Abstract
Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.
Highlights
Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca2؉ release channel
The aim of this study was to evaluate the impact of RyR1 mutations that produce MH and CCD in humans on cellular Ca2ϩ homeostasis
Earlier work in which wt and mutant forms of RyR1 were expressed in heterologous and homologous cells focused on measurements of caffeine and halothane sensitivity, on the amplitude of Ca2ϩ release from the sarcoplasmic reticulum (SR), on resting Ca2ϩ levels and on retrograde and orthograde interactions between mutant forms of RyR1 and DHPR (8, 14 –21, 33, 34)
Summary
Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca2؉ release channel. The correct functioning of the contraction/relaxation cycle demands a precise balance between Ca2ϩ release and re-uptake Disturbances of this balance, such as those that result from mutations in the proteins involved in the uptake and release processes, lead to muscle diseases, including Brody disease, malignant hyperthermia (MH), central core disease (CCD), and cardiomyopathy [2,3,4,5,6]. Some CCD mutations have been found to generate constitutively active channels, which would reduce the size of the SR Ca2ϩ pool [14, 15, 20] This has led to the “leaky channel” hypothesis for the formation of central cores [2, 13]. Conflicting results, in particular on the ability of EC-uncoupling mutants to elevate resting Ca2ϩ concentration, have been attributed to differences in experimental conditions, including the cell type employed [8]
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