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Detection and localization of triadin in rat ventricular muscle

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Summary

Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the Mr 102K Ca2+ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same Mr as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.

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References

  • Ashraf, M., Park, W.H., Grupp, I., Schwartz, A. 1986. Distribution of3H nitrendipine in the isolated perfused rat heart as revealed by electron microscopic autoradiography.J. Mol. Cell Cardiol. 18:265–272

    PubMed  Google Scholar 

  • Bers, D.M. 1979. Isolation and characterization of cardiac sarcolemma.Biochem. Biophys. Acta 55:131–146

    Google Scholar 

  • Brandt, N. 1985. Identification of two populations of cardiac microsomes with nitrendipine receptors: Correlation of the distribution of dihydropyridine receptors with organelle specific markers.Arch. Biochem. Biophys. 242:306–319

    Article  PubMed  Google Scholar 

  • Brandt, N.R., Kawamoto, R.M., Caswell, A.H. 1985. Dihydropyridine binding sites on transverse tubules isolated from triads of rabbit skeletal muscles.J. Receptor Res. 5:155–170

    Google Scholar 

  • Brandt, N., Bassett, A. 1986. Separation of dihydropyridine binding sites from cardiac junctional sarcoplasmic reticulum.Arch. Biochem. Biophys. 244:872–875

    Article  PubMed  Google Scholar 

  • Brandt, N.R., Caswell, A.H., Wen, S.-R., Talvenheimo, J.A. 1990. Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads.J. Membrane Biol. 113:237–251

    Article  Google Scholar 

  • Brandt, N.R., Caswell, A.H., Brunschwig, J.-P., Kang, J.-J., Antoniu, B., Ikemoto, N. 1992a. Effects of anti-triadin antibody on Ca2+ release from sarcoplasmic reticulum.FEBS Lett. 299:57–59

    Article  PubMed  Google Scholar 

  • Brandt, N.R., Caswell, A.H., Brandt, T., Brew, K., Mellgren, R.L. 1992b. Mapping of the calpain proteolyses products of the junctional foot protein of the skeletal muscle triad junction.J. Membrane Biol. 127:35–47

    Article  Google Scholar 

  • Campbell, K.P., MacLennan, D.H., Jorgensen, A.O. 1982. Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye “Stains-All”.J. Biol. Chem. 258:11267–11273

    Google Scholar 

  • Caswell, A.H., Lau, Y.H., Brunschwig, J.-P. 1976. Ouabainbinding vesicles from skeletal muscle.Arch. Biochem. Biophys. 176:417–430

    Article  PubMed  Google Scholar 

  • Caswell, A.H., Brandt, N.R., Brunschwig, J.-P., Kawamoto, R.M. 1988. Isolation of transverse tubule membranes from skeletal muscle: Ion transport activity, reformation of triad junctions and isolation of junctional spanning proteins of triads.Methods in Enzymol. 157:68–84

    Google Scholar 

  • Caswell, A.H., Brandt, N.R., Brunschwig, J.-P., Purkerson, S. 1991. Localization and partial characterization of the oligomeric disulfide-linked molecular weight 95,000 protein (triadin) which binds the ryanodine and dihydropyridine receptors in skeletal muscle triadic vesicles.Biochem. 30:7507–7513

    Article  Google Scholar 

  • Cleemann, L., Morad, M. 1991. Role of Ca2+ channel in cardiac excitation-contraction coupling in the rat: Evidence from Ca2+ transients and contraction.J. Physiol. 432:283–312

    PubMed  Google Scholar 

  • Dombradi, V.K., Silberman, S.R., Lee, E.Y.C., Caswell, A.H., Brandt, N.R. 1984. The association of phosphorylatase kinase with rabbit muscle T-tubules.Arch. Biochem. Biophys. 230(2):615–630

    Article  PubMed  Google Scholar 

  • Doyle, D.A., Kamp, T.J., Palfrey, H.C., Miller, R.J., Page, E. 1986. Separation of cardiac plasmalemma into cell surface and T-tubular components.J. Biol. Chem. 261:6556–6564

    PubMed  Google Scholar 

  • Fabiato, A. 1989. Appraisal of the physiological relevance of two hypotheses for the mechanism of calcium release from mammalian cardiac sarcoplasmic reticulum: calcium-induced release versus charge-coupled release.Mol. Cell. Biochem. 89:135–140

    Article  PubMed  Google Scholar 

  • Fosset, M., Jaimovich, B., Delpont, E., Lazdunski, M. 1982. [3H]nitrendipine receptors in skeletal muscle: Properties and preferential localization in transverse tubules.J. Biol. Chem. 258:6080–6092

    Google Scholar 

  • Gage, P.W., Eisenberg, R.S. 1969. Action potentials after potentials and excitation-contraction coupling in frog sartorias muscle without transverse tubules.J. Gen. Physiol. 53:298–310

    Article  PubMed  Google Scholar 

  • Heilmann, C., Lindt, T., Müller, W., Pette, D. 1980. Characterization of cardiac microsome from spontaneously hypertonic rats.Basic Res. Cardiol. 75:92–96

    Article  PubMed  Google Scholar 

  • Inui, M., Saito, A., Fleischer, S. 1987. Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures.J. Biol. Chem. 262:15637–15642

