A component of excitation-contraction coupling triggered in the absence of the T671-L690 and L720-Q765 regions of the II-III loop of the dihydropyridine receptor α1s pore subunit

We conducted a deletion analysis of two regions identified in the II-III loop of alpha (1S) residues 671-690, which were shown to bind to ryanodine receptor type 1 (RyR1) and stimulate RyR1 channels in vitro, and residues 720-765 or the narrower 724-743 region, which confer excitation-contraction (EC) coupling function to chimeric dihydropyridine receptors (DHPRs). Deletion mutants were expressed in dysgenic als-null myotubes and analyzed by voltage-clamp and confocal fluo-4 fluorescence. Immunostaining of the mutant subunits using an N-terminus tag revealed abundant protein expression in all cases. Furthermore, the maximum recovered charge movement density was > 80% of that recovered by full-length als in all cases. Delta 671-690 had no effect on the magnitude of voltage-evoked Ca2+ transients or the L-type Ca2+ current density. In contrast, Delta 720-765 or Delta 724-743 abolished Ca2+ transients entirely, and L-type Ca2+ current was reduced or absent. Surprisingly, Ca2+ transients and Ca2+ currents of a moderate magnitude were recovered by the double deletion mutant Delta 671-690/Delta 720-765. A simple explanation for this result is that Delta 720-765 induces a conformation change that disrupts EC coupling, and this conformational change is partially reverted by Delta 671-690. To test for Ca 21 -entry independent EC coupling, a pore mutation (E1014K) known to entirely abolish the inward Ca 21 current was introduced. a,, Delta 671-690/Delta 720-765/ E1014K expressed Ca2+ transients with Boltzmann parameters identical to those of the Ca2+-conducting double deletion construct. The data strongly suggest that skeletal-type EC coupling is not uniquely controlled by alpha (1S) 720-765. Other regions of alpha (1S) or other DHPR subunits must therefore directly contribute to the activation of RyR1 during EC coupling.