Involvement of the Carboxy-Terminus Region of the Dihydropyridine Receptor β1a Subunit in Excitation-Contraction Coupling of Skeletal Muscle

Abstract

Skeletal muscle knockout cells lacking the β 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 β1a subunit critical for the recovery of excitation-contraction. Experiments were performed in β1-null myotubes expressing deletion mutants of the skeletal muscle-specific β1a, the cardiac/brain-specific β2a, or β2a/ β1a chimeras. Immunostaining was used to determine that all β 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 β1a. In addition, all constructs except β2a mutants recovered charge movements with a density similar to full-length β1a. Thus, all β constructs became integrated into a skeletal-type DHPR and, except for β2a 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 β1a or the central linker region of β1a connecting two highly conserved domains. Also, replacement of the N-terminus half of β1a with that of β2a had no effect. However, deletion of 35 residues of β1a 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 β1a. The identified domain at the C-terminus of β1a 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 β in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.