L-type Ca2+ Channel Modulation of Beta Cell Function

L 型 Ca2 通道对 β 细胞功能的调节

• 批准号: 7173793
• 负责人: GREGORY Howard HOCKERMAN
• 金额: $ 20.38万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7173793
• 关键词: Adenovirus Vector; Affinity; Beta Cell; Biological Models; Blood Glucose; C-terminal; Cell Line; Cell Nucleus; Cell membrane; Cell model; Cell physiology; Cells; Class; Confocal Microscopy; Coupled; Coupling; Data; Dependence; Dihydropyridines; Distal; Drug usage; Electrophysiology (science); Etiology; Event; Gene Expression; Glucose; Goals; Hyperglycemia; Insulin; Islets of Langerhans; Knock-in Mouse; L-Type Calcium Channels; L-type calcium channel alpha(1C); Lead; Localized; Mediating; Membrane; Methods; Microscopy; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Pharmaceutical Preparations; Play; Prevention strategy; Proteins; Rattus; Role; Signal Transduction; Specificity; Structure of beta Cell of islet; System; Tail; Techniques; Testing; Tissues; Vesicle; Work; channel blockers; dihydropyridine; insulin secretion; insulinoma; mutant; novel; patch clamp; prevent; research study; trafficking; voltage

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.

描述（由申请人提供）：在II型糖尿病中，胰岛的胰岛素分泌β细胞不能分泌足量的胰岛素以维持正常的血糖水平。高血糖会导致很多严重的并发症。因此，了解介导胰岛素分泌的机制可能会导致新的治疗方法，以预防II型糖尿病的发病和并发症。L型钙通道的两个亚类Cav1.2和Cav1.3在胰腺β细胞中表达。已经描述了将对二氢吡啶（DHP）类L型通道阻断剂不敏感的Cav1.2和Cav1.3突变通道引入胰岛素瘤细胞系INS-1的“敲入”方法。在该系统中，内源性L-型通道可以用DHP药物“关闭”，从而间接分离Cav1.2或Cav1.3通道。使用该系统，它表明，Cav1.3，但不是Cav1.2通道可以介导葡萄糖刺激的胰岛素分泌，但这两个通道可以介导KCl刺激的胰岛素分泌。此外，Cav1.3而不是Cav1.2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。为了阐明Cav1.3偶联INS-1细胞中葡萄糖刺激的胰岛素分泌的特异性的机制和结构决定因素，本提议将：1.确定葡萄糖刺激的胰岛素分泌与Cav1.3特异性偶联的机制。 2.确定Cav1.3与葡萄糖刺激的胰岛素分泌优先偶联的分子决定因素。3.鉴定与Cav1.3胞内结构域相互作用并介导葡萄糖诱导的胰岛素分泌特异性的蛋白质。 4.定义INS-1细胞中Cav1.2和Cav1.3远端C末端尾部的功能作用。虽然这项工作的大部分将在INS-1细胞模型中完成，但腺病毒载体将用于将突变通道和通道片段引入原代大鼠β细胞中，以在这个生理学相关性更高的系统中重复关键实验。这项建议将利用技术，如膜片钳全细胞电生理学，共聚焦显微镜，全内反射显微镜。这一提议与PI了解L型钙通道调节和细胞功能的长期目标一致。