Isolation of T‐Tubules from Skeletal Muscle

Antonio Zorzano1, Marta Camps1

1 Departament de Bioquimica i Biologia, Molecular, Universitat de Barcelona, and Institute for Research in Biomedicine, Barcelona Science Park, Barcelona
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.24
DOI:  10.1002/0471143030.cb0324s31
Online Posting Date:  July, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The transverse tubules (T‐tubules) of mammalian cardiac and skeletal muscles are invaginations of the sarcolemma. They play a crucial role in excitation‐contraction coupling as well as in intracellular signaling and in regulation of glucose transport. The biochemical purification of T‐tubule membranes is a difficult task, and membrane fractions enriched in transverse tubules are usually contaminated with other cell‐surface and intracellular membranes. This unit includes methods that permit the isolation and purification of T‐tubules from skeletal muscle.

Keywords: T‐tubules; transverse tubules; vesicle immunoisolation; wheat germ agglutination; skeletal muscle; dihydropyridine receptors; tt28 protein

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Subcellular Fractionation of Rat Skeletal Muscle Membranes
  • Basic Protocol 2: Separation of T‐Tubules from Sarcolemmal Vesicles by Wheat Germ Agglutination
  • Alternate Protocol 1: Purification of T‐Tubules by Vesicle Immunoisolation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Subcellular Fractionation of Rat Skeletal Muscle Membranes

  Materials
  • Rats (Wistar, male, ∼250 g)
  • Homogenization buffer (see recipe), ice cold
  • 4 M KCl
  • Ca2+‐loading buffer 1 (see recipe)
  • 1 M ATP
  • 150 mM CaCl 2
  • Ca2+‐loading buffer 2 (see recipe).
  • 50 mM Tris·Cl, pH 7.2 ( appendix 2A)
  • Solution C (see recipe)
  • 35%, 29%, 26%, and 23% (w/v) sucrose in sucrose gradient buffer (see recipe for buffer)
  • Tris‐sucrose buffer (see recipe)
  • HEPES‐sucrose buffer (see recipe)
  • Liquid nitrogen
  • Petri dish
  • Dissecting instruments
  • Polytron homogenizer with 1‐cm‐diameter probe
  • Sorvall refrigerated centrifuge with SA‐600 rotor (or equivalent fixed‐angle rotor)
  • Sorvall refrigerated high‐speed centrifuge with T‐647.5 rotor (or equivalent fixed‐angle rotor)
  • Orbital shaker
  • Sorvall refrigerated high‐speed centrifuge with TA‐641 rotor (or equivalent swinging‐bucket rotor) and 13‐ml ultracentrifuge tubes
  • Additional reagents and equipment for Bradford protein assay ( appendix 3H)

Basic Protocol 2: Separation of T‐Tubules from Sarcolemmal Vesicles by Wheat Germ Agglutination

  Materials
  • F2 fraction 23 ( protocol 1)
  • 50 mM potassium phosphate buffer, pH 7.4 ( appendix 2A), containing 160 mM NaCl
  • 1 mg/ml wheat germ agglutinin (WGA; lectin from Triticum vulgaris, lyophilized powder, Sigma) in 50 mM phosphate buffer (pH 7.4)/160 mM NaCl
  • Tris‐sucrose buffer (see recipe)
  • Deagglutination buffer (see recipe)
  • Liquid nitrogen
  • Beckman ultracentrifuge with TLS‐55 rotor (or equivalent)
  • 10‐ml ultracentrifuge tubes for TLS‐55 rotor

Alternate Protocol 1: Purification of T‐Tubules by Vesicle Immunoisolation

  Materials
  • Goat anti–mouse IgG coupled to agarose beads, saline suspension (5 to 10 mg antibody/ml bead suspension; Sigma)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Anti‐tt28 antibody, monoclonal (Rosemblatt and Scales, ), or other antibody directed against tt‐28 or against other proteins such as DHPR (Morton and Froehner, , ; Flucher et al., )
  • Blocking solution: 1% (w/v) bovine serum albumin (BSA) in PBS, pH 7.4 (prepare fresh)
  • T‐tubule‐enriched fraction (typically fraction 23 from F2; see protocol 1)
  • 2× immunoadsorption solution (see recipe)
  • 3× Laemmli sample buffer (see recipe)
  • Orbital shaker
  • 95°C water bath or heat block
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Angelica, C.M., Sierralta, J., and Hidalgo, C. 1993. Phospholipase C activity in membranes and a soluble fraction isolated from frog skeletal muscle. Biochim. Biophys. Acta 1152:44‐48.
   Barchi, R.L., Weigele, J.B., Chalikian, D.M., and Murphy, L.E. 1979. Muscle surface membranes: Preparative methods affect apparent chemical properties and neurotoxin binding. Biochim. Biophys. Acta 550:59‐76.
   Brette, F. and Orchard, C. 2003. T‐tubule function in mammalian cardiac myocytes. Circ. Res. 92:1182‐1192.
   Burdett, E., Beeler, T., and Klip, A. 1987. Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle. Arch. Biochem. Biophys. 253:279‐286.
   Carl, S.L., Felix, K., Caswell, A.H., Brandt, N.R., Ball, W.J. Jr., Vaghy, P.L., Meissner, G., and Ferguson, D.G. 1995. Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium. J. Cell Biol. 129:672‐682.
   Caswell, A.H., Baker, S.P., Boyd, H., Potter, L.T., and Garcia, M. 1978. Beta‐adrenergic receptor and adenylate cyclase in transverse tubules of skeletal muscle. J. Biol. Chem. 253:3049‐3054.
   Charuk, J.H., Howlett, S., and Michalak, M. 1989. Subfractionation of cardiac sarcolemma with wheat‐germ agglutinin. Biochem. J. 264:885‐892.
   Di Virgilio, F., Salviati, G., Pozzan, T., and Volpe, P. 1986. Is a guanine nucleotide‐binding protein involved in excitation‐contraction coupling in skeletal muscle? EMBO J. 5:259‐262.
   Dunn, S.M. 1989. Voltage‐dependent calcium channels in skeletal muscle transverse tubules: Measurements of calcium efflux in membrane vesicles. J. Biol. Chem. 264:11053‐11060.
   Fischer, Y., Thomas, J., Sevilla, L., Munoz, P., Becker, C., Holman, G., Kozka, I.J., Palacin, M., Testar, X., Kammermeier, H., and Zorzano, A. 1997. Insulin‐induced recruitment of glucose transporter 4 (GLUT4) and GLUT1 in isolated rat cardiac myocytes: Evidence of the existence of different intracellular GLUT4 vesicle populations. J. Biol. Chem. 272:7085‐7092.
   Flucher, B.E., Terasaki, M., Chin, H., Beeler, T., and Daniels, M.P. 1991. Biogenesis of transverse tubules in skeletal muscle in vitro. Dev. Biol. 145:77‐90.
   Gao, T., Puri, T.S., Gerhardstein, B.L., Chien, A.J., Green, R.D., and Hosey, M.M. 1997. Identification and subcellular localization of the subunits of L‐type calcium channels and adenylyl cyclase in cardiac myocytes. J. Biol. Chem. 272:19401‐19407.
   Hidalgo, C., Gonzalez, M.E., and Lagos, R. 1983. Characterization of the Ca2+‐ or Mg2+‐ATPase of transverse tubule membranes isolated from rabbit skeletal muscle. J. Biol. Chem. 258:13937‐13945.
   Hidalgo, C., Jorquera, J., Tapia, V., and Donoso, P. 1993. Triads and transverse tubules isolated from skeletal muscle contain high levels of inositol 1,4,5‐trisphosphate. J. Biol. Chem. 268:15111‐15117.
   Horgan, D.J. and Kuypers, R. 1987. Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion‐free sucrose density gradients. Arch. Biochem. Biophys. 253:377‐387.
   Laflamme, M.A. and Becker, P.L. 1999. G(s) and adenylyl cyclase in transverse tubules of heart: Implications for cAMP‐dependent signaling. Am. J. Physiol. 277:H1841‐H1848.
