Muscarinic agonists activate Ca2+ store-operated and -independent ionic currents in insulin-secreting HIT-T15 cells and mouse pancreatic β-cells

The neurotransmitter acetylcholine, a muscarinic receptor agonist, augments glucose-induced insulin secretion from pancreatic beta-cells by depolarizing the membrane to enhance voltage-gated Ca2+ influx. To clarify the electrical events involved in this process, we measured ionic currents from a clonal beta-cell line (HIT-TI5) and mouse pancreatic beta-cells. In whole-cell recordings, the muscarinic agonist carbachol (CCh) dose-dependently and reversibly activated a voltage-independent, nonselective current (whole-cell conductance 24 pS/pF, reversal potential similar to-15 mV). The current, which we refer to as I-musc, was blocked by atropine, a muscarinic receptor antagonist, and SKF 96365, a nonspecific ion channel blocker. The magnitude of the current decreased by 52% when extracellular Na+ was removed, but was not affected by changes in extracellular Ca2+ confirming that I-musc is a nonselective current. To determine if activates following release of Ca2+ from an intracellular store, we blocked intracellular IP3 receptors with heparin. Carbachol still activated a current in the presence of heparin, demonstrating the presence of a Ca2+ store-independent, muscarinic agonist-activated ionic current in HIT cells. However, the store-independent current was smaller and had a more positive reversal potential(similar to0 mV) than the current activated by CCh under control conditions. This result indicates that heparin had blocked a component of I-musc, which likely activates following release of stored Ca2+. Depleting IP3-sensitive calcium stores with thapsigargin also activated a non-selective, SKF 96365-blockable current in HIT cells. The properties of this putative store-operated current were similar to the component of I-musc that was blocked by heparin, being voltage-independent and reversing near -30 mV. We conclude that I-musc consists of store-operated and store-independent components, both of which may contribute to the depolarizing action of muscarinic agonists on pancreatic beta-cells.