Involvement of the carboxy-terminus region of the dihydropyridine receptor β1a 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 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 beta 1a subunit critical for the recovery of excitation-contraction. Experiments were performed in beta-null myotubes expressing deletion mutants of the skeletal muscle-specific pla, the cardiac/brain-specific beta 2a, or beta 2a/beta 1a chimeras. Immunostaining was used to determine that all beta 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 beta 1a. In addition, all constructs except beta 2a mutants recovered charge movements with a density similar to full-length beta 1a. Thus, all beta constructs became integrated into a skeletal-type DHPR and, except for beta 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 pla or the central linker region of pla connecting two highly conserved domains. Also, replacement of the N-terminus half of beta 1a with that of beta 2a had no effect. However, deletion of 35 residues of pla 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 beta 1a. The identified domain at the C-terminus of beta 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 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.