Chloride Channel Involvement in Diabetes

描述(由申请人提供)：我们之前已经证明，β细胞颗粒的启动和分泌需要通过氯通道ClC3的颗粒氯-通量。当ClC-3在质膜上表达时，它的门控需要多功能的钙，钙调蛋白依赖的蛋白激酶II(CaMKII)的磷酸化。在这一应用中的实验将探讨CaMKII通过相同的途径调节胰岛β细胞胞吐的假设，即颗粒氯通道ClC3的磷酸化依赖的门控。来自其他实验室的数据显示，胰岛素促分泌剂激活了CaMKII，促进了钙离子跨质膜内流以及通过释放细胞内存储。此外，CaMKII的激活与胰岛灌流后产生的胰岛素在时间上存在相关性。拟议的研究将研究ClC-3在CaMKII依赖的胰岛素分泌调节中的作用：首先，我们将检测ClC-3基因敲除小鼠的表型，并确定它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岛β细胞中表明，药物抑制ClC-3可以阻止胰岛素的分泌。在本申请中提出的实验中，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存在缺陷。其次，为了证实CaMKII的活性对胰岛素分泌的增强是必需的，我们将检查颗粒酸化是否依赖于CaMKII。颗粒酸化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是确定β细胞功能所必需的。拟议的研究将表征一个调控步骤和参与β细胞分泌的相关蛋白质。与公共健康相关：这项研究将解决在胰腺β细胞中氯通道活性是如何调节的问题，以及氯通道活性调节与胰岛素分泌之间是否存在联系。更好地了解氯通道活动是如何调节的，以及它与胰岛素分泌不足的关系可能会导致新的治疗方法的发展，以改善2型糖尿病的病程。

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.