Role of some unconserved residues in the C region of the skeletal DHPR II-III loop

The actions of the recombinant skeletal dihydropyridine receptor II-III loop (SDCL), and the C region peptide ( CS) on native skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) have been examined. Three non conserved residues in the C region of the skeletal DHPR II-III loop were replaced by the equivalent cardiac residues in SDCLAFP-PTT (A739P, F741T and P742T) and single substitutions made in SDCLA-P, SDCLF-T and SDCLP-T. Wild type SDCL as well as SDCLF-T and SDCLP-T activated RyR1 in lipid bilayers with high affinity ( 10 nM to 1 microM). Wild type SDCL at higher concentrations inhibited RyR1. In contrast, SDCLAFP-PTT and SDCLA-P inhibited the channels at = 10 nM. The inhibitory actions of these two skeletal loop mutants were distinctly different from the cardiac II-III loop (CDCL) which, like the wild-type SDCL, activated channels. In contrast to the full loop, the triple A739P, F741T and P742T mutation in peptide C-S converted the peptides' function from skeletal-like to cardiac-like. The individual A739P mutation, but not F741T or P742T, reduced the functional efficacy of C-S. None of the mutations significantly altered the NMR-based secondary structure of the C residues in SDCLAFP-PTT or C-S. The C-S peptide and its mutants, like the cardiac C-C peptide, were all partially alpha helical at low temperatures. The results show that residue A739 is critical for the functional consequences of interactions between RyR1 and either the skeletal II-III loop or CS, but that none of A739, F741 or P742 are critical determinants of the structure of the C region.