sorcin antibody immunoprecipitated endogenously expressed alpha 1C and alpha 1S from heart tissue and C2C12 cell myotubes, respectively, and heterologously expressed alpha 1 subunits from Sf9 and HEK 293 cells. A, Western blots with Card I antibody. Immunoprecipitation of 200-µg samples of membrane proteins from rat heart (lane 1), alpha 1C-expressing Sf9 (lane 2), uninfected Sf9 (lane 3), alpha 1C-expressing Sf9 (lane 4), untransfected HEK 293 (lane 5), and alpha 1C-expressing HEK (lane 6) was carried out with an antibody directed against a peptide from the C terminus of sorcin (19) (antibody to a peptide from the N terminus of sorcin produced equivalent results) for lanes 1 and 3-6 and with preimmune serum for lane 2. Nitrocellulose membranes containing immunoprecipitated proteins were blocked, incubated with Card I in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 1 h at room temperature, washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody in TBST before detection by chemiluminescence. B, Western blot with antibody directed against a peptide from the C terminus of sorcin. Aliquots containing 60 µg of protein from soluble fractions of rat heart (lane 1), HEK 293 cells (lane 2), and Sf9 cells (lane 3) lysed in buffer A were fractionated on 11% polyacrylamide gels before Western analysis as described previously (19). The arrow indicates 22-kDa sorcin, which comigrated with recombinant sorcin. C, Western blots with SKN antibody (lanes 1-4 and 7-11) or SKC (directed against the alpha 1S C terminus) (lanes 5 and 6). Lanes 1, 2, 5, and 6 show soluble proteins (60 µg/lane) from C2C12 cells lysed in buffer A (see "Materials and Methods"). Myoblasts (lanes 1 and 5) and fully differentiated myotubes (lanes 2 and 6) were analyzed. Lanes 3 (blasts) and 4 (myotubes) show proteins immunoprecipitated with sorcin antibody from 200 µg of C2C12 cell protein. Immunoprecipitation of 200-µg samples of membrane proteins from uninfected Sf9 (lane 7), alpha 1C-expressing Sf9 (lane 8), alpha 1S-expressing Sf9 (lane 9), untransfected HEK 293 (lane 10), or alpha 1S-expressing HEK cells (lane 11) with sorcin antibody is depicted. See the Fig. 1A legend for Western blot conditions.

To confirm the identity of the cardiac protein recognized by Card I and Card C, we examined whether alpha 1 subunits could be immunoprecipitated with sorcin antibody from Sf9 cells infected with a recombinant baculovirus directing expression of alpha 1C or from HEK 293 cells transiently expressing full-length alpha 1C. This ability would require the presence of endogenous sorcin species in those nonmuscle cells. We found that sorcin antibody recognized an 18-kDa protein in HEK 293 cells (Fig. 1B, lane 2), similar in size to the 18-kDa protein in heart, and an ~30-kDa protein in Sf9 cells (Fig. 1B, lane 3). The 18-, 22-, and ~30-kDa bands in Fig. 1B were not detected on Western blots with antibody preincubated with the antigenic sorcin peptide (data not shown). Card I recognized a protein immunoprecipitated by anti-sorcin antibody from alpha 1C-expressing Sf9 (Fig. 1A, lane 4) or HEK 293 cells (Fig. 1A, lane 6). The immunoprecipitated bands co-migrated with bands detected by Card I by direct Western blot of proteins from Sf9 and HEK 293 cells heterologously expressing the alpha 1C subunit (data not shown). No specific proteins identified by Card I were recovered by immunoprecipitation of alpha 1C-expressing Sf9 cells with preimmune serum (Fig. 1A, lane 2) or by immunoprecipitation of uninfected Sf9 (Fig. 1A, lane 3) or untransfected HEK cells (Fig. 1A, lane 5) with anti-sorcin antibody. These results strongly suggested that a heretofore unidentified protein immunoprecipitated with sorcin antibody from metabolically labeled cardiac myocytes (19) was alpha 1C.

The cardiac alpha 1C subunit has been previously reported to exist in two forms in isolated cardiac membranes (29, 43). A minor fraction of the cardiac alpha 1C is the full-length protein of ~240 kDa that can be recognized by both the Card I and Card C antibodies (29), while the major fraction of alpha 1C in cardiac membranes is a C-terminal truncated protein of ~190 kDa that reacts with Card I but not Card C (29). Interestingly, in the experiments described here, only the full-length form of the alpha 1C was immunoprecipitated by the anti-sorcin antibody (Fig. 1A), suggesting a potential role of the C terminus for the alpha 1C/sorcin interaction.

Sorcin Antibody Immunoprecipitates Full-length alpha 1S-- We next addressed the question of whether the alpha 1C/sorcin association was alpha 1 isoform-specific. SKN, an alpha 1S-specific antibody generated against the N terminus of alpha 1S and shown to recognize full-length (214-kDa) and C-terminal truncated (170-190-kDa) forms of alpha 1S (31, 36, 44), recognized three proteins in C2C12 myotubes (Fig. 1C, lane 2). None of these proteins were detected in undifferentiated C2C12 myoblasts (Fig. 1C, lane 1); up-regulation of the alpha 1S subunit during skeletal muscle development has been established (45, 46). The anti-sorcin antibody immunoprecipitated only the most slowly migrating form that was detected in myotubes, while lower Mr forms that were reactive with SKN were not recovered (Fig. 1C, lane 4). The SKC antibody, directed against the alpha 1S C terminus, recognizes the full-length 214-kDa alpha 1S subunit but does not detect the C-terminal truncated forms of alpha 1S that are commonly observed in skeletal muscle (44, 47). In the present experiments, SKC recognized only the full-length subunit in C2C12 myotubes (Fig. 1C, lane 6), a band of similar size recovered by sorcin antibody (Fig. 1C, lane 4). The Mr of full-length alpha 1S has been established by Ferguson plot analysis to be 214 kDa, although under most conditions it migrates anomalously as an ~190-kDa protein (44). From these data, we concluded that the lower Mr bands in C2C12 myotubes that were immunoreactive with SKN, but not with SKC, were C-terminal truncations of alpha 1S and that the sorcin antibody only immunoprecipitated full-length alpha 1S. These results suggested that an intact C terminus in alpha 1S was necessary for interaction with sorcin. As in the alpha 1C studies, we examined whether sorcin antibody would recover alpha 1S heterologously expressed in Sf9 and HEK 293 cells. SKN (and SKC, not shown) recognized a single protein immunoprecipitated by sorcin antibody from alpha 1S-expressing Sf9 (Fig. 1C, lane 9) and HEK 293 (Fig. 1C, lane 11) cells. The bands co-migrated with proteins recognized by SKN and SKC by direct Western blot (data not shown). No specific proteins identified by SKN were recovered by sorcin antibody immunoprecipitation of uninfected Sf9 (Fig. 1C, lane 7), Sf9 infected with alpha 1C (Fig. 1C, lane 8), or untransfected HEK cells (Fig. 1C, lane 10). The results are consistent with the interpretation that sorcin associates with the alpha 1S and alpha 1C subunits and that an intact C terminus in each protein is necessary for this interaction.

Association of Sorcin with alpha 1 C Terminus Domains-- In order to test the hypothesis that sorcin interacts with C-terminal domains of alpha 1C and alpha 1S, HIS6-tagged C terminus fragments of alpha 1C and alpha 1S were immobilized on Ni2+ resin and incubated with recombinant sorcin protein. As shown in Fig. 2, recombinant sorcin (22 kDa) specifically bound to immobilized alpha 1S (Fig. 2, lane 4) and alpha 1C (Fig. 2, lane 6) C termini (57 and 70 kDa, respectively). In contrast, no specific association of sorcin with the irrelevant HIS6-tagged p27 protein was observed (Fig. 2, lane 2).


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Fig. 2.   Recombinant sorcin bound to HIS6-tagged alpha 1C and alpha 1S C termini (amino acids 1622-2171 and amino acids 1497-1873, respectively) immobilized on Ni2+ resin. Western blots with anti-polyhistidine (upper panel) and sorcin antibody (lower panel) are shown. Recombinant sorcin was incubated with bacterially expressed HIS6-tagged proteins bound to Ni2+ resins as described under "Materials and Methods." Resins were washed and eluted with 0.5 M imidazole. Final wash fractions before elution are shown in lanes 1, 3, and 5, and eluted proteins are shown in lane 2 (negative control protein HIS6-tagged p27), lane 4 (alpha 1S C terminus (C-term) and sorcin), and lane 6 (alpha 1C C terminus and sorcin). Eluted proteins were fractionated on 11% acrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked, incubated with anti-polyhistidine, washed, incubated with horseradish peroxidase-conjugated goat anti-mouse antibody, and treated with materials for chemiluminescence detection. The membranes were then reblocked and probed with sorcin antibody followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody and detection by chemiluminescence as previously described (19). Sorcin signals are separated by a space that slightly exaggerates the appropriate distance between the 22-kDa recombinant sorcin signal and p27.

