Molecular Physiology of Store-Dependant Calcium Entry in Pancreatic Beta Cells.

描述（由申请人提供）：本提案的总体目标是更好地了解2型糖尿病（T2DM）的病理生理学。增加细胞质Ca2+浓度（[Ca2+]c）在胰腺¿-细胞刺激胰岛素分泌。钾离子和电压门控Ca2+电流对Ca2+依赖性信号转导和胰岛素分泌有很大的影响；相对而言，对调节细胞Ca2+信号动力学的其他Ca2+通道知之甚少。我们发表的研究表明，储存操作阳离子（SOC）通道调节葡萄糖刺激的细胞[Ca2+]c和胰岛素分泌的变化。初步证据还表明，从T2DM小鼠模型中分离的朗格汉斯胰岛中储存操作的Ca2+进入（SOCE）异常。关于细胞中SOC通道（ISOC）的知识是有限的：我们既不知道它们的分子身份，也不完全了解控制ISOC激活的生物物理特性或机制。我们将专注于确定小鼠和人类细胞中SOCE的分子基础。我们将使用一种新颖和创新的实验方法组合，包括生物传感器成像技术，膜片钳电生理学，带有RNA干扰（RNAi）的分子工程和转基因小鼠的条件基因缺失：[A]确定细胞中ISOC的分子基础和生物物理特性，[B]确定激活SOCE的分子机制，[C]确定SOCE在细胞功能中的作用，[D]确定SOCE缺陷是否导致与T2DM相关的细胞缺陷。我们提出的研究将提供令人兴奋的新信息，有助于进一步了解细胞中刺激-分泌耦合机制，对T2DM的细胞衰竭有新的见解，并为治疗T2DM提供新的分子靶点。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to better understand the pathophysiology of Type 2 diabetes mellitus (T2DM). Increased cytoplasmic Ca2+ concentration ([Ca2+]c) in pancreatic ¿- cells stimulates insulin secretion. Much is known about K+ and voltage-gated Ca2+ currents that contribute to Ca2+-dependent signal transduction and insulin secretion; relatively little is known about other Ca2+ channels that regulate ¿-cell Ca2+ signaling dynamics. Our published work suggests that store-operated cation (SOC) channels regulate glucose-stimulated changes in ¿-cell [Ca2+]c and insulin secretion. Preliminary evidence also suggests that store-operated Ca2+ entry (SOCE) is abnormal in islets of Langerhans isolated from a mouse model of T2DM. Knowledge about SOC channels (ISOC) in ¿-cells is limited: we neither know their molecular identity, nor have complete understanding of the biophysical properties or mechanisms that control ISOC activation. We will focus on defining the molecular basis of SOCE in mouse and human ¿-cells. We will use a novel and innovative combination of experimental approaches that includes biosensor imaging technology, patch-clamp electrophysiology, molecular engineering with RNA interference (RNAi) and conditional gene deletion in transgenic mice to: [A] define the molecular basis and biophysical properties of ISOC in ¿-cells, [B] determine the molecular mechanisms that activate SOCE, [C] define the roles of SOCE in ¿-cell function, and [D] determine whether defects in SOCE contribute to ¿-cell defects associated with T2DM. Our proposed studies will provide exciting new information essential for advancing understanding of stimulus-secretion coupling mechanisms in ¿-cells, novel insights into ¿-cell failure in T2DM, and suggest new molecular targets for treatment of T2DM. PUBLIC HEALTH RELEVANCE: Type 2 diabetes mellitus is a significant and growing health problem in the United States. Insulin secretion failure plays a major role in the initiation and progression of diabetes. Insulin secretion is dependent on metabolism and ionic signals in insulin-secreting cells. Here, we propose to study the molecular mechanisms that regulate these ionic signals in mouse and human insulin-secreting cells.