L-type Ca2+ Channel Modulation of Beta Cell Function

描述(申请人提供)：在II型糖尿病中，胰岛分泌胰岛素的β细胞不能分泌足够数量的胰岛素来维持正常的血糖水平。由此产生的高血糖会导致许多严重的并发症。因此，了解调节胰岛素分泌的机制可能会导致新的治疗方法来预防II型糖尿病的发生和并发症。L类钙通道的两个亚类Cav1.2和Cav1.3在胰岛β细胞中表达。本文报道了一种将对L类二氢吡啶类通道阻断剂不敏感的Cav1.2和Cav1.3突变通道导入胰岛素瘤细胞系INS-1的“敲入”方法。在这个系统中，内源性的L类通道可以被DHP药物“关闭”，从而从药理上隔离Cav1.2或Cav1.3的任一个通道。结果表明，Cav1.3通道可介导葡萄糖刺激的胰岛素分泌，而Cav1.2通道不能介导葡萄糖刺激的胰岛素分泌，但两个通道均可介导KCI刺激的胰岛素分泌。此外，当在INS-1细胞中过表达时，Cav1.3而不是Cav1.2的同源结构域III和IV之间的细胞内环(II-III环)完全抑制葡萄糖刺激的胰岛素分泌。为了阐明Cav1.3偶联葡萄糖刺激的INS-1细胞胰岛素分泌的机制和结构决定因素，本研究将：1.确定葡萄糖刺激的胰岛素分泌与Cav1.3偶联的机制。2.确定Cav1.3优先偶联葡萄糖刺激的胰岛素分泌的分子决定因素。3.确定与Cav1.3胞内结构域相互作用的蛋白质，并介导葡萄糖诱导的胰岛素分泌的特异性。4.确定Cav 1.2和Cav1.3的C末端末端在INS-1细胞中的功能。虽然这项工作的大部分将在INS-1细胞模型中完成，但腺病毒载体将被用于将突变通道和通道片段引入原代大鼠β细胞，以在这个更具生理相关性的系统中重复关键实验。这项提议将利用膜片钳全细胞电生理学、共聚焦显微镜和全内反射显微镜等技术。这一建议与PI了解L型钙通道调节和细胞功能的长期目标是一致的。

DESCRIPTION (provided by applicant): In type II diabetes, the insulin secreting beta cells of pancreatic islets fail to secrete insulin in sufficient quantities to maintain normal blood glucose levels. The resulting hyperglycemia can Iead to many serious complications. Therefore, understanding the mechanisms that mediate insulin secretion could lead to new therapies to prevent the onset and complications of Type II diabetes. Two Sub-classes of L-type Calcium channels, Cav1.2 and Cav1.3 are expressed in pancreatic beta cells. A "knock in" method to introduce Cav1.2 and Cav1.3 mutant channels that are insensitive to the dihydropyridine (DHP) class of L-type channel blockers into the insulinoma cell line INS-1 has been described. In this system, the endogenous L-type channels can be "shut off" with DHP drugs, thus pharmacologically isolating either Cav1.2 of Cav1.3 channels. Using this system, it is shown that Cav1.3 but not Cav1.2 channels can mediate glucose-stimulated insulin secretion, but that both channels can mediate KCI stimulated insulin secretion. Furthermore, the intracellular loop between homologous domains III and IV (II-III loop) of Cav1.3 but not Cav1.2 completely inhibits glucose-stimulated insulin secretion when over-expressed in INS-1 cells. To elucidate the mechanism and structural determinants of the specificity of Cav1.3 coupling glucose-stimulated insulin secretion in INS-1 cells, this proposal will: 1. Determine the mechanism whereby glucose stimulated insulin secretion is specifically coupled to Cav1.3. 2. Identify the molecular determinants of the preferential coupling of Cav1.3 to glucose-stimulated insulin secretion. 3. Identify proteins that interact with intracellular domains of Cav1.3 and mediate the specificity for glucose-induced insulin secretion. 4. Define functional roles for the distal C-terminal tails of Cav 1.2 and Cav1.3 in INS-1 cells. Although much of this work will be done in the INS-1 cell model, adenovirus vectors will be used to introduce mutant channels and channel fragments into primary rat beta cells to repeat key experiments in this more physiologically relevant system. This proposal will utilize techniques such as patch clamp whole-cell electrophysiology, confocal microscopy, and total internal reflection microscopy. This proposal is consistent with the PI's long-term goal of understanding L-type calcium channel modulation and cellular function.