title: 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 alpha(1s) pore subunit.
abstract: 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 alpha(1S)-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 alpha(1S) in all cases. Delta671-690 had no effect on the magnitude of voltage-evoked Ca(2+) transients or the L-type Ca(2+) current density. In contrast, Delta720-765 or Delta724-743 abolished Ca(2+) transients entirely, and L-type Ca(2+) current was reduced or absent. Surprisingly, Ca(2+) transients and Ca(2+) currents of a moderate magnitude were recovered by the double deletion mutant Delta671-690/Delta720-765. A simple explanation for this result is that Delta720-765 induces a conformation change that disrupts EC coupling, and this conformational change is partially reverted by Delta671-690. To test for Ca(2+)-entry independent EC coupling, a pore mutation (E1014K) known to entirely abolish the inward Ca(2+) current was introduced. alpha(1S) Delta671-690/Delta720-765/E1014K expressed Ca(2+) transients with Boltzmann parameters identical to those of the Ca(2+)-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.
authors.affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Ahern
authors.forename: D
authors.initials: D
authors.lastname: Bhattacharya
authors.forename: L
authors.initials: L
authors.lastname: Mortenson
authors.forename: R
authors.initials: R
authors.lastname: Coronado
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004095:Dihydropyridines / Q000737:chemistry; D004594:Electrophysiology; D017353:Gene Deletion; D006579:Heterozygote; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009132:Muscles / Q000166:cytology; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D020746:Calcium Channels, L-Type; D018076:DNA, Complementary; D004095:Dihydropyridines; C038806:1,4-dihydropyridine; D002118:Calcium