This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sheridan, D. C. Articles by Coronado, R. Search for Related Content PubMed PubMed Citation Articles by Sheridan, D. C. Articles by Coronado, R.

Ca²⁺-Dependent Excitation-Contraction Coupling Triggered by the Heterologous Cardiac/Brain DHPR ß2a-Subunit in Skeletal Myotubes

Department of Physiology, University of Wisconsin, School of Medicine, Madison, Wisconsin 53706 USA

Correspondence: Address reprint requests to Roberto Coronado, Dept. of Physiology, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-263-7487; Fax: 608-265-5512; E-mail: coronado{at}physiology.wisc.edu.

Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) 1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR ß1a-subunit. It is unknown whether EC coupling domains present in the ß-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit 1S is paired with the heterologous heart/brain DHPR ß2a-subunit. Studies were conducted in primary cultured myotubes from ß1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The ß2a-mediated Ca²⁺ current recovered in ß1 KO myotubes lacking the endogenous DHPR ß1a-subunit verified formation of the 1S/ß1a pair. In myotube genotypes which express no or low-density L-type Ca²⁺ currents, namely ß1 KO and RyR1 KO, ß2a overexpression recovered a wild-type density of nifedipine-sensitive Ca²⁺ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca²⁺ current recovery, there was a drastic reduction of voltage–dependent, skeletal-type EC coupling and emergence of Ca²⁺ transients triggered by the Ca²⁺ current. A comparison of ß2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca²⁺-dependent Ca²⁺ release triggered by the ß2a-subunit. In ß1 KO and RyR1 KO myotubes, the Ca²⁺-dependent EC coupling promoted by ß2a overexpression had the following characteristics: 1), L-type Ca²⁺ currents had a wild-type density; 2), Ca²⁺ transients activated much slower than controls overexpressing ß1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca²⁺ current; 3), the voltage dependence of the Ca²⁺ transient was bell-shaped and the maximum was centered at +30 mV, consistent with the voltage dependence of the Ca²⁺ current; and 4), Ca²⁺ currents and Ca²⁺ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by ß2a was inferred by the drastic reduction in maximal Ca²⁺ fluorescence at large positive potentials (F/F_max) in double dysgenic/ß1 KO myotubes overexpressing the pore mutant 1S (E1014K) and ß2a. The data indicate that ß2a, upon interaction with the skeletal pore subunit 1S, overrides critical EC coupling determinants present in 1S. We propose that the 1S/ß pair, and not the 1S-subunit alone, controls the EC coupling signal in skeletal muscle.

This article has been cited by other articles:

				R. A. Bannister, H. M. Colecraft, and K. G. Beam Rem Inhibits Skeletal Muscle EC Coupling by Reducing the Number of Functional L-Type Ca2+ Channels Biophys. J., April 1, 2008; 94(7): 2631 - 2638. [Abstract] [Full Text] [PDF]

				S. A. Goonasekera, N. A. Beard, L. Groom, T. Kimura, A. D. Lyfenko, A. Rosenfeld, I. Marty, A. F. Dulhunty, and R. T. Dirksen Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation Contraction Coupling J. Gen. Physiol., September 24, 2007; 130(4): 365 - 378. [Abstract] [Full Text] [PDF]

				S. A. Goonasekera, S. R. W. Chen, and R. T. Dirksen Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6 Am J Physiol Cell Physiol, December 1, 2005; 289(6): C1476 - C1484. [Abstract] [Full Text] [PDF]

				L. Carbonneau, D. Bhattacharya, D. C. Sheridan, and R. Coronado Multiple Loops of the Dihydropyridine Receptor Pore Subunit Are Required for Full-Scale Excitation-Contraction Coupling in Skeletal Muscle Biophys. J., July 1, 2005; 89(1): 243 - 255. [Abstract] [Full Text] [PDF]

				A. M. Payne, Z. Zheng, E. Gonzalez, Z.-M. Wang, M. L. Messi, and O. Delbono External Ca2+-dependent excitation-contraction coupling in a population of ageing mouse skeletal muscle fibres J. Physiol., October 1, 2004; 560(1): 137 - 155. [Abstract] [Full Text] [PDF]

				D. C. Sheridan, W. Cheng, L. Carbonneau, C. A. Ahern, and R. Coronado Involvement of a Heptad Repeat in the Carboxyl Terminus of the Dihydropyridine Receptor {beta}1a Subunit in the Mechanism of Excitation-Contraction Coupling in Skeletal Muscle Biophys. J., August 1, 2004; 87(2): 929 - 942. [Abstract] [Full Text] [PDF]