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 (C_S).

Mutants SDCL_A-P, SDCL_F-T and SDCL_P-T contained individual substitutions A737P, F741T and P742T and SDCL_AFP PTT contained the triple substitution. Wild type SDCL activated RyR1 with high affinity (≥10 nM). In contrast SDCL_AFP-PTT inhibited channels (≥100 nM). SDCL_A-P mimicked the inhibitory action of the triple mutant, while activation by SDCL_F-T or SDCL_P-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 C_S (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.