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ISABELLE MARTY*, MYLtNE ROBERT*, MICHEL VILLAZ*, KAREN S. DE JONGHt, YVONNE LAIt, ..... Striessnig, J., Murphy, B. J. & Catterall, W. A. (1991) Proc.
Proc. Natl. Acad. Sci. USA Vol. 91, pp. 2270-2274, March 1994

Physiology

Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle (exdtatlon-contractlon couplng)-

ISABELLE MARTY*, MYLtNE ROBERT*, MICHEL VILLAZ*, KAREN S. DE JONGHt, YVONNE LAIt, WILLIAM A. CATTERALLt, AND MICHEL RONJAT*f *Laboratoire de Biophysique Mol6culaire et Cellulaire, D6partement de Biologie Mol6culaire et Structurale, CENG 85X, F-38041 Grenoble Cedex, France; and tDepartment of Pharmacology SJ-30, University of Washington, Seattle, WA 98195

Contributed by William A. Catterall, September 23, 1993

for the Ca2+ release from the sarcoplasmic reticulum (16). Ryanodine binding stoichiometry (one ryanodine binding site per tetramer of 565-kDa monomers), as well as sedimentation and structural data, indicates that the active form of the RyR is a homotetramer with an apparent mass close to 2000 kDa (14, 17, 18). Three different mechanisms have been proposed for excitation-contraction coupling in skeletal muscle: Ca2 -induced Ca2+ release, inositol 1,4,5-trisphosphate-induced Ca2W release, and direct physical coupling (for review see refs. 19 and 20). Current evidence favors a model in which a voltagedriven conformational change of the al subunit of the DHP-R activates Ca2+ release by the RyR through direct physical interaction (11, 21, 22). However, although this model is supported by freeze-fracture studies of muscle fibers showing regularly arranged particles (presumably the DHP-R) in the T-tubule membrane facing the RyR (23), a direct interaction between these two proteins has not been demonstrated. Using solubilized triad membranes and different antibodies directed against the al, a2, or (3 subunits of the DHP-R, or against the RyR, we demonstrate here the existence of a complex involving the DHP-R and RyR. We show specific immunoprecipitation of the RyR by antibodies directed against the DHP-R as well as immunoprecipitation of the DHP-R by antibodies directed against the RyR. The identification of the immunoprecipitated proteins was achieved either by Western blot analysis or by previous labeling of the proteins with [3H]ryanodine or with the high-affinity dihydropyridine antagonist [3H]PN200-110. The results support a model of direct physical linkage of DHP-R and RyR in excitation-contraction coupling.

Membrane vesicles enriched in both ryanoABSTRACT dine receptor and dihydropyridine receptor were obtained from rabbit skeletal muscle and solubilized with 3-[(3-

cholamidopropyl)dimethylammonioJ-1-propanesulfonate. Analysis of the sedimentatI behavior of the solublized proteins showed the existence of a population of al subunits of the dihydropyridine receptor which cosedimented with the ryanodine receptor. Solubilized proteins were lmmuno tated with antibodies died aist either the ryanodine receptor or the al, a2, or (3 subunIts of the dihydropyridine receptor. lInmunoprecipitated pr s were idenfied by Western blot analysis and by specific labeling with [3H]ryanoine or [3HJlPN200-110. Immupreipitation of the soubile proteIns with antibodies directed against the dihydroyridine receptor led to the comunoprecipitation of the ryanodine recepr. Conversely, Imngopecipitation with antide directed against the ryanodine receptor led to an immune both receptors, but these antibodies were complex cont unable to pcipate p dihydropyridine receptor. These results demonstrate that ryan e receptor and dihydropyridine receptor are present In the triad membrane preparation in ac which may play an important role in excitationcontraction coupling.

