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. 1999 Apr;76(4):1744–1756. doi: 10.1016/S0006-3495(99)77336-4

Differential regulation of skeletal muscle L-type Ca2+ current and excitation-contraction coupling by the dihydropyridine receptor beta subunit.

M Beurg 1, M Sukhareva 1, C A Ahern 1, M W Conklin 1, E Perez-Reyes 1, P A Powers 1, R G Gregg 1, R Coronado 1
PMCID: PMC1300153  PMID: 10096875

Abstract

The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams B. A., Beam K. G. A novel calcium current in dysgenic skeletal muscle. J Gen Physiol. 1989 Sep;94(3):429–444. doi: 10.1085/jgp.94.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beam K. G., Knudson C. M. Calcium currents in embryonic and neonatal mammalian skeletal muscle. J Gen Physiol. 1988 Jun;91(6):781–798. doi: 10.1085/jgp.91.6.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beurg M., Sukhareva M., Strube C., Powers P. A., Gregg R. G., Coronado R. Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor beta 1 subunit transfected with beta 1 cDNA. Biophys J. 1997 Aug;73(2):807–818. doi: 10.1016/S0006-3495(97)78113-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bourinet E., Zamponi G. W., Stea A., Soong T. W., Lewis B. A., Jones L. P., Yue D. T., Snutch T. P. The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels. J Neurosci. 1996 Aug 15;16(16):4983–4993. doi: 10.1523/JNEUROSCI.16-16-04983.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Castellano A., Wei X., Birnbaumer L., Perez-Reyes E. Cloning and expression of a neuronal calcium channel beta subunit. J Biol Chem. 1993 Jun 15;268(17):12359–12366. [PubMed] [Google Scholar]
  6. Chaudhari N., Beam K. G. mRNA for cardiac calcium channel is expressed during development of skeletal muscle. Dev Biol. 1993 Feb;155(2):507–515. doi: 10.1006/dbio.1993.1048. [DOI] [PubMed] [Google Scholar]
  7. Chien A. J., Zhao X., Shirokov R. E., Puri T. S., Chang C. F., Sun D., Rios E., Hosey M. M. Roles of a membrane-localized beta subunit in the formation and targeting of functional L-type Ca2+ channels. J Biol Chem. 1995 Dec 15;270(50):30036–30044. doi: 10.1074/jbc.270.50.30036. [DOI] [PubMed] [Google Scholar]
  8. De Waard M., Pragnell M., Campbell K. P. Ca2+ channel regulation by a conserved beta subunit domain. Neuron. 1994 Aug;13(2):495–503. doi: 10.1016/0896-6273(94)90363-8. [DOI] [PubMed] [Google Scholar]
  9. Dirksen R. T., Beam K. G. Single calcium channel behavior in native skeletal muscle. J Gen Physiol. 1995 Feb;105(2):227–247. doi: 10.1085/jgp.105.2.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Felix R., Gurnett C. A., De Waard M., Campbell K. P. Dissection of functional domains of the voltage-dependent Ca2+ channel alpha2delta subunit. J Neurosci. 1997 Sep 15;17(18):6884–6891. doi: 10.1523/JNEUROSCI.17-18-06884.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. García J., Beam K. G. Measurement of calcium transients and slow calcium current in myotubes. J Gen Physiol. 1994 Jan;103(1):107–123. doi: 10.1085/jgp.103.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. García J., Tanabe T., Beam K. G. Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors. J Gen Physiol. 1994 Jan;103(1):125–147. doi: 10.1085/jgp.103.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gregg R. G., Messing A., Strube C., Beurg M., Moss R., Behan M., Sukhareva M., Haynes S., Powell J. A., Coronado R. Absence of the beta subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the alpha 1 subunit and eliminates excitation-contraction coupling. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13961–13966. doi: 10.1073/pnas.93.24.13961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grouselle M., Koenig J., Lascombe M. L., Chapron J., Méléard P., Georgescauld D. Fura-2 imaging of spontaneous and electrically induced oscillations of intracellular free Ca2+ in rat myotubes. Pflugers Arch. 1991 Mar;418(1-2):40–50. doi: 10.1007/BF00370450. [DOI] [PubMed] [Google Scholar]
