Involvement of the carboxy-terminus region of the dihydropyridine receptor beta1a subunit in excitation-contraction coupling of skeletal muscle.
Skeletal muscle knockout cells lacking the beta subunit of the dihydropyridine receptor (DHPR) are devoid of slow L-type Ca(2+) current, charge movements, and excitation-contraction coupling, despite having a normal Ca(2+) storage capacity and Ca(2+) spark activity. In this study we identified a specific region of the missing beta1a subunit critical for the recovery of excitation-contraction. Experiments were performed in beta1-null myotubes expressing deletion mutants of the skeletal muscle-specific beta1a, the cardiac/brain-specific beta2a, or beta2a/beta1a chimeras. Immunostaining was used to determine that all beta constructs were expressed in these cells. We examined the Ca(2+) conductance, charge movements, and Ca(2+) transients measured by confocal fluo-3 fluorescence of transfected myotubes under whole-cell voltage-clamp. All constructs recovered an L-type Ca(2+) current with a density, voltage-dependence, and kinetics of activation similar to that recovered by full-length beta1a. In addition, all constructs except beta2a mutants recovered charge movements with a density similar to full-length beta1a. Thus, all beta constructs became integrated into a skeletal-type DHPR and, except for beta2a mutants, all restored functional DHPRs to the cell surface at a high density. The maximum amplitude of the Ca(2+) transient was not affected by separate deletions of the N-terminus of beta1a or the central linker region of beta1a connecting two highly conserved domains. Also, replacement of the N-terminus half of beta1a with that of beta2a had no effect. However, deletion of 35 residues of beta1a at the C-terminus produced a fivefold reduction in the maximum amplitude of the Ca(2+) transients. A similar observation was made by deletion of the C-terminus of a chimera in which the C-terminus half was from beta1a. The identified domain at the C-terminus of beta1a 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 beta in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.
Beurg
M
M
Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
Ahern
C A
CA
Vallejo
P
P
Conklin
M W
MW
Powers
P A
PA
Gregg
R G
RG
Coronado
R
R
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D014162:Transfection
D020746:Calcium Channels, L-Type; D002118:Calcium