Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel

Abstract

Type 1 ryanodine receptor (RYR1) is a Ca release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca -induced Ca release (CICR), resulting in abnormal Ca homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca content, and an increased resting cytoplasmic Ca level. The three parameters for CICR (Ca sensitivity for activation, Ca sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [ H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Reduced threshold for store overload-induced Ca2+ release is a common defect of RyR1 mutations associated with malignant hyperthermia and central core disease

“…Future studies using patient-specific, induced pluripotent stem cell-derived skeletal muscle cells or mouse models harboring some of these RyR1 mutations will be needed to directly address these questions. Encouragingly, consistent with our findings observed in HEK293 cells that MH/CCD RyR1 mutations reduce the threshold for SOICR and thus the propensity for spontaneous Ca 2+ release, increased incidence of spontaneous Ca 2+ release has also been observed in dyspedic myotubes expressing MH/CCD-RyR1 mutants [42]. Therefore, the HEK293 cell expression system represents an appropriate and valuable experimental approach to investigating the intrinsic defects of disease-associated RyR mutations.…”

“…A reduced SOICR threshold would expect to lead to enhanced spontaneous Ca 2+ release, increased cytosolic Ca 2+ , or decreased store Ca 2+ level. Indeed, some human MH/CCD RyR1 mutations have been shown to increase spontaneous Ca 2+ release, elevate resting cytosolic Ca 2+ concentrations, or decrease resting Ca 2+ store content [42]. …”

A tryptophan residue in the caffeine-binding site of the ryanodine receptor regulates Ca2+ sensitivity

“…Caffeine caused Ca 2+ transients of WT RyR1 cells in a dose-dependent manner with an EC 50 of ~1 mM 11,12 (Supplementary Figure 4). W4716A RyR1 cells did not exhibit caffeine-induced Ca 2+ transients, but they still responded to 4-CmC, another activator of the RyR1 channel 25 .…”

“…I4966A showed higher caffeine sensitivity with a reduced amplitude in caffeine-induced Ca 2+ release, indicating a typical gain-of-function phenotype 11,12 . F3573A was much less responsive to caffeine in releasing Ca 2+ compared with WT.…”

“…The binding of Ca 2+ to the Ca 2+ -binding site is an initial step for channel activation 9 ; therefore, regulation of the Ca 2+ -binding is important for channel activity and is implicated in disease states. Indeed, CICR activity is enhanced by many RYR1 mutations found in malignant hyperthermia and central core disease 10–12 and by RYR2 mutations identified in CPVT 13–15 . The molecular details of the Ca 2+ binding site and its regulation, however, remain largely unknown.…”