Role of Li+-permeable Na+/Ca2+ exchanger, NCLX, in the regulation of cytosolic Ca2+ and exocytosis in pancreatic β-cell

Abstract

Na+/Ca2+ exchangers are key players for Ca2+ clearance in pancreatic β-cells, but their molecular determinants and exact roles in insulin secretion are not fully understood. In the present study, I investigated the role of Na+/Ca2+ exchangers on cytosolic Ca2+ dynamics and exocytosis in rat INS-1 insulinoma cells. I newly discovered that the Li+-permeable Na+/Ca2+ exchanger (NCLX), which was known as mitochondrial Na+/Ca2+ exchanger, contributed to the Na+-dependent Ca2+ movement across the plasma membrane in INS-1 cells. Na+/Ca2+ exchange activity by NCLX was comparable to that by the Na+/Ca2+ exchanger, NCX. I also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. I further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization in small interfering (si)NCLX-transfected INS-1 cells. siRNA-mediated downregulation of NCLX did not affect the initial exocytosis, but significantly suppressed sustained exocytosis and recovery of exocytosis. XIP (NCX inhibitory peptide) or Na+ replacement for inhibiting Na+-dependent Ca2+ clearance also selectively suppressed sustained exocytosis. Consistent with the idea that sustained exocytosis requires ATP dependent-vesicle recruitment, mitochondrial function, assessed by mitochondrial membrane potential (ΔΨ), was impaired by siNCLX or XIP. However, depolarization-induced exocytosis was hardly affected by changes in intracellular Na+ concentration, suggesting a negligible contribution of mitochondrial Na+/Ca2+ exchanger. In addition, I discovered that oligomycin inhibited sustained exocytosis in the presence of intracellular ATP, suggesting that local metabolic control by mitochondria is critical for this phase. Taken together, my data indicate that Na+/Ca2+ exchanger-mediated Ca2+ clearance mediated by NCLX and NCX is crucial for optimizing mitochondrial function, which in turn contributes to vesicle recruitment for sustained exocytosis in pancreatic β-cells.