Depolarization of the transverse tubule (T-tubule) membrane of a skeletal muscle cell causes a rapid release of Ca2+ from the sarcoplasmic reticulum that induces contraction of the cell (1). This Ca2+ release has been studied in both isolated muscle fibers and preparations of sarcoplasmic reticulum vesicles and has been shown to be regulated in vitro by various effectors such as caffeine, ATP, Ca2+, ryanodine, and depolarization of the T-tubule membrane (2-4). Two proteins are known to be directly involved in the excitationcontraction coupling: the dihydropyridine receptor (DHP-R) located in the T-tubule membrane and the ryanodine receptor (RyR) located in the membrane of the terminal cisternae of the sarcoplasmic reticulum. The DHP-R is an L-type Ca2+ channel consisting of five subunits (5, 6): al, a2, (, y, and 8. Many of its functional properties have been assigned to the al subunit, including (i) binding of Ca2+ antagonist drugs (7-9), (ii) formation of a functional voltage-sensitive ion channel (10), and (iii) restoration and determination of the kinetics and cellular mecha-

MATERIALS AND METHODS Materials. Ryanodine was from Calbiochem, nitrendipine was from Sigma, and [ Hiryanodine and [3H1PN200110 were from NEN. Protein A-Sepharose 4B-CL was purchased from Pharmacia, 25I-labeled protein G and '25I-labeled anti-mouse IgG were from Amersham. All chemicals were reagent grade. . Triad vesicles Membrane Pre and were obtained by a modification of the method of Kim et al (24). All membrane preparations were carried out in the presence of the protease inhibitors leupeptin (10 pM), pepstatin (1 pAM), phenylmethanesulfonyl fluoride (200 pM), and EDTA (200 pLM). After low-speed centrifugation, the supernatant was poured through eight layers of cheesecloth, and

nism of excitation-contraction coupling (11). The other subunits increase expression and modulate the properties of the al subunit (for review see refs. 12 and 13). The RyR has been purified as a single polypeptide of 565 kDa (14, 15) and shown to be the Ca2+ channel responsible

Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonioj-1-

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.

propanesulfonic acid; RyR, ryanodine receptor; DHP-R, dihydropyridine receptor. tTo whom reprint requests should be addressed. 2270

Physiology: Marty et al. filtered through Whatman no. 4 paper, and incubated for 1 hr at 40C in the presence of 1 M NaCl. After a second centrifugation (40 min at 10,000 x g) the pellets were homogenized in buffer A (150 mM KCI/300 mM sucrose/20 mM Pipes, pH 7.1) containing 2.5 mM EGTA. The triad vesicles were collected by centrifugation (40 min at 17,000 x g), washed twice with buffer A, resuspended in buffer A at a protein concentration of 20-30 mg/ml, and stored in liquid nitrogen. Protein concentration was measured by the biuret method (25). Triad vesicles were solubilized for 30 min at 40C at a protein concentration of 2 mg/ml in a buffer containing 1.6% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), 0.9 M NaCl, 0.1% phospholipids (egg total phosphatide extract, Avanti Polar Lipids), 100 I&M CaCl2, 50 ,uM EGTA, 1 mM dithiothreitol, 20 mM Pipes (pH 7.1), and protease inhibitors (10 AM leupeptin, 2 ,uM pepstatin, 1 mM diisopropyl fluorophosphate, and 100 ,uM phenylmethanesulfonyl fluoride). Insoluble proteins were removed by centrifugation at 100,000 x g for 30 min at 4°C. Purification of RyR. The RyR was purified on a sucrose gradient as described by Lai et al. (14). The triad vesicles were solubilized, and the solubilized proteins were loaded on the top of a 5-20% sucrose gradient. After a 16-hr centrifugation at 26,000 rpm and 4°C in a Beckman SW28 rotor, the gradient was collected from the bottom. [3H]PN200-l1O Binding. Triad vesicles (protein, 1 mg/ml) were incubated for 2 hr at 23°C in 50 mM Tris-HCl, pH 7.5/50 ,uM CaC12/30 nM 13H]PN200-110. [3H]PN200-110 bound to the triad vesicles was measured by filtration through Whatman GF/B filters followed by three washes with 5 ml of ice-cold buffer containing 200 mM choline chloride and 20 mM Tris-HCl (pH 7.5). [3H]PN200-110 binding to solubilized proteins was determined by PEG precipitation (26). Nonspecific binding was determined in the presence of 50 ,uM nitrendipine. [3H]Ryanodine Binding. Triad vesicles (1 mg/ml) were incubated for 2 hr at 37°C in 15 mM Hepes/0.9 M NaCl/2.02 mM CaCl2/2 mM EGTA (pCa 4.5)/100 nM [3H]ryanodine at pH 7.4. [3H]Ryanodine binding to triad vesicles was measured by filtration through Whatman GF/B filter followed by three washes with 5 ml of ice-cold buffer composed of 15 mM Hepes (pH 7.4) and 0.5 M NaCl. [3H]Ryanodine binding to solubilized proteins was determined by PEG precipitation as for [3H]PN200-110. Nonspecific binding was determined in the presence of 100 ,uM ryanodine. Antibodies. The antibodies directed against RyR were obtained by immunization of a rabbit with purified pig (Pietrain normal) RyR. The rabbit received three intradermal injections at 3-week intervals, each consisting of 200 ,ug of RyR emulsified with Freund's complete adjuvant, followed by three intramuscular injections at 1-day intervals, each consisting of 200 ,ug of RyR emulsified with Freund's incomplete adjuvant. The rabbit was bled 2 weeks after the last

