Abstract
The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca2+ release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca2+ release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca2+ signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca2+ release, with no significant change in SR Ca2+ content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1), indicating that the reduced Ca2+ release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca2+ release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.
Highlights
Skeletal muscle cells contract in response to a rise in cytosolic Ca2+ due to the opening of the type 1 ryanodine receptor (RyR1) Ca2+ release channels in the sarcoplasmic reticulum (SR) membrane; RyR1 opening and closure occur through intimate conformational coupling processes with voltage-sensitive dihydropyridine receptor (DHPR)/Cav1.1 proteins in the transverse tubule (T-tubule) membrane
G4896V-expressing fibers Using muscle fibers transfected with the same approach that was used here, we previously reported that overall RyR1 channel density remained homogeneous even across fiber regions exogenously expressing central core disease (CCD)-associated enhanced green fluorescent protein (EGFP)-I4897T mutant channels [20]
The human equivalents of the N-terminal EGFP-tagged G4896V and R4892W mutations in rabbit RyR1 characterized here are both associated with CCD
Summary
Skeletal muscle cells contract in response to a rise in cytosolic Ca2+ due to the opening of the type 1 ryanodine receptor (RyR1) Ca2+ release channels in the SR membrane; RyR1 opening and closure occur through intimate conformational coupling processes with voltage-sensitive dihydropyridine receptor (DHPR)/Cav1.1 proteins in the transverse tubule (T-tubule) membrane. The fact that several types of muscular disorders (e.g. malignant hyperthermia, central core disease, multiminicore disease) result from either inherited or de novo mutations in the RYR1 gene underscores the critical role that proper RyR1 Ca2+ release channel function plays in coordinating skeletal muscle performance (for review, see [1,2,3]). Among these disorders, central core disease (CCD) is associated with more than 100 RYR1 mutations [4]. There is a critical need to provide a more comprehensive understanding of how specific mutations alter RyR1 function in skeletal muscle
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