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 i ntroduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which w as then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 tha n in cells expressing homotetrameric MH mutant RyR1, Cells expressing homot etrameric CCD or MR mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggest ing that MR and CCD mutants might be "weaky." In cells expressing homotetra meric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2 + release was correlated significantly with the amplitude of carbachol- or thapsigarsn-induced Ca2+ release, indicating that maximal drug-induced Ca2 release depends on the size of the endoplasmic reticulum Ca2+ store. The c ontent of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (S ERCA2b), measured by enzyme-linked immunosorbent assay, Ca-45(2+) uptake, a nd confocal microscopy, was increased in HEK-293 cells expressing mild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storag e capacity is increased as a compensatory response to an enhanced Ca2+ leak . When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type R yR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the ampli tude of Ca2+ release in response to low concentrations of caffeine and halo thane 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 wi th a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellul ar Ca2+ concentration.