  15. Heinemann S. H., Conti F. Nonstationary noise analysis and application to patch clamp recordings. Methods Enzymol. 1992;207:131–148. doi: 10.1016/0076-6879(92)07009-d. [DOI] [PubMed] [Google Scholar]
  16. Hullin R., Singer-Lahat D., Freichel M., Biel M., Dascal N., Hofmann F., Flockerzi V. Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain. EMBO J. 1992 Mar;11(3):885–890. doi: 10.1002/j.1460-2075.1992.tb05126.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Josephson I. R., Varadi G. The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels. Biophys J. 1996 Mar;70(3):1285–1293. doi: 10.1016/S0006-3495(96)79685-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kamp T. J., Pérez-García M. T., Marban E. Enhancement of ionic current and charge movement by coexpression of calcium channel beta 1A subunit with alpha 1C subunit in a human embryonic kidney cell line. J Physiol. 1996 Apr 1;492(Pt 1):89–96. doi: 10.1113/jphysiol.1996.sp021291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lacerda A. E., Kim H. S., Ruth P., Perez-Reyes E., Flockerzi V., Hofmann F., Birnbaumer L., Brown A. M. Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature. 1991 Aug 8;352(6335):527–530. doi: 10.1038/352527a0. [DOI] [PubMed] [Google Scholar]
  20. Lamb G. D. DHP receptors and excitation-contraction coupling. J Muscle Res Cell Motil. 1992 Aug;13(4):394–405. doi: 10.1007/BF01738035. [DOI] [PubMed] [Google Scholar]
  21. Lory P., Varadi G., Schwartz A. The beta subunit controls the gating and dihydropyridine sensitivity of the skeletal muscle Ca2+ channel. Biophys J. 1992 Nov;63(5):1421–1424. doi: 10.1016/S0006-3495(92)81705-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nakai J., Dirksen R. T., Nguyen H. T., Pessah I. N., Beam K. G., Allen P. D. Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor. Nature. 1996 Mar 7;380(6569):72–75. doi: 10.1038/380072a0. [DOI] [PubMed] [Google Scholar]
  23. Nakai J., Sekiguchi N., Rando T. A., Allen P. D., Beam K. G. Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels. J Biol Chem. 1998 May 29;273(22):13403–13406. doi: 10.1074/jbc.273.22.13403. [DOI] [PubMed] [Google Scholar]
  24. Neely A., Wei X., Olcese R., Birnbaumer L., Stefani E. Potentiation by the beta subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel. Science. 1993 Oct 22;262(5133):575–578. doi: 10.1126/science.8211185. [DOI] [PubMed] [Google Scholar]
  25. Neuhuber B., Gerster U., Mitterdorfer J., Glossmann H., Flucher B. E. Differential effects of Ca2+ channel beta1a and beta2a subunits on complex formation with alpha1S and on current expression in tsA201 cells. J Biol Chem. 1998 Apr 10;273(15):9110–9118. doi: 10.1074/jbc.273.15.9110. [DOI] [PubMed] [Google Scholar]
  26. Nishimura S., Takeshima H., Hofmann F., Flockerzi V., Imoto K. Requirement of the calcium channel beta subunit for functional conformation. FEBS Lett. 1993 Jun 21;324(3):283–286. doi: 10.1016/0014-5793(93)80135-h. [DOI] [PubMed] [Google Scholar]
  27. Noceti F., Baldelli P., Wei X., Qin N., Toro L., Birnbaumer L., Stefani E. Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels. J Gen Physiol. 1996 Sep;108(3):143–155. doi: 10.1085/jgp.108.3.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Olcese R., Neely A., Qin N., Wei X., Birnbaumer L., Stefani E. Coupling between charge movement and pore opening in vertebrate neuronal alpha 1E calcium channels. J Physiol. 1996 Dec 15;497(Pt 3):675–686. doi: 10.1113/jphysiol.1996.sp021799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Olcese R., Qin N., Schneider T., Neely A., Wei X., Stefani E., Birnbaumer L. The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit's effect on activation. Neuron. 1994 Dec;13(6):1433–1438. doi: 10.1016/0896-6273(94)90428-6. [DOI] [PubMed] [Google Scholar]
