Biophys J, December 1999, p. 2953-2967, Vol. 77, No. 6

Involvement of the Carboxy-Terminus Region of the Dihydropyridine Receptor 1a Subunit in Excitation-Contraction Coupling of Skeletal Muscle

 ^*Department of Physiology, University of Wisconsin School of Medicine, and  ^#Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706; and  ^§Department of Biochemistry, University of Louisville, Louisville, Kentucky 40202 USA

Skeletal muscle knockout cells lacking the  subunit of the dihydropyridine receptor (DHPR) are devoid of slow L-type Ca²⁺ current, charge movements, and excitation-contraction coupling, despite having a normal Ca²⁺ storage capacity and Ca²⁺ spark activity. In this study we identified a specific region of the missing 1a subunit critical for the recovery of excitation-contraction. Experiments were performed in 1-null myotubes expressing deletion mutants of the skeletal muscle-specific 1a, the cardiac/brain-specific 2a, or 2a/1a chimeras. Immunostaining was used to determine that all  constructs were expressed in these cells. We examined the Ca²⁺ conductance, charge movements, and Ca²⁺ transients measured by confocal fluo-3 fluorescence of transfected myotubes under whole-cell voltage-clamp. All constructs recovered an L-type Ca²⁺ current with a density, voltage-dependence, and kinetics of activation similar to that recovered by full-length 1a. In addition, all constructs except 2a mutants recovered charge movements with a density similar to full-length 1a. Thus, all  constructs became integrated into a skeletal-type DHPR and, except for 2a mutants, all restored functional DHPRs to the cell surface at a high density. The maximum amplitude of the Ca²⁺ transient was not affected by separate deletions of the N-terminus of 1a or the central linker region of 1a connecting two highly conserved domains. Also, replacement of the N-terminus half of 1a with that of 2a had no effect. However, deletion of 35 residues of 1a at the C-terminus produced a fivefold reduction in the maximum amplitude of the Ca²⁺ transients. A similar observation was made by deletion of the C-terminus of a chimera in which the C-terminus half was from 1a. The identified domain at the C-terminus of 1a may be responsible for colocalization of DHPRs and ryanodine receptors (RyRs), or may be required for the signal that opens the RyRs during excitation-contraction coupling. This new role of DHPR  in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.

Biophys J, December 1999, p. 2953-2967, Vol. 77, No. 6
© 1999 by the Biophysical Society   0006-3495/99/12/2953/15  $2.00

This article has been cited by other articles:

				R. A. Bannister, M. Grabner, and K. G. Beam The {alpha}1S III-IV Loop Influences 1,4-Dihydropyridine Receptor Gating but Is Not Directly Involved in Excitation-Contraction Coupling Interactions with the Type 1 Ryanodine Receptor J. Biol. Chem., August 22, 2008; 283(34): 23217 - 23223. [Abstract] [Full Text] [PDF]

				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]

				N. M. Lorenzon and K. G. Beam Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling J. Gen. Physiol., September 24, 2007; 130(4): 379 - 388. [Abstract] [Full Text] [PDF]

				A. A. Anderson, X. Altafaj, Z. Zheng, Z.-M. Wang, O. Delbono, M. Ronjat, S. Treves, and F. Zorzato The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca2+ channel Cav1.1 J. Cell Sci., May 15, 2006; 119(10): 2145 - 2155. [Abstract] [Full Text] [PDF]

				V. Leuranguer, S. Papadopoulos, and K. G. Beam Organization of Calcium Channel beta1a Subunits in Triad Junctions in Skeletal Muscle J. Biol. Chem., February 10, 2006; 281(6): 3521 - 3527. [Abstract] [Full Text] [PDF]

				M. C. Garcia, E. Carrillo, J. M. Galindo, A. Hernandez, J. A. Copello, M. Fill, and J. A. Sanchez Short-Term Regulation of Excitation-Contraction Coupling by the {beta}1a Subunit in Adult Mouse Skeletal Muscle Biophys. J., December 1, 2005; 89(6): 3976 - 3984. [Abstract] [Full Text] [PDF]

				A. M. Hurne, J. J. O'Brien, D. Wingrove, G. Cherednichenko, P. D. Allen, K. G. Beam, and I. N. Pessah Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion J. Biol. Chem., November 4, 2005; 280(44): 36994 - 37004. [Abstract] [Full Text] [PDF]

