Ca2+ clearance by plasmalemmal NCLX, Li+-permeable Na+/Ca2+ exchanger, is required for the sustained exocytosis in rat insulinoma INS-1 cells
- 348 Downloads
Na+/Ca2+ exchangers are key players for Ca2+ clearance in pancreatic β-cells, but their molecular determinants and roles in insulin secretion are not fully understood. In the present study, we newly discovered that the Li+-permeable Na+/Ca2+ exchangers (NCLX), which were known as mitochondrial Na+/Ca2+ exchangers, contributed to the Na+-dependent Ca2+ movement across the plasma membrane in rat INS-1 insulinoma cells. Na+/Ca2+ exchange activity by NCLX was comparable to that by the Na+/Ca2+ exchanger, NCX. We also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. We further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization. Small interfering (si)RNA-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. Taken together, our 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.
KeywordsCa2+ transport Na+/Ca2+ exchanger Exocytosis Capacitance Pancreatic β-cell
This work was supported by the National Research Foundation of Korea (NRF) grant (No. 2014051826) funded by the Korea government Ministry of Science, ICT and Future Planning (MSIP)
Conflict of interest
The authors declare that they have no competing interest.
- 4.Barg S, Ma X, Eliasson L, Galvanovskis J, Gopel SO, Obermuller S, Platzer J, Renstrom E, Trus M, Atlas D, Striessnig J, Rorsman P (2001) Fast exocytosis with few Ca2+ channels in insulin-secreting mouse pancreatic B cells. Biophys J 81:3308–3323. doi: 10.1016/s0006-3495(01)75964-4 PubMedCentralCrossRefPubMedGoogle Scholar
- 20.Nita II, Hershfinkel M, Fishman D, Ozeri E, Rutter GA, Sensi SL, Khananshvili D, Lewis EC, Sekler I (2012) The mitochondrial Na+/Ca2+ exchanger upregulates glucose dependent Ca2+ signalling linked to insulin secretion. PLoS One 7, e46649. doi: 10.1371/journal.pone.0046649 PubMedCentralCrossRefPubMedGoogle Scholar
- 23.Palty R, Silverman WF, Hershfinkel M, Caporale T, Sensi SL, Parnis J, Nolte C, Fishman D, Shoshan-Barmatz V, Herrmann S, Khananshvili D, Sekler I (2010) NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. Proc Natl Acad Sci U S A 107:436–441. doi: 10.1073/pnas.0908099107 PubMedCentralCrossRefPubMedGoogle Scholar
- 24.Park S-H, Ryu S-Y, Yu W-J, Han YE, Ji Y-S, Oh K, Sohn J-W, Lim A, Jeon J-P, Lee H, Lee K-H, Lee S-H, Berggren P-O, Jeon J-H, Ho W-K (2013) Leptin promotes KATP channel trafficking by AMPK signaling in pancreatic b-cells. Proc Natl Acad Sci U S A 110:12673–12678. doi: 10.1073/pnas.1216351110 PubMedCentralCrossRefPubMedGoogle Scholar
- 28.Voronina SG, Barrow SL, Gerasimenko OV, Petersen OH, Tepikin AV (2004) Effects of secretagogues and bile acids on mitochondrial membrane potential of pancreatic acinar cells: comparison of different modes of evaluating ΔΨm. J Biol Chem 279:27327–27338. doi: 10.1074/jbc.M311698200 CrossRefPubMedGoogle Scholar