title: Role of some unconserved residues in the "C" region of the skeletal DHPR II-III loop.
abstract: 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 >or=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 CS converted the peptides' function from skeletal-like to cardiac-like. The individual A739P mutation, but not F741T or P742T, reduced the functional efficacy of CS. None of the mutations significantly altered the NMR-based secondary structure of the C residues in SDCLAFP-PTT or CS. The CS peptide and its mutants, like the cardiac CC 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.
authors.affiliation: Division of Molecular Bioscience, JCSMR and Research School of Chemistry, ANU, Canberra, ACT 2601, Australia. angela.dulhunty@anu.edu.au <angela.dulhunty@anu.edu.au>
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.forename: Yamuna
authors.initials: Y
authors.lastname: Karunasekara
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Curtis
authors.forename: Peta J
authors.initials: PJ
authors.lastname: Harvey
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
authors.forename: Maroco G
authors.initials: MG
authors.lastname: Casarotto
mesh_terms: D015220:Calcium Channels / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel