Differential Ca2+ sensitivity of RyR2 mutations reveals distinct mechanisms of channel dysfunction in sudden cardiac death.
Arrhythmogenic point mutations in RyR2 result in abnormal Ca(2+) release following cardiac stimulation, leading to sudden cardiac death (SCD). Recently, we have demonstrated that significant functional differences exist between SCD-linked RyR2 mutations. Here, we investigated the molecular basis of this heterogeneity and determined the sensitivity of mutant RyR2 channels to cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in living cells. Using streptolysin-O permeabilised human embryonic kidney cells, [Ca(2+)](c) was clamped in cells expressing GFP-tagged wild-type (WT) or SCD-linked RyR2 mutants (L(433)P, N(2386)I, and R(176)Q/T(2504)M). Although resting [Ca(2+)](c) was comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca(2+)-dependent inhibition following caffeine stimulation when compared with WT channels. The ER Ca(2+) store was not perturbed in these experiments. Our findings support the hypothesis that SCD-linked mutational loci may be an important mechanistic determinant of RyR2 dysfunction and indicate that there is unlikely to be a unifying mechanism for channel dysfunction in SCD.
Thomas
N Lowri
NL
Department of Cardiology, Wales Heart Research Institute, Cardiff University School of Medicine, Cardiff, Wales CF14 4XN, UK.
Lai
F Anthony
FA
George
Christopher H
CH
D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D016757:Death, Sudden, Cardiac; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D017354:Point Mutation; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism
D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium