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The variably spliced ASI region of RyR1 contains a basic α-helix domain that modifies EC coupling

A.F. Dulhunty,1 T. Kimura,2 J.D. Lueck,3 P.J. Harvey,1 S.M. Pace,1 N. Ikemoto,4 M.G. Casarotto1 and R.T. Dirksen,3 1JCSMR, ANU, PO Box 334, ACT, 2601, Australia, 2Hyogo College of Medicine, 1-1 Mukogawa-cho Nishinomiya, HYOGO 663-8501, Japan, 3University of Rochester Medical Center, PO Box 711, 601 Elmwood Avenue, Rochester, NY, 14642 USA and 4Boston Biomedical Research Institute, Watertown, MA, USA.

The function of the alternatively spliced ASI residues (Ala3481-Gln3485) in the skeletal muscle ryanodine receptor (RyR1) Ca2+ release channel has been examined. The residues are present in the adult (ASI(+)RyR1) isoform but absent in the juvenile (ASI(-)RyR1) splice variant. ASI(−)RyR1 is over-expressed in myotonic dystrophy type 1 (DM1) and is less active than ASI(+) (Kimura et al., 2005). The ASI region contributes to an inhibitory inter-domain interaction which is stronger in ASI(−)RyR1 since its interruption by ASI domain peptides causes greater activation in AS1(−)RyR1 (Kimura et al., 2007). We predicted that interruption of this ASI interaction may contribute to excitation-contraction (EC) coupling and if it did, EC coupling would be stronger in AS1(−)RyR1. The ASI domain peptides include the ASI residues and a sequence of 5 contiguous positively charged residues which bind to the β1a subunit of the skeletal dihydropyridine receptor (DHPR) and whose deletion depresses EC coupling (Cheng et al., 2005). We predicted that this sequence may contribute to ASI peptide activity. Interruption of an inhibitory inter-domain interaction by domain peptide DP4 also activates RyR1 (Yamamoto et al., 2000). Based on their positions in the RyR1 sequence, we predicted that the ASI and DP4 regions must support different inter-domain interactions.

EC coupling was examined in RyR-null myotubes injected with cDNA for ASI(−)RyR1 or ASI(+)RyR1. Intracellular Ca2+ was measured in intact myotubes loaded with fura-FF AM. Voltage-gated L-channel activity and SR Ca2+ release were measured simultaneously using whole cell patch clamp. The structure of the ASI peptides was examined using nuclear magnetic resonance (NMR). Ca2+ release from isolated skeletal SR vesicles was measured using spectrophotometry. RyR activity was assessed from [3H] ryanodine binding (which increases when channel open probability increases) or from RyR channels incorporated into artificial lipid bilayers.

Ca2+ release during EC coupling was greater in myotubes expressing ASI(−)RyR1 than in those expressing ASI(+)RyR1. The L-type Ca2+ current was similar in both ASI(−)RyR1 and ASI(+)RyR1 expressing myotubes (indicating similar DHPR expression, function and alignment with RyRs). As with caffeine (Kimura et al., 2005), maximal 4-chloro-m-cresol induced Ca2+ release was less for ASI(−)RyR1. Total SR Ca2+ load and resting cytoplasmic Ca2+ concentrations were the same in both cases. These results were consistent with ASI(−) region supporting a stronger inter-domain interaction with greater inhibition, which then allowed greater activation when the interaction was interrupted during EC coupling.

The NMR-derived structures of ASI(−) and ASI(+) peptides both have random coil N-terminal regions bracketing (Ala3481-Gln3485) and an α-helical C-terminal part spanning the 5 contiguous basic residues. The activity of the ASI peptides on RyR1 activity was critically dependent on the basic residues and their inclusion in an α-helical structure. The structure and action of the ASI peptides mimicked that of the basic α-helical A region of the DHPR α1s II-III loop. In addition, the ASI peptides competed with peptide A for RyR1 activation. It remains to be determined whether this similarity between the DHPR α1s II-III loop and the ASI region is co-incidental or whether it has a functional significance in the intact cell. RyR activation by the ASI peptides and DP4 exhibited different Ca2+-dependence and the effects of the two domain peptides were additive, suggesting that they acted at separate sites on RyR1 and interrupted distinctly different inter-domain interactions.

The results show that the ASI residues have a strong influence on the efficacy of EC coupling which is stronger when they are deleted. Our findings further indicate that disruption of an inter-domain interaction involving the ASI region may play a role in EC coupling. Overall, the data suggest that enhanced Ca2+ release during EC coupling may contribute both to developmental changes in Ca2+ release and to the myopathy in DM1.

Kimura T, Nakamori M, Lueck JD, Pouliquin P, Aoike F, Fujimura H, Dirksen RT, Takahashi MP, Dulhunty AF & Sakoda S. (2005) Human Molecular Genetics, 14: 2189-200.

Kimura T, Pace SM, Wei L, Beard NA, Dirksen RT & Dulhunty AF. (2007) Biochemical Journal, 401: 317-24.

Yamamoto T, El-Hayek R & Ikemoto N. (2000) Journal of Biological Chemsitry, 275: 11618-25.

Cheng W, Altafaj X, Ronjat M & Coronado R. (2005) Proceedings of the National Academy of Science USA, 102: 19225-30.