Advertisement

Purification of the putative calcium channel from skeletal muscle with the aid of [3H]-nimodipine binding

  • Hartmut Glossmann
  • David R. Ferry
  • C. Bruce Boschek
Article

Summary

High affinity (KD=1.5 nmol/l) binding sites for the potent 1.4-dihydropyridine calcium channel blocker [3H]-nimodipine have been found in guinea-pig skeletal muscle homogenates. These sites could be enriched by differential centrifugation in a crude microsomal fraction. In the microsomal fraction the density of binding sites was 2pMol per mg of protein. The pharmacological profile of the [3H]-nimodipine binding sites suggests that they are part of the putative calcium channel and that [3H]-nimodipine binding can be used as a marker for these ionic pores.

In guinea-pig brain membranes [3H]-nimodipine labels a single class of sites with a KD of 0.6 nmol/l and d-cis-diltiazem decreases the KD by a factor of three without a change in the maximum number of receptors. In contrast d-cis-diltiazem (at the optimal concentration of 10 μmol/l) increases the density of the sites in guinea-pig skeletal muscle available for [3H]-nimodipine with high affinity and the KD decreases marginally to 1.0 nmol/l. These effects of d-cis-diltiazem are stereospecific since the pharmacologically inactive diastereoisomer l-cis-diltiazem does not stimulate [3H]-nimodipine binding, but is inhibitory, albeit at much higher concentrations. It is concluded that a significant fraction of the putative calcium channels has a KD of >50 nmol/l for [3H]-nimodipine, and that d-cis-diltiazem can increase the affinity of this subpopulation for [3H]-nimodipine so that they are detectable in ligand binding experiments.

The binding sites of [3H]-nimodipine have been purified from the crude microsomal pellet by means of sucrose gradient centrifugation. [3H]-nimodipine binding copurifies with (Na+, K+)-ATP' ase and [3H]-ouabain binding and is enriched in a vesicular fraction (by a factor of 30–60 fold) of low bouyant density [<25% (w/w) sucrose], with a ratio of (Na+, K+)-ATP'ase to Ca2+-ATP'ase activity of 0.77.

Biochemical and electron microscopic examination suggests that a specialized structure of the sarcolemma, possibly the transverse tubule, is the subcellular locus for the [3H]-nimodipine binding site. Since the density of the drug receptors in this purified preparation is extremely high and exceeds that reported for [3H]-saxitoxin binding sites (a specific sodium channel label) by a factor of 4–10 (with respect to most highly purified skeletal muscle membrane isolated), the isolation and purification of the putative calcium channel from skeletal muscle is feasable.

Our results confirm recent findings with biophysical methods, on the presence of calcium channels which are blocked by (±) D-600, d-cis-diltiazem and the 1,4-dihydropyridine nifedipine in skeletal muscle. The data are discussed in the context of the possible physiological role of calcium channels located in transverse tubules.

