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

Abstract

AbstractThe 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 RyR2KOcells compared to controls, but was unchanged in IRBITKOcells. 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 RyR2KOcells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KOand IRBITKOcells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2levels were increased and cortical f-actin levels were reduced in RyR2KOcells compared to controls. Whole-cell Cavchannel current density was increased by 65% in RyR2KOcells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KOcells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KOcells 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 PIP2levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Cavcurrent density, via regulation of PIP2levels, and SK channel activation.