An association between sorcin and the alpha 1 subunit C terminus was confirmed and extended in a mammalian expression system. Several different HIS6-tagged fragments of the alpha 1C C terminus were generated and co-transfected with full-length sorcin into COS-1 cells or transfected alone into HEK cells for further delineation of the alpha 1C sorcin-binding domain. Sorcin antibody consistently immunoprecipitated fragment A, extending from amino acid 1622 to the carboxyl end of alpha 1C at amino acid 2171 (70 kDa) (Fig. 3, lane A). Two domains of fragment A, one extending from amino acid 1622 to 1978 and one from amino acid 1979 to 2171, were prepared and tested for sorcin association. The proximal fragment and not the distal C-terminal region was immunoprecipitated by anti-sorcin antibody (data not shown). The proximal region was then further examined by generating fragments B and C. Fragment B, extending from amino acid 1622 to 1872 (37 kDa), was readily recovered by sorcin antibody immunoprecipitation (Fig. 3, lane B). Fragment C, extending from amino acid 1748 to 1978 (39 kDa), was consistently unrecoverable by sorcin antibody (Fig. 3, lane C). Sorcin antibody immunoprecipitated fragments D (amino acids 1622-1772) (31 kDa) and E (amino acids 1622-1748) (26 kDa) (Fig. 3). Both direct Western (left lanes A-E) and immunoprecipitation/Western (right lanes A-E) analyses are shown in Fig. 3. We concluded that sorcin was associated with a domain of the alpha 1C C terminus within amino acids 1622-1748 (Fig. 3). While we did not construct similar fusion proteins derived from the alpha 1S C terminus, it is noteworthy that the cardiac and skeletal alpha 1 isoforms are highly homologous (40) in the domain implicated in the alpha 1/sorcin interaction.


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Fig. 3.   Sorcin antibody recovered HIS6-tagged alpha 1C C terminus fragments. COS-1 cells transiently co-transfected with full-length sorcin and alpha 1C fragments A, B, or C and HEK cells (expressing endogenous sorcin, Fig. 1B) transfected with fragments D and E were lysed in buffer A. Aliquots containing 30 µg of the soluble materials were fractionated on 11% acrylamide gels along with proteins immunoprecipitated from 100 µg of soluble cell proteins with antibody raised against peptides from the N or C terminus of sorcin (C terminus peptide antibody immunoprecipitation is shown here). After electrophoresis, proteins were transferred to nitrocellulose, and the membranes were incubated with anti-polyhistidine antibody as described above. Direct Western blot is shown in the first set of lanes labeled A-E, indicating the fragments depicted in the panel under the Western blot. Immunoprecipitated proteins are in the second set of lanes A-E. The fragments A (amino acids 1622-2171) (70 kDa), B (amino acids 1622-1872) (37 kDa), D (amino acids 1622-1772) (31 kDa), and E (amino acids 1622-1748) (26 kDa) were recovered by sorcin antibody. The shaded arrowhead indicates an IgG artifact. Sorcin's putative alpha 1C binding domain is within the margins of fragment E (amino acids 1622-1748). The data are representative of results of a group of experiments in which sorcin antibody immunoprecipitated the fragments shown from both COS and HEK transfected cells.

Increase in Sorcin Expression during Muscle Development-- To further probe potential relationships between sorcin and L-type Ca2+ channel alpha 1 subunits, we examined the developmental expression of sorcin during physiological maturation of E-C coupling in developing muscle. It is well established that the expression of sarcolemmal and SR channel proteins is regulated in concordance with a program of muscle maturation (1, 45, 46, 48-52). Sorcin abundance in postnatal rat heart substantially increased from day 1 to day 12 after birth (Fig. 4A). Both the 22- and 18-kDa species, depicted in Fig. 1B, increased in parallel. The alpha 1C subunit was readily detectable as early as postnatal day 1 and also increased in abundance during this time period (Fig. 4A), in agreement with previous reports (45).


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Fig. 4.   Sorcin expression during postnatal rat heart maturation. A, Western blots with Card C and sorcin antibodies. Rat cardiac membranes prepared from postnatal tissues on day 1 (lane 1), day 2 (lane 2), day 6 (lane 3), or day 12 (lane 4) were analyzed for full-length alpha 1C with Card C antibody, and soluble fractions were analyzed for sorcin with antibody raised against a peptide from the sorcin C terminus. Aliquots containing 60 µg of protein were analyzed in each lane. Membrane proteins were fractionated on 6% acrylamide and soluble proteins on 11% gels. The arrows indicate 22-kDa sorcin and the 18-kDa form. B, Western blot with sorcin antibody. Aliquots containing 60 µg of protein from soluble fractions of isolated rat cardiac myocytes lysed in buffer A 1 day (lanes 1 and 2), 2 days (lanes 3 and 4), and three days after plating (lanes 5 and 6) were analyzed. Lanes 2, 4, and 6 contain proteins from cells maintained in the presence of 10 mM verapamil (VRPL). The arrow indicates 22-kDa sorcin.

