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.

Central core disease (CCD) is a human congenital myopathy characterized by fetal hypotonia and proximal muscle weakness that is linked to mutations in the gene encoding the type-1 ryanodine receptor (RyR1). CCD is thought to arise from Ca ²⁺ -induced damage stemming from mutant RyR1 proteins forming “leaky” sarcoplasmic reticulum (SR) Ca ²⁺ release channels. A novel mutation in the C-terminal region of RyR1 (I4898T) accounts for an unusually severe and highly penetrant form of CCD in humans [Lynch, P. J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J. J., Vaughan, P., Zafra, G., MacLennan, D. H. & McCarthy, T. V. (1999) Proc. Natl. Acad. Sci. USA 96, 4164–4169]. We expressed in skeletal myotubes derived from RyR1-knockout (dyspedic) mice the analogous mutation engineered into a rabbit RyR1 cDNA (I4897T). Here we show that homozygous expression of I4897T in dyspedic myotubes results in a complete uncoupling of sarcolemmal excitation from voltage-gated SR Ca ²⁺ release without significantly altering resting cytosolic Ca ²⁺ levels, SR Ca ²⁺ content, or RyR1-mediated enhancement of dihydropyridine receptor (DHPR) channel activity. Coexpression of both I4897T and wild-type RyR1 resulted in a 60% reduction in voltage-gated SR Ca ²⁺ release, again without altering resting cytosolic Ca ²⁺ levels, SR Ca ²⁺ content, or DHPR channel activity. These findings indicate that muscle weakness suffered by individuals possessing the I4898T mutation involves a functional uncoupling of sarcolemmal excitation from SR Ca ²⁺ release, rather than the expression of overactive or leaky SR Ca ²⁺ release channels.

L-type Ca²⁺ channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H.T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72–75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and “RyR1-knockout” (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q_max, 7.5 ± 0.8 and 4.5 ± 0.4 nC/μF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G_max/Q_max). Thus, RyR1 enhances both the expression level and Ca²⁺ conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A_fast and A_slow) and time constants (τ_slow and τ_fast) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A_fast/[A_fast + A_slow] ∼ 0.25). However, both τ_fast and τ_slow were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (τ_fast, 8.5 ± 1.2 and 4.4 ± 0.5 ms; τ_slow, 79.5 ± 10.5 and 34.6 ± 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (−) Bay K 8644 (5 μM) caused a hyperpolarizing shift (∼10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A_slow and A_fast in normal myotubes, but a large increase in only A_fast in dyspedic myotubes. Equimolar substitution of Ba²⁺ for extracellular Ca²⁺ increased both A_fast and A_slow in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A_fast or A_slow. These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca²⁺ channel accessory subunit.