RyR2 regulates store-operated Ca²⁺ entry, phospholipase C activity, and electrical excitability in the insulinoma cell line INS-1

by Kyle E. Harvey, Shiqi Tang, Emily K. LaVigne, Evan P. S. Pratt, Gregory H. Hockerman

The ER Ca²⁺ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca²⁺entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca²⁺ entry (SOCE) stimulated with thapsigargin was reduced in RyR2^KO cells compared to controls, but was unchanged in IRBIT^KO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 μM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2^KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2^KO and IRBIT^KO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP₂ levels were increased and cortical f-actin levels were reduced in RyR2^KO cells compared to controls. Whole-cell Ca_v channel current density was increased in RyR2^KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2^KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2^KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP₂ levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Ca_v current density and SK channel activation.