injection. Preparation and characterization of the monoclonal antibodies MAC9 and MANC-1, directed against the al and the a2 subunits of the DHP-R (anti-al DHP-R and anti-a2 DHP-R), respectively, were described previously (27, 28). The polyclonal anti-peptide antibody against the 8 subunit (anti-CB-1) of the DHP-R was generated by immunizing rabbits with a peptide corresponding to residues 203-219 of the rabbit skeletal muscle (3 subunit (29) plus N-terminal lysine and tyrosine residues. The methods for peptide conjugation, immunization, and affinity purification of the antibody were as described (30). Immunoprecipitation. Triad vesicles were solubilized as described above except that dithiothreitol was omitted from the solubilization buffer. The solubilized proteins were diluted with 1 volume of phosphate-buffered saline (0.14 M NaCl/2.7 mM KCI/1.5 mM KH2PO4/8.1 mM Na2HPO4, pH

Proc. Nati. Acad. Sci. USA 91 (1994)

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7.4) and 1 volume of water. To eliminate nonspecific binding to protein A-Sepharose, the solubilized proteins were incubated 30 min at 40C in the presence of Sepharose 4B-CL and the Sepharose was discarded by centrifugation. Supernatants were then incubated for 2 hr at 40C with the antibodies (dilution, 1:20 to 1:50) and then allowed to react with protein A-Sepharose for 2 hr more at 40C under agitation. For immunoprecipitation with monoclonal antibodies, the protein A-Sepharose was preincubated with rabbit anti-mouse IgG. After three washes with phosphate-buffered saline, the immune complexes were dissociated from protein A-Sepharose by incubation with Laemmli sample buffer. When the triad vesicles were previously labeled with [3H]ryanodine or [3H]PN200-110, the Sepharose beads were subjected directly to liquid scintillation counting. Western Blot Analysis. Triad vesicle proteins or proteins dissociated from protein A-Sepharose were analyzed in a 5-15% polyacrylamide gel. After electrophoretic separation, the proteins were transferred to a nitrocellulose sheet by transverse electrophoresis for 4 hr at 1.5 A (31). After saturation of the remaining binding sites with bovine serum albumin, the nitrocellulose sheets were incubated overnight at 40C with the antibodies (dilution, 1:100) and then with 25I-labeled protein G (anti-RyR) or with 1251-labeled antimouse IgG (MAC9), and the reactive proteins were detected by autoradiography.

