Uncoupling of Cav1.2 From Ca2+-Induced Ca2+ Release and SK Channel Regulation in Pancreatic β-Cells

We investigated the role of Ca(v)1.2 in pancreatic beta-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Ca(v)1.2/ II-III cells) to disrupt channel-protein interactions. Neither block of K-ATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Ca-v current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 mu M) stimulated insulin secretion and Ca2+ transients in INS-1 cells, and Ca(v)1.2/II-III cells were completely blocked by nicardipine (2 mu M), but thapsigargin (1 mu M) blocked tolbutamide-stimulated secretion and Ca2+ transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca2+] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca2+ transients in both INS-1 cells and Cav1.2/ II-III cells were significantly potentiated by 8-pCPT-2 '-O-Me-cAMP (5 mu M), FPL-64176 (0.5 mu M), or replacement of extracellular Ca2+ with Sr2+. Glucose (10 mM) + glucagon-like peptide- 1 (10 nM) stimulated discrete spikes in [ Ca2+](i) in the presence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM) stimulated more frequent action potentials in Ca(v)1.2/II-III cells and primary rat beta-cells expressing the Ca(v)1.2/II-II loop than in control cells. Further, apamin (1 mu M) increased glucose-stimulated action potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channels were not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-III loop of Cav1.2 as a key molecular determinant that couples the channel to Ca2+ -induced Ca2+ release and activation of SK channels in pancreatic beta-cells.