L-type Ca2+ Channel Modulation of Beta Cell Function

描述（由申请人提供）：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型糖尿病的发病和并发症。l型钙通道Cav1.2和Cav1.3在胰腺β细胞中表达。本文描述了一种“敲入”方法，将对二氢吡啶（DHP）类l型通道阻滞剂不敏感的Cav1.2和Cav1.3突变通道引入胰岛素瘤细胞系INS-1。在该系统中，内源性l型通道可以被DHP药物“关闭”，从而在药理学上分离Cav1.2或Cav1.3通道。利用该系统，我们发现Cav1.3通道介导葡萄糖刺激的胰岛素分泌，而Cav1.2通道不介导，但这两个通道都能介导KCI刺激的胰岛素分泌。此外，Cav1.3的同源结构域III和IV之间的胞内环（II-III环）在INS-1细胞中过表达时完全抑制葡萄糖刺激的胰岛素分泌，而Cav1.2则不完全抑制。为了阐明Cav1.3偶联葡萄糖刺激的INS-1细胞胰岛素分泌特异性的机制和结构决定因素，本课题将：1。确定葡萄糖刺激胰岛素分泌与Cav1.3特异性偶联的机制。2. 确定Cav1.3优先偶联到葡萄糖刺激胰岛素分泌的分子决定因素。3. 鉴定与细胞内Cav1.3结构域相互作用并介导葡萄糖诱导胰岛素分泌特异性的蛋白。4. 明确INS-1细胞中Cav 1.2和Cav1.3远端c端尾的功能作用。虽然大部分工作将在INS-1细胞模型中完成，但腺病毒载体将用于将突变通道和通道片段引入原代大鼠β细胞，以便在这个更生理相关的系统中重复关键实验。该方案将利用膜片钳全细胞电生理、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.