RESULTS Triad membrane vesicles obtained from skeletal muscle bound ryanodine and PN200-110 with B. values of 15 and 12 pmol/mg, respectively. The presence of RyR and DHP-R in this membrane preparation was also detected by immunoblotting with antibodies directed against the RyR (Fig. 1, lane 1) or against the al subunit of the DHP-R (lane 2). A major high molecular weight band (apparent molecular mass > 400 kDa) was recognized by the antibodies raised against the RyR, and a single band of 170 kDa by the antibodies directed against the al subunit of the DHP-R. In addition to the major polypeptide, a minor band of 400 kDa was recognized by the anti-RyR antibodies. This peptide corresponds to the degradation product of the RyR already described by other groups (15, 32, 33), as confirmed by the increase in the intensity of the corresponding band in absence of protease inhibitors and by the presence of this peptide in purified RyR 1

2 3 kDa

- 200

- 97.4 - 69 -

46

FIG. 1. Western blot analysis of solubilized membrane vesicles with anti-RyR or anti-al DHP-R antibodies. Solubilized protein (20 ,ug) was electrophoresed in a 5-15% gradient polyacrylamide gel and electroblotted to nitrocellulose. After saturation with bovine serum albumin, the nitrocellulose sheets were incubated with the anti-RyR serum (1:100) (lane 1) or with anti-al DHP-R monoclonal antibody (MAC9, 1:100) (lane 2). After incubation with ml'I-protein G (lane 1) or with 125I-anti-mouse IgG (lane 2), the immunoreactive proteins were detected by autoradiography. Lane 3, 14C-Rainbow molecular weight standard (Amersham).

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preparations. These results clearly show that the polyclonal anti-RyR antibodies do not crossreact with the al subunit of the DHP-R and that MAC9 antibodies do not crossreact with RyR. This membrane preparation was solubilized in presence of CHAPS, NaCI, and exogenous lipids, and the solubilized proteins were separated by sedimentation through a sucrose gradient. RyR migration was followed by measuring the specific binding of [3Hryanodine in each fraction of the gradient. In a parallel experiment, the protein content of each fraction was analyzed by SDS/PAGE and immunoblotting with anti-al DHP-R antibodies. RyR solubilized in its tetrameric form sediments homogenously in the lower part of the gradient, as indicated by a single peak of bound [3H]ryanodine observed in fractions 8-12 (Fig. 2A). Similar behavior of the solubilized RyR has been previously described (14, 33). In contrast, analysis of the sedimentation profile of the DHP-R (Fig. 2B) clearly shows the existence of two populations of al subunits. While the majority of solubilized al subunit is recovered in the fractions near the top of the gradient (fractions 19-25), a significant amount of al subunit comigrates with the RyR in fractions 9-11. Using -catalase and P-galactosidase as sedimentation markers, we measured a sedimentation coefficient of 30 S for the RyR (fractions 9-11) and 15 S for the DHP-R (fractions 19-25). These values are in good agreement with those obtained by Lai et al. (14, 17) for the tetrameric form of the RyR and by Home et al. (34) for the DHP-R solubilized in the presence of CHAPS. These results suggest that the portion of al subunit which migrates in fractions 9-11 is associated with the RyR. 2 55

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FIG. 3. Immunoprecipitation of the solubilized proteins with anti-RyR antibodies. Proteins were solubilized from membrane vesicles and immunoprecipitated with anti-RyR antibodies. The composition of the immune complex was analyzed by Western blotting with anti-RyR (1:100) (lane 1) or anti-al DHP-R (1:100) Qane 2).