    PubMed  Google Scholar 

  • Inui, M., Wang, S., Saito, A., Fleischer, S. 1986. Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle.J. Biol. Chem. 263:10843–10859

    Google Scholar 

  • Janis, R.A., Maurer, S.C., Sarmiento, J.G., Bolger, G.T., Triggle, D.J. 1982. Binding of [3H]Nimodipine to cardiac and smooth muscle membranes.Eur. J. Pharmacol. 82:191–194

    Article  PubMed  Google Scholar 

  • Jones, L.R., Besch, H.R., Jr., Fleming, J.W., McConnaughey, M.M., Watanabe, A.M. 1979. Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum.J. Biol. Chem. 254:530–539

    PubMed  Google Scholar 

  • Jorgensen, A.O., Shen, A.C.-Y., Denney, D.H., Jr., Campbell, K.P. 1983. Immunoelectron microscopical localization of calsequestrin in rat ventricular muscle.J. Cell Biol. 97:260a

    Article  Google Scholar 

  • Kim, K.C., Caswell, A.H., Brunschwig, J.-P., Brandt, N.R. 1990a. Identification of a new subpopulation of triad junctions isolated from skeletal muscle; morphological correlations with intact muscle.J. Membrane Biol. 113:221–235

    Article  Google Scholar 

  • Kim, K.C., Caswell, A.H., Talvenheimo, J.A., Brandt, N.R. 1990b. Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle.Biochemistry 29:9281–9289

    Article  PubMed  Google Scholar 

  • Limas, C.J., Spier, S.S., Kahlon, J. 1980. Enhanced calcium transport by sarcoplasmic reticulum in mild cardiac hypertrophy.J. Molec. Cell Cardiol. 12:1103–1116

    Article  Google Scholar 

  • Manalan, A.S., Jones, L.R. 1982. Characterization of the intrinsic cAMP-dependent protein kinase activity and endogenous substrates in highly purified cardiac sarcolemma vesicles.J. Biol. Chem. 257:10052–10062

    PubMed  Google Scholar 

  • Mansier, P, Charlemange, D., Rossi, B., Pretesoille, M., Swynghedauw, B., Lelievre, L. 1983. Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart.J. Biol. Chem. 208:6628–6635

    Google Scholar 

  • Murphy, K.M.M., Snyder, S.H. 1982. Calcium antagonist receptor binding sites labelled with [3H]Nitrendipine.Eur. J. Pharmacol. 77:201–202

    Article  PubMed  Google Scholar 

  • Nayler, W.G., Dillon, J.S., Elz, J.S., McKelvie, M. 1985. An effect of ischemia on myocardial dihydropyridine binding sites.Eur. J. Pharmacol. 115:81–89

    Article  PubMed  Google Scholar 

  • Otsu, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, Í.M., MacLennan, D.H. 1991. Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum.J. Biol. Chem. 265:13472–13483

    Google Scholar 

  • Rios, E., Brum, G. 1987. Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle.Nature 325:717–720

    Article  PubMed  Google Scholar 

  • Rios, E., Ma, J., Gonzalez, A. 1991. The mechanical hypothesis of excitation-contraction (EC) coupling in skeletal muscle.J. Muscle Res. & Cell Motil. 12:127–135

    Google Scholar 

  • Schneider, M.F., Chandler, W.K. 1973. Voltage dependent charge movement in skeletal muscles: a possible step in excitation contraction coupling.Nature 242:244–246

    Article  PubMed  Google Scholar 

  • Seifert, J., Cassida, J.E. 1986. Ca2+-dependent ryanodine binding site: Soluble preparation from rabbit cardiac sarcoplasmic reticulum.Biochim. Biophys. Acta 861:399–405

    PubMed  Google Scholar 

  • Seiler, S., Wegener, A.D., Whang, D.D., Hathaway, D.R., Jones, L.R. 1984. High molecular weight proteins in cardiac and skeletal muscle. Junctional sarcoplasmic reticulum vesicles bind calmodulin are phosphorylated, and are degraded by Ca2+-activated protease.J. Biol. Chem. 259:8550–8557

    PubMed  Google Scholar 

  • Tanabe, T., Beam, K.G., Adams, B.A., Niidome, T., Numa, S. 1990. Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling.Nature 346:567–5722

    Article  PubMed  Google Scholar 

  • Wei, J.W., Janis, R.A., Daniel, E.E. 1976. Calcium accumulation and enzymatic activities of subcellular fractions from aortas and ventricles of genetically hypertensive rats.Circ. Res. 39:133–140

    PubMed  Google Scholar 

  • Wibo, M., Bravo, G., Godfraind, T. 1991. Postnatal maturation of excitation-contraction coupling in rat ventricle in relation to the subcellular density of 1,4-dihydropyridine and ryanodine receptors.Circ. Res. 68:662–673

    PubMed  Google Scholar 

  • Wientzek, M., Katz, S. 1991. Isolation and characterization of purified sarcoplasmic reticulum membranes from isolated adult rat ventricular myocytes.J. Mol. Cell Cardiol. 23:1149–1163

    Article  PubMed  Google Scholar 

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Brandt, N.R., Caswell, A.H., Lewis Carl, S.A. et al. Detection and localization of triadin in rat ventricular muscle. J. Membrain Biol. 131, 219–228 (1993). https://doi.org/10.1007/BF02260110

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