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

描述(申请人提供)：这项建议的总体目标是为了更好地了解2型糖尿病(T2 DM)的病理生理学。胰腺�细胞胞浆内钙浓度([Ca~(2+)]_c)升高可刺激胰岛素分泌。K+和电压门控钙电流参与了钙依赖的信号转导和胰岛素分泌，但对调节�细胞钙信号动力学的其他钙通道知之甚少。我们发表的工作表明，储存操作的阳离子通道调节葡萄糖刺激的�细胞[Ca~(2+)]c和胰岛素分泌的变化。初步证据还表明，从T2 DM小鼠模型分离的朗格汉斯胰岛中，存储操作的钙离子进入(SOCE)是异常的。关于�细胞中的SOC通道的知识有限：我们既不知道它们的分子身份，也不完全了解控制ISOC激活的生物物理性质或机制。我们将着重于确定小鼠和人类�细胞中SOCE的分子基础。我们将使用一种新颖而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干扰的分子工程和转基因小鼠的条件性基因缺失：[A]确定�细胞中ISOC的分子基础和生物物理性质，[B]确定激活SOCE的分子机制，[C]定义SOCE在�细胞功能中的作用，以及[D]确定SOCE中的缺陷是否导致与T2 DM相关的�细胞缺陷。我们提出的研究将提供令人振奋的新信息，促进对�细胞刺激-分泌耦合机制的了解，对T2 DM中�细胞失效的新见解，并为T2 DM的治疗提供新的分子靶点。

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.