Measurement of resting cytosolic Ca²⁺ concentrations and Ca²⁺ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca²⁺ release channels

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca²⁺ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca²⁺ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca²⁺ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be 'leaky.' In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca²⁺ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca²⁺ release, indicating that maximal drug-induced Ca²⁺ release depends on the size of the endoplasmic reticulum Ca²⁺ store. The content of endogenous sarco(endo)plasmic reticulum Ca²⁺-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca²⁺ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca²⁺ storage capacity is increased as a compensatory response to an enhanced Ca²⁺ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca²⁺ reuptake, the amplitude of Ca²⁺ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca²⁺-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca²⁺ concentration.