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

Abstract

The neurotransmitter acetylcholine, a muscarinic receptor agonist, augments glucose-induced insulin secretion from pancreatic beta-cells by depolarizing the membrane to enhance voltage-gated Ca(2+) influx. To clarify the electrical events involved in this process, we measured ionic currents from a clonal beta-cell line (HIT-T15) 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 of approximately -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 Ca(2+), confirming that I(musc) is a nonselective current. To determine if I(musc) activates following release of Ca(2+) from an intracellular store, we blocked intracellular IP(3) receptors with heparin. Carbachol still activated a current in the presence of heparin, demonstrating the presence of a Ca(2+) store-independent, muscarinic agonist-activated ionic current in HIT cells. However, the store-independent current was smaller and had a more positive reversal potential (approximately 0 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 Ca(2+). Depleting IP(3)-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.