Key words

Skeletal muscle Calcium channel blocker [3H]-Nimodipine Isolation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Akaike N, Fishman HM, Lee KS, Moore LE, Brown AM (1978) The units of calcium conductance in Helix neurones. Nature 273:379–382Google Scholar
  2. Almers W, Fink R, Palade PT (1981) Calcium depletion in frog muscle tubules: The decline of calcium currents under maintained depolarization. J Physiol 312:177–207Google Scholar
  3. Atwater I, Darrson CM, Eddlestone GT, Rojas E (1981) Voltage noise measurements across the pancreatic β-cell membrane: Calcium channel characteristics. J Physiol 314:195–212Google Scholar
  4. Barchi RL, Weigele JB, Chalikian DM, Murphy LE (1979) Muscle surface membranes. Preparative methods affect apparent chemical properties and neurotoxin binding. Biochem Biophys Acta 550:56–79Google Scholar
  5. Barett JN, Barett EF (1978) Excitation-contraction coupling in skeletal muscle: Blockade by high extracellular concentrations of calcium buffers. Science 200:1270–1272Google Scholar
  6. Beaty GN, Stefani E (1976) Inward calcium current in twitch muscle fibres of the frog. J Physiol 260:27Google Scholar
  7. Bennett JP (1978) Methods in binding studies. In: Yamamura HI, Enna SJ, Kuhar MJ (eds) Neurotransmitter receptor binding. Raven Press, New York, pp 57–90Google Scholar
  8. Betz H, Bourgeois J-P, Changeux J-P (1977) Evidence for degradation of the acetylcholine (nicotinic) receptor in skeletal muscle during the development of the chick embryo. FEBS Lett 77:219–224Google Scholar
  9. Bonacker O, Glossmann H (1981) Mammalian β-adrenoceptors: concomitant biospecific elution with protein kinase activity from sepharosealprenolol. Eur J Pharmacol 75:197–204Google Scholar
  10. Chiriandini DJ, Stefani E (1983) Calcium action potentials in rat fast-and slow-twitch muscle fibres. J Physiol (in press)Google Scholar
  11. DeLean A, Munson PJ, Rodbard D (1978) Simultaneous analysis of families of sigmoid curves: Application to bioassay, radioligand assay and physiological dose-response curves. Am J Physiol 4:E97-E102Google Scholar
  12. Ehlert FJ, Roeske WR, Hoga E, Yamamura HI (1982) The binding of [3H]-nitrendipine to receptors for calcium antagonists in the heart, cerebral cortex and ileum of rats. Life Sci 30:2191–2202Google Scholar
  13. Erdmann E, Phillipp G, Tanner G (1976) Ouabain-receptor interactions in (Na+, K+)-ATP'ase preparations. A contribution to the problem of non-linear Scatchard plots. Biochem Biophys Acta 455:287–296Google Scholar
  14. Ferry DR, Glossmann H (1982a) Evidence of multiple drug receptor sites within the putative calcium channel. Naunyn-Schmiedeberg's Arch Pharmacol 321:80–83Google Scholar
  15. Ferry DR, Glossmann H (1982b) Identification of putative calcium channels in skeletal muscle microsomes. FEBS Lett 148:331–337Google Scholar
  16. Festoff BW, Engel WK (1974) In vitro analysis of the general properties and junctional receptor characteristics of skeletal muscle membranes. Isolation, purification and partial characterisation of sarcolemmal fragments. Proc Natl Acad Sci (USA) 71:2435–2439Google Scholar
  17. Glossmann H, Ferry DR, Lübbecke F, Mewes R, Hoffmann F (1982a) Identification of voltage operated calcium channels by binding studies: Differentiation of subclasses of calcium antagonist drugs with [3H]-nimodipine radioligand binding. J Receptor Res (in press)Google Scholar
  18. Glossmann H, Ferry DR, Lübbecke F, Mewes R, Hoffmann F (1982b) Calcium channels: Direct identification with radioligand binding studies. Trends Pharm Sci 3:431–437Google Scholar
  19. Gonzales-Serratos H, Valle-Aguilera R, Lathrop DA, Garcia M del C (1982) Slow inward calcium currents have no obvious role in muscle excitation-contraction coupling. Nature 298:292–294Google Scholar
  20. Jones LR, Besch HR, Fleming JW, McConnaughey MM, Watanabe AG (1979) Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmatic reticulum. J Biol Chem 254:530–539Google Scholar
  21. Lau YH, Caswell AH, Brunschwig J-P (1977) Isolation of transverse tubules by fractionation of triad junctions of skeletal muscle. J Biol Chem 252:5565–5574Google Scholar
  22. Lüttgau HC, Spiecker W (1979) The effects of calcium deprivation upon mechanical and electrophysiological parameters in skeletal muscle fibres of the frog. J Physiol 296:411–429Google Scholar
  23. Lux HD, Nagy K (1981) Single channel Ca2+ currents in Helix pomatia neurones. Pflügers Arch 391:252–254Google Scholar
  24. Maurer HR (1968) Disk-Elektrophorese. Walter de Gruyter & Co, BerlinGoogle Scholar
  25. McCleskey E, Almers W (1981) Pharmacological comparison of E.C. coupling and the skeletal muscle Ca2+ channel. Biophys J 33:33aGoogle Scholar
  26. McLennan DH, Holland PC (1975) Ca2+-transport in sarcoplasmic reticulum. Annu Rev Biophys Bioeng 4:373–404Google Scholar
  27. McLennan DH (1974) Isolation of a second form of calcequestrin. J Biol Chem 249:980–984Google Scholar
  28. Mobley BA, Eisenberg BE (1975) Sizes of components in frog skeletal muscle measured by methods of stereology. J Gen Physiol 66:31–45Google Scholar
  29. Nagao T, Sato M, Iwasawa Y, Takada T, Ishida R, Nakajima H, Kiyomoto A (1972) Studies on a new 1,5-benzothiazepine derivate (CRD-401) II. Effects of optical isomers of CRD-401 on smooth muscle and other pharmacological properties. Jpn J Pharmacol 22:467–478Google Scholar
  30. Osterrieder W, Yang Q-F, Trautwein W (1982) Conductance of the slow inward channel in the rabbit sinoatrial node. Pflügers Arch 394:85–89Google Scholar
  31. Ritchie JM, Rogart RB (1977) The binding of labelled saxitoxin to the sodium channels in normal and denerved mammalian muscle and in amphibian muscle. J Physiol 269:341–354Google Scholar
  32. Sanchez JA, Stefani E (1978) Inward calcium current in twitch muscle fibres of the frog. J Physiol 283:197–209Google Scholar
  33. Siri LN, Sanchez JA, Stefani E (1980) Effect of glycerol treatment on the calcium current of skeletal muscle. J Physiol 305:87–96Google Scholar
  34. Spiecker W, Metzer W, Lüttgau HC (1979) Extracellular Ca2+ and excitation-contractraction coupling. Nature 280:158–160Google Scholar
  35. Stefani E, Chiriandini DJ (1982) Ionic channels in skeletal muscle. Ann Rev Physiol 44:357–372Google Scholar
  36. Vander AJ, Sherman JH, Luciano DS (1970) Human physiology. The mechanism of body function. McGraw-Hill, NY, p 219Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • Hartmut Glossmann
    • 1
  • David R. Ferry
    • 1
  • C. Bruce Boschek
    • 2
  1. 1.Rudolf-Buchheim-Institut für Pharmakologie der Justus-Liebig-Universität, GießenGießenFederal Republic of Germany
  2. 2.Fachbereich Humanmedizin, GießenInstitut für Virologie der Justus-Liebig-UniversitätGießenFederal Republic of Germany

Personalised recommendations