   Lau, Y.H., Caswell, A.H., and Brunschwig, J.P. 1977. Isolation of transverse tubules by fractionation of triad junctions of skeletal muscle. J. Biol. Chem. 252:5565‐5574.
   Luise, M., Presotto, C., Senter, L., Betto, R., Ceoldo, S., Furlan, S., Salvatori, S., Sabba dini, R.A., and Salviati, G. 1993. Dystrophin is phosphorylated by endogenous protein kinases. Biochem. J. 293:243‐247.
   Marette, A., Burdett, E., Douen, A., Vranic, M., and Klip, A. 1992. Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle. Diabetes 41:1562‐1569.
   Milting, H., Heilmeyer, L.M. Jr., and Thieleczek, R. 1994. Phosphoinositides in membranes that build up the triads of rabbit skeletal muscle. FEBS Lett. 345:211‐218.
   Morton, M.E. and Froehner, S.C. 1987. Monoclonal antibody identifies a 200‐kDa subunit of the dihydropyridine‐sensitive calcium channel. J Biol Chem. 262:11904‐11907.
   Morton, M.E. and Froehner, S.C. 1989. The α1 and α2 polypeptides of the dihydropyridine‐sensitive calcium channel differ in developmental expression and tissue distribution. Neuron 2:1499‐1506.
   Munoz, P., Rosemblatt, M., Tes tar, X., Palacin, M., Thoidis, G., Pilch, P.F., and Zorzano, A. 1995a. The T‐tubule is a cell‐surface target for insulin‐regulated recycling of membrane proteins in skeletal muscle. Biochem. J. 312:393‐400.
   Munoz, P., Rosemblatt, M., Tes tar, X., Palacin, M., and Zorzano, A. 1995b. Isolation and characterization of distinct domains of sarcolemma and T‐tubules from rat skeletal muscle. Biochem. J. 307:273‐280.
   Munoz, P., Mora, S., Sevilla, L., Kaliman, P., Tomas, E., Guma, A., Tes tar, X., Palacin, M., and Zorzano, A. 1996. Expression and insulin‐regulated distribution of caveolin in skeletal muscle: Caveolin does not colocalize with GLUT4 in intracellular membranes. J. Biol. Chem. 271:8133‐8139.
   Ohlendieck, K., Ervasti, J.M., Snook, J.B., and Campbell, K.P. 1991. Dystrophin‐glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. J. Cell Biol. 112:135‐148.
   Petrecca, K., Atanasiu, R., Grinstein, S., Orlowski, J., and Shrier, A. 1999. Subcellular localization of the Na+/H+ exchanger NHE1 in rat myocardium. Am. J. Physiol. 276:H709‐H717.
   Rosemblatt, M.S. and Scales, D.J. 1989. Morphological, immunological and biochemical characterization of purified transverse tubule membranes isolated from rabbit skeletal muscle. Mol. Cell Biochem. 87:57‐69.
   Rosemblatt, M., Hidalgo, C., Vergara, C., and Ike moto, N. 1981. Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle. J. Biol. Chem. 256:8140‐8148.
   Salvatori, S., Furlan, S., Millikin, B., Sabbadini, R., Betto, R., Margreth, A., and Salviati, G. 1993. Localization of protein kinase C in skeletal muscle T‐tubule membranes. Biochem. Biophys. Res. Commun. 196:1073‐1080.
   Toutant, M., Gabrion, J., Vandaele, S., Peraldi‐Roux, S., Barhanin, J., Bockaert, J., and Rouot, B. 1990. Cellular distribution and biochemical characterization of G proteins in skeletal muscle: Comparative location with voltage‐dependent calcium channels. EMBO J. 9:363‐369.
   Wang, W., Hansen, P.A., Marshall, B.A., Holloszy, J.O., and Mueckler, M. 1996. Insulin unmasks a COOH‐terminal Glut4 epitope and increases glucose transport across T‐tubules in skeletal muscle. J. Cell Biol. 135:415‐430.
   Zorzano, A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinkski, B.E., Ruoho, A.E., and Pilch, P.F. 1989. Insulin‐regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J. Biol. Chem. 264:12358‐12363.
   Zorzano, A., Munoz, P., Camps, M., Mora, C., Tes tar, X., and Palacin, M. 1996. Insulin‐induced redistribution of GLUT4 glucose carriers in the muscle fiber: In search of GLUT4 trafficking pathways. Diabetes 45:S70‐S81.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library