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。我们开发了一种“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，我们已经证明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。胰岛素分泌被激素GLP-1增强，通过它与GLP-1受体的结合。我们已经从GLP-1受体一级氨基酸序列中鉴定出一种短肽，它可以模仿GLP-1的一些作用，但不是所有的作用。我们假设在受体的情况下，这种肽包括一个在配体结合时被揭露的自激活结构域。在这个项目的目的1中，我们将描述这种肽作为小分子激动剂的活性，以及它在GLP-1受体结构背景下对GLP-1受体激活的贡献。在

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.