PIP2 in pancreatic β-cells regulates voltage-gated calcium channels by a voltage-independent pathway.

Phosphatidylinositol-4,5-bisphosphate (PIP) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (Ca) channels promotes insulin secretion in pancreatic β-cells. However, whether Ca channels are regulated by PIP, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether Ca channels are regulated by PIP depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). Ca channel currents were recorded by the patch-clamp technique. The Ca current amplitude was reduced by activation of the muscarinic receptor 1 (MR) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced Ca inhibition was diminished by a high concentration of Ca chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP dialysis. Localization of PIP in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP hydrolysis and Ca channel inhibition. Furthermore, the depletion of PIP by a voltage-sensitive phosphatase reduced Ca currents in a way similar to that observed following MR activation. These results indicate that activation of the MR pathway inhibits the Ca channel via PIP depletion by a Ca-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.