Cav1.3 Is Preferentially Coupled to Glucose-Stimulated Insulin Secretion in the Pancreatic -Cell Line INS-1

L-Type Ca channel blockers inhibit glucose and KCl-stimulated insulin secretion by pancreatic cells. However, the role of the two distinct L-type channels expressed by cells, Cav1.2 and Cav1.3, in this process is not clear. Therefore, we stably transfected INS-1 cells with two mutant channel constructs, Cav1.2DHPi or Cav1.3 DHPi. Whole-cell patch-clamp recordings demonstrated that both mutant channels are insensitive to dihydropyridines (DHPs), but are blocked by diltiazem. INS-1 cells expressing Cav1.3/DHPi maintained glucoseand KClstimulated insulin secretion in the presence of DHPs, whereas cells expressing Cav1.2/DHPi demonstrated DHP resistance to only KCl-induced secretion. INS-1 cells were also stably transfected with cDNAs encoding the intracellular loop between domains II and III of either Cav1.2 or Cav1.3 (Cav1.2/II-III or Cav1.3/II-III). Glucoseand KCl-stimulated insulin secretion in Cav1.2/II-III cells were not different from untransfected INS-1 cells. However, glucose-stimulated insulin secretion was completely inhibited and KCl-stimulated secretion was substantially resistant to inhibition by DHPs, but sensitive to -agatoxin IVA in Cav1.3/II-III cells. Moreover, the L-type channel agonist FPL 64176 markedly enhanced KCl-stimulated secretion by Cav1.3/ II-III cells. Together, our results suggest that Ca influx via Cav1.3 is preferentially coupled to glucose-stimulated insulin secretion in INS-1 cells. Calcium influx through voltage-dependent Ca channels (VDCCs) plays a crucial role in insulin secretion from pancreatic cells (Wollheim and Sharp, 1981). Glucose metabolism in cells causes an increase in ATP/ADP ratio, which closes ATP-dependent potassium channels (Rajan et al., 1990). The resulting membrane depolarization opens VDCCs, and calcium influx triggers insulin secretion. Several distinct VDCCs have been detected in cells and insulinsecreting cell lines, including high-voltage-activated subtypes (Seino et al., 1992; Ligon et al., 1998) and the lowvoltage-activated channels (Zhuang et al., 2000). L-Type VDCCs play a major role in the function of pancreatic cells because L-type-specific blockers significantly inhibit glucose or depolarization-induced insulin secretion (Devis et al., 1975). Two distinct L-type channels, Cav1.2 and Cav1.3, are expressed in human pancreatic islets (Seino et al., 1992) and in insulin-secreting cells lines (Horvath et al., 1998). However, Cav1.2 and Cav1.3 are both blocked by the same smallmolecule drugs (Hockerman et al., 1997; Bell et al., 2001). Thus, the relative contribution of calcium flux through Cav1.2 and/or Cav1.3 to Ca 2 -dependent insulin secretion is