A component of excitation-contraction coupling triggered in the absence ofthe T671-L690 and L720-Q765 regions of the II-III loop of the dihydropyridine receptor alpha(1s) pore subunit

We conducted a deletion analysis of two regions identified in the II-III lo op of alpha (1S) residues 671-690, which were shown to bind to ryanodine re ceptor type 1 (RyR1) and stimulate RyR1 channels in vitro, and residues 720 -765 or the narrower 724-743 region, which confer excitation-contraction (E C) coupling function to chimeric dihydropyridine receptors (DHPRs). Deletio n mutants were expressed in dysgenic als-null myotubes and analyzed by volt age-clamp and confocal fluo-4 fluorescence. Immunostaining of the mutant su bunits 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 ef fect on the magnitude of voltage-evoked Ca2+ transients or the L-type Ca2current density. In contrast, Delta 720-765 or Delta 724-743 abolished Ca2 transients entirely, and L-type Ca2+ current was reduced or absent. Surpri singly, Ca2+ transients and Ca2+ currents of a moderate magnitude were reco vered by the double deletion mutant Delta 671-690/Delta 720-765. A simple e xplanation for this result is that Delta 720-765 induces a conformation cha nge 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 curre nt was introduced. a,, Delta 671-690/Delta 720-765/ E1014K expressed Ca2+ t ransients with Boltzmann parameters identical to those of the Ca2+-conducti ng 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.