Chloride Channel Involvement in Diabetes

氯离子通道参与糖尿病

• 批准号: 8098817
• 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
• 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/8098817
• 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

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）：我们之前已经表明，通过氯通道 ClC3 的颗粒 Cl 通量是 β 细胞颗粒启动和分泌所必需的。当 ClC-3 在质膜中表达时，其门控需要多功能钙、钙调蛋白依赖性蛋白激酶 II (CaMKII) 的磷酸化。本应用中的实验将研究 CaMKII 通过相同途径（即颗粒氯离子通道 ClC3 的磷酸化依赖性门控）调节胰腺 β 细胞中的胞吐作用的假设。其他实验室的数据表明，胰岛素促分泌剂可激活 CaMKII，促进 Ca2+ 跨质膜流入以及通过细胞内储备释放。此外，CaMKII 的激活与灌注胰岛的胰岛素在时间上相关。拟议的研究将检查 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 敲除小鼠的表型并确定其是否表现出糖尿病表型。我们之前已经在从野生型小鼠分离的胰腺β细胞中证明，药物抑制ClC-3可以阻止胰岛素分泌。在本申请提出的实验中，我们将确定从 ClC3 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（颗粒融合和胰岛素释放的先决条件）是否依赖于 CaMKII。最后，我们将确定 CaMKII 在胰腺 β 细胞中的亚细胞定位，并检查颗粒状 ClC3 是否被 CaMKII 磷酸化，就像该通道在质膜上表达时的情况一样。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）对于确定 β 细胞功能是不可或缺的。拟议的研究将描述参与β细胞分泌的调节步骤和相关蛋白质。公共健康相关性：本研究将解决胰腺β细胞中氯离子通道活性如何调节以及氯离子通道活性调节与胰岛素分泌之间是否存在联系的问题。更好地了解氯离子通道活性如何调节及其与胰岛素分泌缺乏的关系可能会导致开发出改善 2 型糖尿病病程的新疗法。