Store-operated influx of Ca2+ in pancreatic β-cells exhibits graded dependence on the filling of the endoplasmic reticulum

The store-operated pathway for Ca2+ entry was studied in individual mouse pancreatic beta -cells by measuring the cytoplasmic concentrations of Ca2+ ([Ca2+](i)) and Mn2+ ([Mn2+](i)) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca2+ sequestration. To avoid interference with voltage-dependent Ca2+ entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca2+ depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca2+](i) or from the rate of increase in [Mn2+](i) due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn2+ depended on store filling in a graded manner. The store-operated influx of Ca2+/Mn2+ was inhibited by Gd3+ and 2-aminoethoxydiphenyl berate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the beta -cell has direct implications for the control of membrane potential and insulin secretion.