Wang Yuchen, Jarrard Rachel E, Pratt Evan P S, Guerra Marcy L, Salyer Amy E, Lange Allison M, Soderling Ian M, Hockerman Gregory H
Purdue University Life Sciences Graduate Program (R.E.J., E.P.S.P., A.M.L.) and Department of Medicinal Chemistry and Molecular Pharmacology (Y.W., M.L.G., A.E.S., I.M.S., G.H.H.), Purdue University, West Lafayette, Indiana 47907-2091.
Mol Endocrinol. 2014 Apr;28(4):458-76. doi: 10.1210/me.2013-1094. Epub 2014 Feb 7.
We investigated the role of Cav1.2 in pancreatic β-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block of KATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Cav current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 μM) stimulated insulin secretion and Ca(2+) transients in INS-1 cells, and Cav1.2/II-III cells were completely blocked by nicardipine (2 μM), but thapsigargin (1 μM) blocked tolbutamide-stimulated secretion and Ca(2+) transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca(2+)] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca(2+) transients in both INS-1 cells and Cav1.2/II-III cells were significantly potentiated by 8-pCPT-2'-O-Me-cAMP (5 μM), FPL-64176 (0.5 μM), or replacement of extracellular Ca(2+) with Sr(2+). Glucose (10 mM) + glucagon-like peptide-1 (10 nM) stimulated discrete spikes in [Ca(2+)]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 Cav1.2/II-III cells and primary rat β-cells expressing the Cav1.2/II-II loop than in control cells. Further, apamin (1 μM) 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 Ca(2+)-induced Ca(2+) release and activation of SK channels in pancreatic β-cells.
我们通过在INS-1细胞（Cav1.2/II-III细胞）中表达Cav1.2 II-III环/绿色荧光蛋白融合蛋白以破坏通道-蛋白相互作用，研究了Cav1.2在胰腺β细胞功能中的作用。与Cav1.2/II-III细胞相比，INS-1细胞中KATP通道的阻断或甲苯磺丁脲对膜去极化的刺激均无差异，但Cav1.2/II-III细胞中的全细胞Cav电流密度显著增加。甲苯磺丁脲（200μM）刺激INS-1细胞中的胰岛素分泌和Ca(2+)瞬变，尼卡地平（2μM）可完全阻断Cav1.2/II-III细胞中的该作用，但毒胡萝卜素（1μM）仅阻断INS-1细胞中甲苯磺丁脲刺激的分泌和Ca(2+)瞬变。与INS-1细胞相比，Cav1.2/II-III细胞中甲苯磺丁脲刺激的内质网[Ca(2+)]降低有所减少。然而，INS-1细胞和Cav1.2/II-III细胞中的Ca(2+)瞬变均被8-pCPT-2'-O-Me-cAMP（5μM）、FPL-64176（0.5μM）或用Sr(2+)替代细胞外Ca(2+)显著增强。在维拉帕米存在的情况下，葡萄糖（10mM）+胰高血糖素样肽-1（10nM）刺激INS-1细胞中[Ca(2+)]i的离散尖峰频率高于Cav1.2/II-II细胞。葡萄糖（18mM）刺激表达Cav1.2/II-II环的Cav1.2/II-III细胞和原代大鼠β细胞产生比对照细胞更频繁的动作电位。此外，蜂毒明肽（1μM）增加了INS-1细胞中葡萄糖刺激的动作电位频率，但未增加Cav1.2/II-III细胞中的频率，这表明在表达Cav1.2/II-III环的细胞中，在这些条件下SK通道未被激活。我们提出Cav1.2的II-III环是将通道与胰腺β细胞中Ca(2+)诱导的Ca(2+)释放和SK通道激活相偶联的关键分子决定因素。
