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。由此产生的高血糖可导致许多严重的并发症。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。我们已经开发了一种“敲入”方法，将Cav1.2和Cav1.3突变体通道引入胰岛素瘤细胞系INS-1中，所述突变体通道对二氢吡啶（DHP）类L型通道阻断剂不敏感。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。使用这个系统，我们已经表明，Cav1.3，而不是Cav1.2通道可以介导葡萄糖刺激的胰岛素分泌。激素GLP-1通过与GLP-1受体结合而增强胰岛素分泌。我们已经确定了一个短肽，来自GLP-1受体的一级氨基酸序列，可以模拟GLP-1的一些，但不是所有的行动。我们假设，在受体的情况下，这种肽包含一个自激活结构域，其在配体结合时被揭露。在本项目的目标1中，我们将表征该肽作为小分子激动剂的活性及其在GLP-1受体结构背景下对GLP-1受体活化的贡献。在 目的2，我们将进一步研究L型钙通道与胰岛素分泌和β细胞增殖的耦合机制，以及GLP-1受体激活如何调节它们。虽然大部分工作将在INS-1细胞模型中完成，但已经开发了病毒载体以将突变通道和通道片段引入大鼠原代β细胞。该提案将利用诸如膜片钳全细胞电生理学、荧光寿命成像、全内反射荧光显微镜、胰岛素分泌测定、免疫沉淀测定和蛋白质印迹测定等技术。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。公共卫生相关性：该项目将探索一组新的分子，这些分子在葡萄糖存在的情况下刺激胰腺的β细胞分泌胰岛素，并且还可能促进β细胞的增殖。它还将检查Ca 2+通过特定类型的Ca 2+通道流入β细胞调节胰岛素分泌的机制。这些研究结果可能为开发治疗2型糖尿病的新药提供信息。

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 lead 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. We have developed 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. 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, we have shown that Cav1.3 but not Cav1.2 channels can mediate glucose-stimulated insulin secretion. Insulin secretion is potentiated by the hormone GLP-1, by its binding to the GLP-1 receptor. We have identified a short peptide, derived from the GLP-1 receptor primary amino acid sequence, that can mimic some, but not all of the actions of GLP-1. We hypothesize that in the context of the receptor this peptide comprises an autoactivation domain that is unmasked upon ligand binding. In Aim 1 of this project, we will characterize both the activity of this peptide as a small molecule agonist, and its contribution to GLP-1 receptor activation in the context of the GLP-1 receptor structure. In Aim 2, we will further examine the mechanisms that couple L-type calcium channels to insulin secretion and beta cell proliferation, and how they are modulated by GLP-1 receptor activation. Although most of this work will be done in the INS-1 cell model, viral vectors have been developed to introduce mutant channels and channel fragments into rat primary beta cells. This proposal will utilize techniques such as patch clamp whole-cell electrophysiology, Fluorescence Lifetime Imaging, Total Internal Reflection Fluorescence Microscopy, insulin secretion assays, immunoprecipitation assays, and western blot assays. This proposal is consistent with the PI's long term goal of understanding L-type calcium channel modulation and cellular function. PUBLIC HEALTH RELEVANCE: This project will explore a set of novel molecules that stimulate beta cells of the pancreas to secret insulin in the presence of glucose, and may also promote the proliferation of beta cells. It will also examine the mechanisms by which the influx of Ca2+ into beta cells through specific types of Ca2+ channels regulates insulin secretion. The results of these studies may provide information for the development of new drugs to treat type 2 diabetes.