Abstract
Ca2+ homeostasis is a vital cellular control mechanism in which Ca2+ release from intracellular stores plays a central role. Ryanodine receptor (RyR)-mediated Ca2+ release is a key modulator of Ca2+ homeostasis, and the defective regulation of RyR is pathogenic. However, the molecular events underlying RyR-mediated pathology remain undefined. Cells stably expressing recombinant human RyR2 (Chinese hamster ovary cells, CHOhRyR2) had similar resting cytoplasmic Ca2+ levels ([Ca2+]c) to wild-type CHO cells (CHOWT) but exhibited increased cytoplasmic Ca2+ flux associated with decreased cell viability and proliferation. Intracellular Ca2+ flux increased with human RyR2 (hRyR2) expression levels and determined the extent of phenotypic modulation. Co-expression of FKBP12.6, but not FKBP12, or incubation of cells with ryanodine suppressed intracellular Ca2+ flux and restored normal cell viability and proliferation. Restoration of normal phenotype was independent of the status of resting [Ca2+]c or ER Ca2+ load. Heparin inhibition of endogenous inositol trisphosphate receptors (IP3R) had little effect on intracellular Ca2+ handling or viability. However, purinergic stimulation of endogenous IP3R resulted in apoptotic cell death mediated by hRyR2 suggesting functional interaction occurred between IP3R and hRyR2 Ca2+ release channels. These data demonstrate that defective regulation of RyR causes altered cellular phenotype via profound perturbations in intracellular Ca2+ signaling and highlight a key modulatory role of FKBP12.6 in hRyR2 Ca2+ channel function.
Highlights
Ca2؉ homeostasis is a vital cellular control mechanism in which Ca2؉ release from intracellular stores plays a central role
We determined a marked reduction in the viability of resting CHOhRyR2 when compared with CHOWT, which was proportional to the expression levels of human RyR2 (hRyR2), i.e. viability of CHOH Ͻ CHOL ϽϽ CHOWT (Fig. 1C, panel b)
The normal modulation of RyR2 requires a multitude of accessory proteins, some of which are not endogenously expressed in CHO cells [21, 29] and our CHOhRyR2 cell model provides a powerful platform to determine the impact of defective hRyR2 Ca2ϩ signaling on cell function
Summary
Ca2؉ homeostasis is a vital cellular control mechanism in which Ca2؉ release from intracellular stores plays a central role. Normal cellular phenotype was restored by modulating hRyR2-mediated Ca2ϩ release via co-expression of FKBP12.6, which highlighted the role of FKBP12.6 in homeostatic Ca2ϩ signaling and cell function.
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