Sorcin was not present in neonatal rat cardiac myocytes 24 h after establishment in culture (Fig. 4B, lanes 1 and 2) but was detected on subsequent days as the cells commenced beating (Fig. 4B, lanes 3 and 5). In contrast to the intact heart (Fig. 1B, lane 1, and Fig. 4A) and freshly isolated adult cardiac myocytes (data not shown), only the 22-kDa form was observed in neonatal myocytes. We next examined whether contractile arrest, produced by treating cells with verapamil (51), would affect sorcin expression. As shown in Fig. 4B, lanes 4 and 6, the level of sorcin was substantially reduced in verapamil-arrested cells.

The 22-kDa sorcin (the 18-kDa form was not detected) also accumulated in differentiating mouse skeletal muscle C2C12 cells (Fig. 5). RyR expression increased in parallel with sorcin, whereas myosin heavy chain was only detected after a 48-h period in mitogen-depleted medium (Fig. 5), consistent with previous reports (33, 52). Full-length and C-terminal truncated forms of the alpha 1S subunit were expressed in early stages of differentiation and were up-regulated in parallel during progression to fully differentiated myotubes (Fig. 5, lanes 3 and 4).


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Fig. 5.   Western blots of C2C12 cells with antibodies raised against RyR (565 kDa), MHC (200 kDa), alpha 1S (214-kDa and truncated forms), and sorcin (22 kDa). Cells were lysed in buffer A, and 60 µg of protein were applied to each lane for each antibody. SKN antibody was used to detect alpha 1S, and other antibodies were described under "Materials and Methods." Myoblasts (lane 1), cells in mitogen-depleted medium for 6 or 24 h (lanes 2 and 3, respectively), and fully differentiated myotubes 48 h after mitogen depletion (lane 4) were analyzed. RyR, MHC, and alpha 1S were analyzed on 6% gels, and sorcin was analyzed on 11% gels. The legend to Fig. 1A and Ref. 19 describe additional Western blot conditions.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The voltage-sensing L-type Ca2+ channels in T-tubules play a major role in E-C coupling in both heart and skeletal muscle (1, 28). The close apposition of the L-type channels and RyRs in specific spatial relationships is required to accurately bridge the gap between SR and the sarcolemma for interchannel cross-talk (12-14). Indeed, a change in the relationship between SR Ca2+-release channels and sarcolemmal Ca2+ channels may be a defect in cardiac hypertrophy that contributes to contractile failure (53). Functional coupling between L-type Ca2+ channels and RyRs in neurons has also been demonstrated (54), and sensors for synaptic signaling just beneath sites of Ca2+ entry in the hippocampus have been proposed (55). In normal cardiac myocytes, the opening of voltage-activated L-type channels results in the development of a high local intracellular [Ca2+] in the microenvironment of the junctional space surrounding the sarcolemmal and SR membranes (56-59). How the local L-type channel-generated signals are transmitted to and activate RyRs is incompletely understood as is the potential retrograde signal (60) by which RyRs may affect L-type channel function. Proteins or factors interposed between the L-type Ca2+ channels and RyRs that might facilitate the relay of signals between them have been postulated (1, 8, 12). The results of the present study indicate that the Ca2+-binding protein, sorcin, associates with voltage-dependent Ca2+ channels. These new data, together with previous studies, which established that sorcin binds to and modulates RyR, suggest a role for sorcin in interchannel communication and E-C coupling.

Sorcin binds Ca2+ (Kd ~1 µM) and undergoes both a Ca2+-dependent decrease in intrinsic fluorescence and a Ca2+-mediated intracellular translocation from soluble to membrane components (41, 61). The protein inhibits binding of ryanodine to cardiac RyR and is a modulatory ligand for RyR gating (22). In contrast to sorcin's effect on cardiac RyR, sorcin increases binding of ryanodine to skeletal muscle RyRs, perhaps reflecting structural differences between the cardiac and skeletal muscle RyR isoforms (22).

Our finding that sorcin associates with the cytoplasmically oriented C-terminal domains of L-type Ca2+ channel alpha 1 subunits together with our previous demonstration that the addition of sorcin to the cytoplasmic side of cardiac RyRs in planar bilayers decreases the single channel open probability (22) places sorcin within the sarcolemmal/SR junctional space, a strategic location for facilitation of interchannel communication. The alpha 1C domain from amino acids 1622-1748 appeared to be sufficient for sorcin binding in vitro; however, the sorcin/alpha 1 interaction may be more complex in vivo. Shortened forms of alpha 1 subunits, reportedly cleaved at C-terminal residues distal to the delineated sorcin binding domain (43, 44), were not immunoprecipitated by sorcin antibody. This suggests that an intact C-terminal is required for sorcin interaction with the subunits in vivo and that the distal C-terminal regions may participate in stabilization of the sorcin/alpha 1 interaction.

Sorcin modulates RyR gating in planar bilayers, but whether it affects L-type Ca2+ channel function is not known. We have no evidence that sorcin antibody recovered functional channels. The cytoplasmically oriented C terminus of alpha 1C has been shown to be involved in Ca2+-sensitive channel inactivation, and a 142-amino acid segment from amino acids 1572-1717 in the alpha 1C C terminus has been suggested to be required for inactivation (62). The putative sorcin-binding domain delineated in this report is within the margins of the potential inactivation domain and suggests that sorcin's involvement in this aspect of L-type Ca2+ channel regulation should be investigated.

We found that sorcin expression increases in abundance in both developing heart and differentiating muscle cells. The contractile machinery in developing rodent heart is activated largely by Ca2+ entering the cell through L-type channels (1, 49). As the T-tubule/SR system gradually develops, contraction becomes more dependent on SR Ca2+ release activated by Ca2+ entry through sarcolemmal channels (49). The increase in sorcin expression in postnatal rat heart coincides with the developing Ca2+-induced Ca2+ release mechanism, consistent with a putative role for sorcin in L-type channel/RyR interchannel communication. The increase in sorcin expression during C2C12 myotube formation, commensurate with L-type Ca2+ channel and contractile apparatus development, is in accord with a role for sorcin in interchannel communication in skeletal muscle as well. A program of coordinate expression of myocyte proteins involved in contraction and Ca2+ regulation has been suggested (51). Treatment of heart cells with verapamil initiates divergent expression of proteins that may be involved in this program along with contractile arrest (51). Whether sorcin, whose expression was substantially reduced in verapamil-arrested myocytes, is a component of that Ca2+ regulation program remains to be determined.

Sorcin may undergo Ca2+-mediated dynamic changes in structure and subcellular localization, allowing it to either simultaneously or sequentially interact with L-type Ca2+ channels and RyR in response to changing Ca2+ levels. [Ca2+] changes in the sarcolemmal/SR microenvironment may result in conformational changes and translocation of sorcin from cytoplasmic to membrane locations (41, 61) in a manner analogous to the Ca2+-mediated conformational modification of recoverin (63). Ca2+-mediated dimerization of sorcin, shown to occur in vitro (60), may also occur in vivo. In addition, the recent demonstration of sorcin binding to annexin VII (64), thought to play a role in E-C coupling in skeletal muscle (65), and our demonstration that calmodulin exerts an additive effect on sorcin's inhibition of ryanodine binding to cardiac RyR (22) suggests that sorcin may function in conjunction with other channel accessory proteins. Whether these proteins might include L-type Ca2+ channel subunits other than alpha 1 is speculative; direct interaction between sorcin and other channel subunits was not in evidence.

Our studies demonstrating that sorcin interacts with both sarcolemmal L-type Ca2+ channels and SR Ca2+ release channels suggest a role for sorcin in interchannel communication. Sorcin may act as a sensor of the T-tubule junctional environment to ultimately participate in regulation of intracellular Ca2+ mobilization and E-C coupling.

    ACKNOWLEDGEMENT

We thank Dr. Richard Pestell of Albert Einstein College of Medicine for bacterial cultures expressing HIS6-tagged p27.

    FOOTNOTES

* This work was supported in part by a grant from the American Heart Association (to G. I. F.), by National Institutes of Health (NIH) Grant HL23306 (to M. M. H.), and by NIMH (NIH) National Research Service Award Grant 1 F30-MH10770 (to A. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Medicine, Cardiovascular Institute, Box 1030, Mount Sinai School of Medicine, One Gustave L. Levy Pl., New York, New York 10029-6574. Tel.: 212-241-5143; Fax: 212-860-7032; E-mail: marian_meyers{at}smtplink.mssm.edu.

parallel Established Investigator of the American Heart Association.

1 The abbreviations used are: SR, sarcoplasmic reticulum; DHPR, dihydropyridine receptor; E-C coupling, excitation-contraction coupling; HEK, human embryonic kidney; MHC, myosin heavy chain; RyR, ryanodine receptor; TBST, Tris-buffered saline with Tween 20; T-tubule, transverse tubule; PBS, phosphate-buffered saline.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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