Functional Effects of Central Core Disease Mutations in the Cytoplasmic Region of the Skeletal Muscle Ryanodine Receptor

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Abstract
Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca2+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH2-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca2+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca2+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 μM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca2+ release was smaller and activated at more negative potentials for myotubes expressing the NH2-terminal CCD mutations. The shift in the voltage dependence of SR Ca2+ release correlated strongly with changes in resting Ca2+, SR Ca2+ store depletion, and peak voltage–gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca2+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca2+ leak. These results demonstrate that the NH2-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca2+ leak, store depletion, and a reduction in voltage-gated Ca2+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.