Characterization of Ryanodine Receptor-mediated Calcium Release in Human B Cells: Relevance to Diagnostic Testing for Malignant Hyperthermia.

Background: Mutations in the ryanodine type 1 receptor (RyR1) are causative for malignant hyperthermia. Studies in human B lymphocytes have shown that measurement of RyR1-mediated intracellular Ca2+ (Ca2+i) release can differentiate between normal and malignant hyperthermia-susceptible individuals. The authors have further developed the B-cell assay by pharmacologically characterizing RyR1-mediated Ca2+ release in two normal human B-cell lines and demonstrating increased sensitivity of lymphocytes to the RyR1 agonist 4-chloro-m-cresol (4-CmC) in the porcine model of MH. Methods: Ca2+i was measured fluorometrically using fura-2 in populations of cells in suspension or with fluo-4 in single cells using confocal microscopy. The Dakiki and PP normal human B cell lines were used, as well as lymphocytes obtained from normal and malignant hyperthermia-susceptible pigs. 4-CmC was used to elicit RyR1-mediated Ca2+ release; all experiments were performed in the absence of external Ca2+. Results: EC50 values for 4-CmC were 0.98 and 1.04 mm for Dakiki and PP cells, respectively, demonstrating reproducibility. The 4-CmC-induced increase in Ca2+i was eliminated by thapsigargin and was unaffected by xestospongin C. The Ca2+i increase was separable from mitochondrial stores and was inhibited by azumolene. Caffeine did not induce Ca2+ i release, but ryanodine depleted intracellular stores by 50%. Lymphocytes from pigs carrying the Arg614Cys mutation in RyR1 showed increased sensitivity to 4-CmC (EC50 =3D 0.47 vs. 0.81 mm for cells derived from normal animals). Conclusions: RyR1-mediated Ca2+ signals can be pharmacologically distinguished from other intracellular sources in human B cells, and alterations of RyR1 function can be successfully detected using Ca2+ release from intracellular stores as an end point