  30. Perez-Reyes E., Castellano A., Kim H. S., Bertrand P., Baggstrom E., Lacerda A. E., Wei X. Y., Birnbaumer L. Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel. J Biol Chem. 1992 Jan 25;267(3):1792–1797. [PubMed] [Google Scholar]
  31. Powers P. A., Liu S., Hogan K., Gregg R. G. Skeletal muscle and brain isoforms of a beta-subunit of human voltage-dependent calcium channels are encoded by a single gene. J Biol Chem. 1992 Nov 15;267(32):22967–22972. [PubMed] [Google Scholar]
  32. Pragnell M., De Waard M., Mori Y., Tanabe T., Snutch T. P., Campbell K. P. Calcium channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha 1-subunit. Nature. 1994 Mar 3;368(6466):67–70. doi: 10.1038/368067a0. [DOI] [PubMed] [Google Scholar]
  33. Pérez-García M. T., Kamp T. J., Marbán E. Functional properties of cardiac L-type calcium channels transiently expressed in HEK293 cells. Roles of alpha 1 and beta subunits. J Gen Physiol. 1995 Feb;105(2):289–305. doi: 10.1085/jgp.105.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Qin N., Olcese R., Zhou J., Cabello O. A., Birnbaumer L., Stefani E. Identification of a second region of the beta-subunit involved in regulation of calcium channel inactivation. Am J Physiol. 1996 Nov;271(5 Pt 1):C1539–C1545. doi: 10.1152/ajpcell.1996.271.5.C1539. [DOI] [PubMed] [Google Scholar]
  35. Ruth P., Röhrkasten A., Biel M., Bosse E., Regulla S., Meyer H. E., Flockerzi V., Hofmann F. Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle. Science. 1989 Sep 8;245(4922):1115–1118. doi: 10.1126/science.2549640. [DOI] [PubMed] [Google Scholar]
  36. Singer D., Biel M., Lotan I., Flockerzi V., Hofmann F., Dascal N. The roles of the subunits in the function of the calcium channel. Science. 1991 Sep 27;253(5027):1553–1557. doi: 10.1126/science.1716787. [DOI] [PubMed] [Google Scholar]
  37. Stea A., Dubel S. J., Pragnell M., Leonard J. P., Campbell K. P., Snutch T. P. A beta-subunit normalizes the electrophysiological properties of a cloned N-type Ca2+ channel alpha 1-subunit. Neuropharmacology. 1993 Nov;32(11):1103–1116. doi: 10.1016/0028-3908(93)90005-n. [DOI] [PubMed] [Google Scholar]
  38. Strube C., Beurg M., Powers P. A., Gregg R. G., Coronado R. Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor beta 1 subunit. Biophys J. 1996 Nov;71(5):2531–2543. doi: 10.1016/S0006-3495(96)79446-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Strube C., Beurg M., Sukhareva M., Ahern C. A., Powell J. A., Powers P. A., Gregg R. G., Coronado R. Molecular origin of the L-type Ca2+ current of skeletal muscle myotubes selectively deficient in dihydropyridine receptor beta1a subunit. Biophys J. 1998 Jul;75(1):207–217. doi: 10.1016/S0006-3495(98)77507-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tanabe T., Adams B. A., Numa S., Beam K. G. Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics. Nature. 1991 Aug 29;352(6338):800–803. doi: 10.1038/352800a0. [DOI] [PubMed] [Google Scholar]
  41. Tanabe T., Beam K. G., Powell J. A., Numa S. Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature. 1988 Nov 10;336(6195):134–139. doi: 10.1038/336134a0. [DOI] [PubMed] [Google Scholar]
  42. Tareilus E., Roux M., Qin N., Olcese R., Zhou J., Stefani E., Birnbaumer L. A Xenopus oocyte beta subunit: evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1703–1708. doi: 10.1073/pnas.94.5.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wei X. Y., Perez-Reyes E., Lacerda A. E., Schuster G., Brown A. M., Birnbaumer L. Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels. J Biol Chem. 1991 Nov 15;266(32):21943–21947. [PubMed] [Google Scholar]

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