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.
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.
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