				R. P. Schuhmeier, E. Gouadon, D. Ursu, N. Kasielke, B. E. Flucher, M. Grabner, and W. Melzer Functional Interaction of CaV Channel Isoforms with Ryanodine Receptors Studied in Dysgenic Myotubes Biophys. J., March 1, 2005; 88(3): 1765 - 1777. [Abstract] [Full Text] [PDF]

				H. Takekura, C. Paolini, C. Franzini-Armstrong, G. Kugler, M. Grabner, and B. E. Flucher Differential Contribution of Skeletal and Cardiac II-III Loop Sequences to the Assembly of Dihydropyridine-Receptor Arrays in Skeletal Muscle Mol. Biol. Cell, December 1, 2004; 15(12): 5408 - 5419. [Abstract] [Full Text] [PDF]

				S. Papadopoulos, V. Leuranguer, R. A. Bannister, and K. G. Beam Mapping Sites of Potential Proximity between the Dihydropyridine Receptor and RyR1 in Muscle Using a Cyan Fluorescent Protein-Yellow Fluorescent Protein Tandem as a Fluorescence Resonance Energy Transfer Probe J. Biol. Chem., October 15, 2004; 279(42): 44046 - 44056. [Abstract] [Full Text] [PDF]

				N. M. Lorenzon, C. S. Haarmann, E. E. Norris, S. Papadopoulos, and K. G. Beam Metabolic Biotinylation as a Probe of Supramolecular Structure of the Triad Junction in Skeletal Muscle J. Biol. Chem., October 15, 2004; 279(42): 44057 - 44064. [Abstract] [Full Text] [PDF]

				C. Paolini, J. D. Fessenden, I. N. Pessah, and C. Franzini-Armstrong Evidence for conformational coupling between two calcium channels PNAS, August 24, 2004; 101(34): 12748 - 12752. [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]

				G. Kugler, R. G. Weiss, B. E. Flucher, and M. Grabner Structural Requirements of the Dihydropyridine Receptor {alpha}1S II-III Loop for Skeletal-type Excitation-Contraction Coupling J. Biol. Chem., February 6, 2004; 279(6): 4721 - 4728. [Abstract] [Full Text] [PDF]

				D. C. Sheridan, L. Carbonneau, C. A. Ahern, P. Nataraj, and R. Coronado Ca2+-Dependent Excitation-Contraction Coupling Triggered by the Heterologous Cardiac/Brain DHPR {beta}2a-Subunit in Skeletal Myotubes Biophys. J., December 1, 2003; 85(6): 3739 - 3757. [Abstract] [Full Text] [PDF]

				C. A. Ahern, David. C. Sheridan, W. Cheng, L. Mortenson, P. Nataraj, P. Allen, M. D. Waard, and R. Coronado Ca2+ Current and Charge Movements in Skeletal Myotubes Promoted by the {beta}-Subunit of the Dihydropyridine Receptor in the Absence of Ryanodine Receptor Type 1 Biophys. J., February 1, 2003; 84(2): 942 - 959. [Abstract] [Full Text] [PDF]

				D. C. Sheridan, W. Cheng, C. A. Ahern, L. Mortenson, D. Alsammarae, P. Vallejo, and R. Coronado Truncation of the Carboxyl Terminus of the DihydropyridineReceptor {beta}1a Subunit Promotes Ca2+ Dependent Excitation-Contraction Coupling in Skeletal Myotubes Biophys. J., January 1, 2003; 84(1): 220 - 237. [Abstract] [Full Text] [PDF]

				K. M.S. O'Connell, N. Yamaguchi, G. Meissner, and R. T. Dirksen Calmodulin Binding to the 3614-3643 Region of RyR1 Is Not Essential for Excitation-Contraction Coupling in Skeletal Myotubes J. Gen. Physiol., August 26, 2002; 120(3): 337 - 347. [Abstract] [Full Text] [PDF]

				C. A. Ahern, J. Arikkath, P. Vallejo, C. A. Gurnett, P. A. Powers, K. P. Campbell, and R. Coronado Intramembrane charge movements and excitation- contraction coupling expressed by two-domain fragments of the Ca2+ channel PNAS, May 18, 2001; (2001) 111001898. [Abstract] [Full Text]

				C. A. Ahern, J. Arikkath, P. Vallejo, C. A. Gurnett, P. A. Powers, K. P. Campbell, and R. Coronado Intramembrane charge movements and excitation- contraction coupling expressed by two-domain fragments of the Ca2+ channel PNAS, June 5, 2001; 98(12): 6935 - 6940. [Abstract] [Full Text] [PDF]