Chloride Channel Involvement in Diabetes

氯离子通道参与糖尿病

基本信息

批准号：
8293392
负责人：
DEBORAH J. NELSON
金额：
$ 38.22万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-28 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8293392
关键词：
Acetylcholine Address Adopted Affinity Animals Area Beta Cell Biological Assay Calcium/calmodulin-dependent protein kinase Cell membrane Cell physiology Cell secretion Cell surface Cells Chemosensitization Chicago Chloride Channels ClC-3 channel Code Coupled Cytoplasmic Granules Cytoplasmic Tail Data Development Diabetes Mellitus Electric Capacitance Exhibits Exocytosis Fasting Fluorescence Fluorescence Microscopy Glucose Glucose tolerance test Goals Health Hyperglycemia Imaging Techniques Immunoprecipitation In Vitro Individual Insulin Intracellular Membranes Ion Channel Kinetics Knock-out Knockout Mice Knowledge Label Laboratories Lead Length Lentivirus Infections Life Link Measurement Mediating Membrane Membrane Potentials Modeling Modification Molecular Mus Mutant Strains Mice Non-Insulin-Dependent Diabetes Mellitus Pathway interactions Peptide antibodies Phenotype Phosphorylation Physiological Play Primary Cell Cultures Process Protein Dephosphorylation Proteins Regulation Role Secretory Vesicles Shunt Device Stimulus Structure of beta Cell of islet Subfamily lentivirinae System Testing Time Total Internal Reflection Fluorescent Transfection Universities Vesicle Wild Type Mouse base calmodulin-dependent protein kinase II carbon fiber cellular imaging diabetic glucose tolerance in vivo insulin granule insulin secretagogues insulin secretion islet knock-down morphometry mutant overexpression prevent research study response vacuolar H+-ATPase

项目摘要

DESCRIPTION (provided by applicant): We have previously shown that granular Cl-flux through the chloride channel ClC3 is required for beta cell granule priming and secretion. When ClC-3 is expressed in the plasma membrane its gating requires phosphorylation by the multifunctional calcium, calmodulin dependent protein kinase II (CaMKII). Experiments in this application will investigate the hypothesis that CaMKII regulates exocytosis in pancreatic beta cells through the same pathway, namely the phosphorylation dependent gating of the granular chloride channel, ClC3. Data from other laboratories have shown activation of CaMKII by insulin secretagogues that promote Ca2+ influx across the plasma membrane as well as via release from intracellular stores. Furthermore, activation of CaMKII is temporally correlated with insulin from perifused islets. The proposed studies will examine the role of ClC-3 in the CaMKII-dependent regulation of insulin secretion: First, we will examine the phenotype of the ClC-3 knockout mouse and determine whether it exhibits a diabetic phenotype. We have previously shown in pancreatic beta cells isolated from wild-type mice that pharmacological inhibition of ClC-3 prevents insulin secretion. In experiments proposed in this application we will determine whether insulin secretion in beta cells isolated from the ClC3 knockout mouse is defective. Second, in order to confirm that the activity of CaMKII is necessary for potentiation of insulin secretion, we will examine whether granule acidification, a prerequisite to granule fusion and insulin release, is CaMKII-dependent. And finally, we will determine the subcellular localization of CaMKII in pancreatic beta cells and examine whether granular ClC3 is phosphorylated by CaMKII as is the case when the channel is expressed at the plasma membrane. Knowledge of the expression and regulation of the channels that control granule acidification, the priming step necessary for secretion is integral to the determination of beta cell function. The proposed studies will characterize a regulatory step and associated proteins that are involved in beta cell secretion. PUBLIC HEALTH RELEVANCE: This study will address the question of how chloride channel activity is regulated in the pancreatic beta cell and whether there is a link between the regulation of chloride channel activity and insulin secretion. A better understanding of how chloride channel activity regulated and its relationship with insulin-secretion deficiency may lead to the development of new therapies that ameliorate the course of type 2 diabetes.

描述（由申请人提供）：我们先前已经表明，通过氯化物通道Clc3的颗粒状Cl升 - β细胞颗粒启动和分泌需要。当Clc-3在质膜中表达时，其门控需要通过多功能钙，钙调蛋白依赖性蛋白激酶II（CAMKII）磷酸化。该应用程序中的实验将研究CAMKII通过相同途径调节胰腺β细胞中的胞吐作用的假设，即颗粒氯化物通道的磷酸化依赖性门控CLC3。来自其他实验室的数据表明，胰岛素促分泌物激活了CAMKII，促进质膜的Ca2+流入以及通过细胞内存储的释放。此外，CAMKII的激活在时间上与Perifused Islet的胰岛素相关。拟议的研究将研究CLC-3在CAMKII依赖性胰岛素分泌调节中的作用：首先，我们将检查CLC-3基因敲除小鼠的表型，并确定其是否表现出糖尿病表型。我们以前已经在从野生型小鼠中分离出的胰腺β细胞中表明，CLC-3的药理抑制可防止胰岛素分泌。在本应用程序中提出的实验中，我们将确定从CLC3基因敲除小鼠分离的β细胞中的胰岛素分泌是否有缺陷。其次，为了确认CAMKII的活性对于增强胰岛素分泌是必要的，我们将检查颗粒酸化是CAMKII依赖性的，是颗粒融合和胰岛素释放的先决条件。最后，我们将确定CAMKII在胰腺β细胞中的亚细胞定位，并检查颗粒CLC3是否被CaMKII磷酸化，就像在质膜上表达通道时的情况一样。了解控制颗粒酸化的通道的表达和调节，分泌所需的启动步骤是确定β细胞功能的组成部分。拟议的研究将表征与β细胞分泌有关的调节步骤和相关蛋白质。公共卫生相关性：这项研究将解决胰腺β细胞中氯化物通道活性如何调节的问题，以及在调节氯化物通道活性和胰岛素分泌之间是否存在联系。更好地了解氯化物通道活性如何调节及其与胰岛素分泌缺乏症的关系可能会导致发展新疗法的发展，从而改善2型糖尿病的病程。