To confirm the existence of a complex involving both the RyR and the DHP-R, we examined their coimnunoprecipitation by specific antibodies. Triad vesicles were solubilized as described above and the soluble proteins were collected after removal of insoluble material by centAgation. Solubilized proteins were incubated with antibodies, and the immune complexes were precipitated with protein A-Sepharose. Immunoprecipitated proteins were then separated by SDS/PAGE and analyzed by immunoblot with the anti-RyR or anti-al subunit antibodies. Following immunoprecipitation with the anti-RyR antibodies, both RyR and the al subunit of DHP-R were precipitated as indicated by labeling with anti-RyR (Fig. 3, lane 1) and anti-al subunit (lane 2) antibodies. In a second set of experiments, membranes labeled with either [3H]PN200-110 or VH]ryanodine were solubilized as described above and the solubilized proteins were immunoprecipitated with the different antibodies. We then measured the amount of [3H]PN200-110 and [3H]ryanodine immuno-

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FIG. 2. Separation of solubilized RyR and al-DHP-R by sucrose gradient sedimentation. Membrane vesicles, labeled with [3H]ryanodine (A) or unlabeled (B), were solubilized and the solubilized proteins were loaded on a 5-20% sucrose gradient. After overnight centrifugation, the gradients were collected from the bottom. (A) Specific ryanodine binding in each fraction. The radioactivity in fractions 6-25 was measured by scintillation counting of 100 Al of each fraction. Nonspecific binding was determined by using the same procedure with vesicles labeled with 100 nM [3Hlryanodine in the presence of 100 ,uM ryanodine and was subtracted from total binding. (B) Immunoblotting with anti-al DHP-R. Samples (0.5 ml) of fractions 6-25 were concentrated, denatured with Laemmli's SDS/ PAGE sample buffer, and loaded on a 5-15% gradient SDS/ polyacrylamide gel. After migration, proteins corresponding to 100200 kDa were electrotransferred to nitrocellulose for Western blotting with anti-al DHP-R (1:100).

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FIG. 4. Immunoprecipitation of [3H~ryanodine or 1H]PN200-110 from labeled triad vesicles with anti-al DHP-R or anti-RyR, respectively. Triad vesicles were labeled with pHIPN200-110 or 3H]ryanodine, solubilized, and immunoprecipitated with various amounts of anti-RyR (m in A) or anti-al DHP-R (M in B). Each point corresponds to the immunoprecipitation of labeled receptors extracted from 1 mg of solubilized triad vesicle protein. Immunopecipitation was carried out with nonimmune serum (b) as a control.

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Proc. Natl. Acad. Sci. USA 91 (1994)

B 2 3 PI RyR al 1

4 a2

or against the subunits of the DHP-R. Our results lead to an approximate stoichiometry of two DHP-R molecules associated with one RyR. We found that, in our detergentsolubilized triad membrane preparations, 6% of the DHP-R and 2% of the RyR molecules are involved in this complex. Electronmicroscopy has shown that isolated triad membrane preparations contain some intact triad structures after purification (35). However, both electron microscopic analysis and studies of depolarization-induced Ca2+ release have shown that intact triad structures represent only 10-20%o of

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,8 subunits of DHP-R. Immunoprecipitation of solubilized proteins was achieved with anti-DHP-R antibodies (anti-al, anti-a2, anti-,8) or preimmune (PI) serum for anti-RyR as a control. After immunoprecipitation and dissociation, the immune complexes were analyzed in Western blot with anti-RyR (A) or anti-al DHP-R (B).

