Hormone Regulation of [Ca2+] in Pancreatic Acinar Cells

描述(申请人提供)：胰腺腺泡细胞的主要功能是分泌消化酶和少量等渗液。这两种功能都受到激素和神经递质的调节，这些激素和神经递质使用钙离子作为第二信使。同时，钙信号的异常也是各种类型胰腺炎的结点。这一假说认为，胰腺炎的发病机制是由钙离子通道(SOCs)引起的钙离子内流异常。因此，了解钙离子内流通道的调节和功能对于了解胰腺炎腺泡细胞的功能和功能障碍至关重要。腺泡细胞有几个钙内流通道，TRPC1、TRPC3、TRPC4、TRPC6和Orai1，所有这些通道都被内质网钙传感器STIM1门控。TRPC也被抑制其活性的支架Hmer 1门控。考虑到SOCs在胰腺炎中的参与，本研究的总体目标是了解STIM1和Hmer 1对胰腺SOCs的门控机制及其在胰腺炎中的作用。这将通过四个目标来实现：目标1：破译STIM1和Hmer 1对TRPC门控的分子机制。Hmer 1与PPXF基序相互作用，使通道保持关闭状态。STIM1通过与Hmer 1结合基序上游只有4个残基的两个保守残基相互作用来打开TRPC。我们建议通过STIM1来确定a)TRPC的门控机制；b)Hmer 1和STIM1如何协同作用来选通TRPC。目的2：探讨STIM1在Orai1门控中的分子机制。激动剂刺激的胰腺钙内流是由TRPC和Orai1的组合介导的，而TRPC和Orai1是由STIM1差异化地门控的。我们发现STIM1(234-)片段足以完全激活Orai1。因此，我们将：a)确定通向Orai1的最小STIM1域；b)STIM1如何打开Orai1；c)原生TRPC和原生Orai1之间的关系是什么。目的：通过对TRPC1-/-、TRPC3-/-、TRPC6-/-、Orai1-/-和STIM1-/-小鼠腺泡细胞钙信号和胞吐的研究，探讨TRPC、Orai1和STIM1在生理和病理性腺泡细胞钙信号和胞吐中的作用。目的：研究TRPC通道、Orai1、STIM1在体外诱导的胰腺炎中的作用，并测量细胞应激和细胞损伤的情况。然后，KO小鼠将被用来评估钙离子内流通道在卡瑞林和胆汁酸胰腺炎模型中的作用。与公共健康相关：胰腺在激素和神经递质的刺激下分泌消化酶和液体。刺激剂通过改变细胞内的钙离子浓度来告诉胰腺细胞如何做到这一点。大部分的钙离子从细胞外环境进入细胞内。当这一过程异常时，消化酶仍被困在细胞内，细胞染色，患者患上称为胰腺炎的疾病。这项建议的目的是了解钙离子是如何进入细胞的，以及异常的钙离子进入是如何导致胰腺炎的。这将通过研究钙离子进入模型细胞系统中的细胞和小鼠胰腺细胞中负责钙离子进入细胞的基因已被删除来实现。

DESCRIPTION (provided by applicant): The chief function of pancreatic acinar cells is the secretion of digestive enzymes and a small amount of isotonic fluid. Both functions are regulated by hormones and neurotransmitters that use Ca2+ as the second messenger. At the same time, aberrant Ca2+ signaling is the nodal point in all forms of pancreatitis. The hypothesis of this proposal is that specifically aberrant Ca2+ influx by store-operated Ca2+ channels (SOCs) is responsible for pancreatitis. Therefore, understanding regulation and function of Ca2+ influx channels is critical for understanding acinar cell function and dysfunction in the context of pancreatitis. Acinar cells have several Ca2+ influx channels, TRPC1, TRPC3, TRPC4, TRPC6 and Orai1, all of which are gated by the ER Ca2+ sensor STIM1. TRPCs are also gated by the scaffold Homer1 that inhibits their activity. Considering the involvement of SOCs in pancreatitis, the overall goal of this proposal is to understand gating mechanism of the pancreatic SOCs by STIM1 and Homer1 and their role in pancreatitis. This will be achieved in four aims: Aim 1: Decipher the molecular mechanism of TRPCs gating by STIM1 and Homer1. Homer1 interacts with a PPXF motif to keep the channels in a close state. STIM1 opens the TRPCs by interacting with two conserved residues that are only 4 aresidues upstream of the Homer1 binding motif. We propose to determine a) the gating mechanism of TRPCs by STIM1; b) how Homer1 and STIM1 function in tandem to gate the TRPCs. Aim 2: Determine molecular mechanism of Orai1 gating by STIM1. Agonist-stimulated pancreatic Ca2+ influx is mediated by a combination of TRPCs and Orai1 that are differentially gated by STIM1. We discovered that the STIM1(234-535) fragment is sufficient for full activation of Orai1. Hence we will: a) determine the minimal STIM1 domain that gates Orai1; b) how STIM1 opens Orai1; c) what is the relationship between the NATIVE TRPCs and NATIVE Orai1. Aim 3: The role of TRPCs, Orai1 and STIM1 in physiological and pathological acinar cells Ca2+ signaling and exocytosis will be determined by characterizing Ca2+ signaling and exocytosis in acinar cells from TRPC1-/-, TRPC3-/-, TRPC6-/-, Orai1-/- and STIM1-/- mice. Aim 4: The role of TRPC channels, Orai1, STIM1 will be studies in vitro-induced pancreatitis and measure cellular reporters of cell stress and cell damage. Then the KO mice will be used to evaluate the role of the Ca2+ influx channels in careluin and bile acid models of pancreatitis. PUBLIC HEALTH RELEVANCE: The pancreas secretes digestive enzymes and fluid in response to stimulation by hormones and neurotransmitters. The stimulants tell the pancreatic cells how to do so by changing the concentration of Ca2+ ions within the cells. Most of the Ca2+ enters the cells from the extracellular environment. When this process is aberrant, the digestive enzymes remain trapped within the cells and the cells dye and the patient develop the disease called pancreatitis. The aims of this proposal are to understand how Ca2+ ions enter the cells and how the aberrant Ca2+ entry causes the pancreatitis. This will be achieved by studying Ca2+ entry into the cells in model cell systems and in the cells of the pancreas of mice from which the genes that are responsible for the entry of Ca2+ into the cells have been deleted.