Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic β-Cells

Ryanodine receptors (RyR) are Ca 2+ channels that mediate Ca 2+ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro- m -cresol (4CmC), agonists of RyR, stimulated Ca 2+ entry that was independent of store-operated Ca 2+ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γ K = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP 3 receptors (IP 3 R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP 3 R3, we showed that RyR2 mediates both the Ca 2+ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic β-cells, where they mediate Ca 2+ entry.