precipitated by the polyclonal anti-RyR antibodies and the monoclonal anti-al DHP-R antibody, respectively. The amount of [3H]PN200-110 or [3H]ryanodine immunoprecipitated increased with the amount of antibodies present in the reaction medium and reached a plateau in presence of high concentrations of antibodies (Fig. 4, U). When immunoprecipitation was performed under identical conditions but with nonimmune serum in place of the specific antibodies (Fig. 4, L), the amount of [3H]ryanodine or [3H]PN200-110 immunoprecipitated did not increase with the amount of antibodies added. After extraction and processing of 1 mg of triad vesicles, the maximum amount of labeled receptors precipitable (PEG precipitation) corresponded to -1.5 pmol of [3H]ryanodine and 0.9 pmol of [3H]PN200-110 specifically bound. Quantification of the [3H]ryanodine immunoprecipitated with anti-al DHP-R and of [3H]PN200-110 immunoprecipitated with anti-RyR indicated that 2% of the solubilized RyR and 6% of the DHP-R precipitable by PEG were specifically coimmunoprecipitated by the anti-al DHP-R and anti-RyR antibodies, respectively. Immunoprecipitation of purified [3H]PN200-ll0-labeled DHP-R with anti-RyR antibodies did not lead to specific precipitation of [3H]PN200-110 (data not shown). These results yield a stoichiometry of about two DHP-R molecules associated with one tetrameric RyR in the immunoprecipitated complex. Immunoprecipitation experiments were also carried out with antibodies directed against the a2 and subunits of the DHP-R. Immunoprecipitation of the al subunit was obtained, as expected, with anti-a2 DHP-R and anti-.8 DHP-R antibodies (Fig. SB, lanes 3 and 4). In these conditions, the immune complexes also contained the RyR (Fig. 5A, lanes 4 and 5). No immunoprecipitation of either the RyR or the al subunit was obtained with nonimmune serum (Fig. 5 A and B, lane 1). Moreover as described above, immunoprecipitation with anti-al DHP-R antibodies resulted in coprecipitation of RyR (Fig. SA, lane 3). However, the amount of RyR immunoprecipitated seemed to be higher for the immunoprecipitation with anti-a2 DHP-R, suggesting that the antigenic site for MANC1 is more accessible than the antigenic sites for the other antibodies in the complex with RyR.

DISCUSSION In this paper we have demonstrated the existence of

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a

complex involving the RyR and the DHP-R in triad membrane preparations obtained from rabbit skeletal muscle. This complex can be immunoprecipitated in a concentrationdependent manner with antibodies directed against the RyR

the total membrane vesicles (36). On these bases, the proportion of RyR and DHP-R associated with each other must represent less than 10-20%6 of the total amount of RyR and DHP-R present. These observations account for the relatively low amount of complex observed. Our results are in agreement with the model proposed by Block et al. (23) describing a close association of the RyR with the DHP-R. Since the identification of the RyR (37, 38) in the sarcoplasmic reticulum membrane and of the DHP-R in the T-tubule membrane (39), various mechanisms have been postulated for excitation-contraction coupling in skeletal muscle (19, 20, 40). At present, the most likely hypothesis suggests that voltage-driven intramembrane charge movement mediated by the DHP-R in the T-tubule membrane leads to activation of Ca2+ release from the sarcoplasmic reticulum mediated by the RyR. Experiments showing that slow Ca2+ currents, charge movement, and excitation-contraction coupling are restored in dysgenic myotubes by expression of the al subunit of the DHP-R (10, 11) strongly suggest that the al subunit is directly involved in the protein-protein interaction between the two receptors. Although various proteins, such as triadin (41) or the 71-kDa protein (42) and the FK506binding protein (43), have been proposed to be responsible for the interaction between RyR and DHP-R, the existence of a complex involving these two receptors has not previously been demonstrated. Our results provide direct biochemical evidence for the existence of such a complex. However, they do not exclude the presence of other proteins in this complex which may be required for stable interaction of the RyR with the DHP-R. Our results support the hypothesis that a direct physical interaction between DHP-R and RyR mediates excitation-contraction coupling in skeletal muscle. The study ofthe regulation of this interaction at a molecular level should allow the determination of its physiological significance in excitation-contraction coupling and its role in differentiation between the skeletal and the cardiac types of excitationcontraction coupling. We thank Dr. Neil Nathanson for critical comments on the manuscript and the SRV/INRA laboratory in Theix (France) for providing Pietrain normal pig muscles. This work was supported by a grant to M.R. and a fellowship to I.M. from the Association Frangaise contre les Myopathies; by research grants from the U.S. National Institutes of Health (NS22625), Miles Laboratory, and the Muscular Dystrophy Association to W.A.C., and by a postdoctoral research fellowship from the American Heart Association, Washington Affiliate, to Y.L.

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