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。这两种功能都是由激素和神经递质调节的，它们使用Ca2+作为第二信使。同时，异常Ca2+信号是所有形式胰腺炎的节点。这一建议的假设是，特别异常的Ca2+内流的存储操作Ca2+通道（soc）是负责胰腺炎。因此，了解Ca2+内流通道的调节和功能对于理解胰腺炎背景下腺泡细胞的功能和功能障碍至关重要。腺泡细胞有几个Ca2+内流通道，TRPC1、TRPC3、TRPC4、TRPC6和Orai1，它们都是由ER Ca2+传感器STIM1门控的。trpc也受到抑制其活性的支架Homer1的门控。考虑到胰腺soc在胰腺炎中的作用，本研究的总体目标是了解STIM1和Homer1对胰腺soc的门控机制及其在胰腺炎中的作用。这将实现四个目标：目标1：破译由STIM1和Homer1控制TRPCs的分子机制。Homer1与PPXF基序相互作用以保持通道处于闭合状态。STIM1通过与Homer1结合基序上游仅4个残基的两个保守残基相互作用打开trpc。我们建议通过STIM1确定a) trpc的门控机制；b) Homer1和STIM1如何串联起门控trpc。目的2：确定STIM1调控Orai1的分子机制。激动剂刺激的胰腺Ca2+内流是由TRPCs和Orai1的组合介导的，它们由STIM1差异门控。我们发现STIM1（234-535）片段足以完全激活Orai1。因此，我们将：a)确定大门Orai1的最小STIM1域；b) STIM1如何打开Orai1；c) NATIVE trpc和NATIVE Orai1之间的关系是什么？目的3：通过表征TRPC1-/-、TRPC3-/-、TRPC6-/-、Orai1-/-和STIM1-/-小鼠的腺泡细胞中的Ca2+信号传导和胞吐，来确定trpc、Orai1和STIM1在生理和病理腺泡细胞Ca2+信号传导和胞吐中的作用。目的4:TRPC通道，Orai1， STIM1的作用将在体外诱导的胰腺炎中进行研究，并测量细胞应激和细胞损伤的细胞报告。然后将KO小鼠用于评估Ca2+内流通道在胰腺炎careluin和胆汁酸模型中的作用。公共卫生相关性：胰腺分泌消化酶和液体以响应激素和神经递质的刺激。兴奋剂通过改变细胞内Ca2+离子的浓度来告诉胰腺细胞如何做到这一点。大多数Ca2+从细胞外环境进入细胞。当这个过程异常时，消化酶就会被困在细胞内，细胞就会染色，病人就会患上胰腺炎。本建议的目的是了解Ca2+离子如何进入细胞以及异常Ca2+进入如何导致胰腺炎。这将通过研究Ca2+进入模型细胞系统和小鼠胰腺细胞中的细胞来实现，这些细胞中负责Ca2+进入细胞的基因已被删除。

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.