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Structural determination of interactions between the the ryanodine receptor and the recombinant DHPR II-III loop and synthetic “C” peptides

A.F. Dulhunty, Y. Karunasekara, S.M. Curtis, P.J. Harvey, P.G. Board, M.G. Casarotto, Molecular Bioscience, JCSMR, Canberra City, ACT, Australia

Skeletal muscle excitation-contraction coupling (S-ECC) is independent of extracellular Ca2+ influx, but requires a skeletal sequences in the II-III loop of the dihydropyridine receptor (DHPR) particularly in “C” region (residues 724-760). It was recently shown that substitution of cardiac for skeletal residues at positions 737, 741 or 742 in the loop abolishes S-ECC in myocytes1. To explore the hypothesis that S-ECC depends on a physical interaction between the skeletal ryanodine receptor (RyR1) and the II-III loop, we examined the effect of these substitutions on functional interactions between native RyR1 channels in lipid bilayers and the recombinant skeletal DHPR II-III loop (SDCL), or skeletal C region peptide (CS).

Mutants SDCLA-P, SDCLF-T and SDCLP-T contained individual substitutions A737P, F741T and P742T and SDCLAFP PTT contained the triple substitution. Wild type SDCL activated RyR1 with high affinity (≥10 nM). In contrast SDCLAFP-PTT inhibited channels (≥100 nM). SDCLA-P mimicked the inhibitory action of the triple mutant, while activation by SDCLF-T or SDCLP-T was indistinguishable from wild-type. Curiously, the cardiac II-III loop (CDCL) activated RyR1 so that the mutations did not functionally convert SDCL to CDCL. The triple A737P, F741T and P742T mutation in peptide CS (a) converted its function from skeletal-like to cardiac-like and (b) changed its structure from skeletal-like random coil to cardiac-like nascent helix (assessed from NMR analysis). The data indicate that SDCL interacts with RyR1 through both it's C region and N-terminal A region. The results show that A373 is critical for the structure of the C region and for functional consequences of interactions between RyR1 and the II-III loop and support the hypothesis that S-ECC depends on a physical interaction between RyR1 and the DHPR.

(1) Kugler, G., Weiss, R.G., Flucher, B.E., Grabner, M. 2004. Structural requirements of the dihydropyridine receptor alpha1S II-III loop for skeletal-type excitation-contraction coupling. J. Biol. Chem. 279: 4721-8.