Differential roles of the L-type calcium channels Cav1.2 and Cav1.3 in modulation of insulin secretion in INS-1 cells
Abstract
L-type Ca2+ channel blockers inhibit glucose- and KCl-stimulated insulin secretion by pancreatic β cells. However, the role of the two distinct L-type channels expressed by β cells, Cav1.2 and Cav1.3, in this process is not clear. Therefore, we stably transfected INS-1 cells with two mutant channel constructs, Cav1.2/DHPi 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 glucose- and KCl-stimulated 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). Glucose- and 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 in Cav1.3/II–III cells. Measurement of [Ca2+]i changes in Ca v1.3/DHPi cells and Cav1.2/DHPi cells revealed that Ca v1.3, but not Cav1.2, was able to stimulate [Ca2+ ]i oscillation in response to glucose. Depletion of ER Ca2+ store by ryanodine did not inhibit glucose-stimulated [Ca2+]i oscillation in INS-1 cells. Moreover, Cav1.3/II–III cells failed to show [Ca2+]i changes upon glucose stimulation. INS-1 cells stably transfected with the chimeric Cav1.2/DHPi channels containing the II–III loop of Cav1.3 (Cav1.2/DHPi/1.3II–III) demonstrated nifedipine-resistant insulin secretion in response to glucose. Our results suggest that Ca2+ influx via Cav1.3 is preferentially coupled to glucose-stimulated insulin secretion and [Ca 2+]i oscillation in INS-1 cells, and the intracellular II–III loop of Cav1.3 plays a role in this coupling. We examined the roles of Cav1.2 and Cav1.3 in the insulinotropic effect of cAMP. Both Cav1.2/DHPi cells and Ca v1.3/DHPi cells exhibited cAMP-potentiated glucose-induced insulin secretion in the presence of nifedipine. The PKA inhibitor H89, thapsigargin, and ryanodine (0.5 μM) inhibited cAMP-potentiated insulin secretion. Moreover, the effect of cAMP was completely blocked in Cav1.2/II–III cells, but normal in Cav1.3/II–III cells. These results suggest that both Cav1.2 and Cav1.3 are able to mediate cAMP-potentiated glucose-induced insulin secretion via a mechanism that involves ER Ca 2+ release.
Degree
Ph.D.
Advisors
Hockerman, Purdue University.
Subject Area
Neurology|Pharmacology
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