Excitation-Contraction Coupling Is Not Affected by Scrambled Sequence in Residues 681–690 of the Dihydropyridine Receptor II-III Loop

A peptide corresponding to residues 681–690 of the II-III loop of the skeletal muscle dihydropyridine receptor α1 subunit (DHPR, α1S) has been reported to activate the skeletal muscle ryanodine receptor (RyR1)in vitro. Within this region of α1S, a cluster of basic residues, Arg681–Lys685, was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact α1S subunit with scrambled sequence in this region of the II-III loop (α1S-scr) to test the importance of residues 681–690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous α1S), α1S-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type α1S. The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mmCd2+ and 0.1 mm La3+. Furthermore, the release of intracellular Ca2+, as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for α1S and α1S-scr. These data suggest that residues 681–690 of the α1S II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.