Molecular Mechanisms Regulating Calcium Flux In Salivary

该项目旨在了解唾液腺细胞中介导和调节Ca-2+信号的机制。唾液腺中液体分泌的神经递质刺激通过胞质[Ca-2+]的双相升高介导;由于内部释放而导致的初始瞬时增加和由于Ca-2+内流而导致的后期持续增加。持续的液体分泌直接依赖于[Ca-2+]的持续升高，从而依赖于Ca-2+内流。最近，我们的工作一直集中在唾液腺细胞中的Ca-2+内流机制，这似乎是一个介导的通过存储操作的Ca-2+内流（SOCE），这是普遍存在于许多其他非兴奋细胞。这种流入的分子机制尚未在任何细胞类型中确定。最近，瞬时受体电位（TRPC）家族的离子通道蛋白已被提出作为分子组成的存储操作的Ca-2+内流通道（SOCC）。然而，目前鉴定的TRPC的生理功能尚未完全确定。通过使用编码hTrp 1的腺病毒（AdHA-hTrp 1）在大鼠SMG和唾液腺细胞系中体内表达TRPC 1，我们先前报道了TRPC 1参与调节唾液腺细胞中的钙池操作的钙内流。在过去的一年中，我们的主要努力继续朝着表征SOCE和确定TRPC 1在唾液腺细胞的SOCE机制中的作用。与我们以前的研究一致，我们现在已经报道了TRPC 1是SOCC的一个组成部分。更重要的是，我们的研究表明，它参与了SOCC的Ca-2+依赖性反馈抑制。我们已经表明，钙调素介导这种反馈抑制结合域，aa 758 -793，在C-末端的TrpC 1。本研究首次揭示了钙依赖性反馈抑制SOCE的可能机制。 尽管TRPC 1似乎是钙库操纵的钙通道（SOCC）功能所必需的，但其在SOCC中的确切作用尚不清楚。基于现有的数据，TRPC 1可能作为SOCC的调节剂或作为孔形成亚基起作用。为了解决这个问题，我们使用了诱变方法来证明TRPC 1直接有助于SOCC活性，并且TRPC 1推定孔结构域（第5和第6 TM区域之间）中的酸性氨基酸残基参与SOCC功能。实验室正在进行的研究旨在确定TRPC 1中决定SOCC钙渗透性的特定氨基酸残基。这些数据首次证明TRPC蛋白是SOCC的成孔亚基。因此，这些研究代表了我们对唾液腺细胞中SOCE机制的理解的重大进展。已经提出SOCC由TRPC二聚体或多聚体组成。在本报告期间，我们已经表明，表位标记的TRPC 1蛋白共免疫沉淀。此外，通过使用酵母双杂交筛选和GST-下拉测定，我们已经确定TRPC 1单体通过其N-末端相互作用，可能在第一个锚定重复结构域。重要的是，TRPC 1 N-末端的表达对SOCC活性产生显性负效应，并破坏TRPC单体的相互作用。因此，我们提出TRPC 1单体的多聚化是SOCC功能所必需的，并且这是通过第一个N-末端锚蛋白重复序列的相互作用来实现的。在接下来的一年里，我们将研究TRPC 1与其他TRPC蛋白的异聚体相互作用。 我们之前已经报道过TRPC 1和3组装在与小窝蛋白-1相关的多分子信号复合物中。我们现在已经确定了在TRPC 1 N-末端的小窝蛋白-1的结合位点，并表明小窝蛋白-1对于TRPC 1和TRPC 3的质膜定位是必需的。此外，酵母双杂交分析表明TRPC 1与SNARE蛋白VAMP 2和SNAP相互作用。进一步的研究表明，VAMP 2参与TRPC 3和小窝蛋白向质膜的运输。用破伤风毒素破坏VAMP 2改变了TRPC 3和小窝蛋白的质膜定位。我们提出TRPC 1和3通过胞吐途径传递到质膜。目前，我们正在研究是否组装成小窝信号复合物发生在质膜或细胞内。我们还将确定TRPC 1信号复合物在质膜中的保留是否需要小窝蛋白-1。我们认为SOCE的调节是通过TRP相关信号复合物中的蛋白质-蛋白质相互作用来实现的。因此，鉴定其蛋白质组分至关重要。为此，我们已经启动了两种方法：酵母双杂交屏幕和蛋白质组学为基础的屏幕。在后者中，我们免疫纯化了TRPC 3，并通过使用2D-MS-MS分析来检查与之相关的蛋白质。这两种方法都产生了新的以及已知的蛋白质。在接下来的一年里，我们将继续这些研究，并通过更详细的实验来证实最初的观察结果。

This project is aimed towards understanding the mechanisms which mediate and regulate Ca-2+ signaling in salivary gland cells. Neurotransmitter stimulation of fluid secretion in salivary glands is mediated via a biphasic elevation in cytosolic [Ca-2+]; an initial transient increase due to internal release and a latter sustained increase due to Ca-2+ influx. Sustained fluid secretion is directly dependent upon the sustained elevation of [Ca-2+] and thus on Ca-2+ influx. Recently, our efforts have been focused on the Ca-2+ influx mechanism in salivary gland cells, which appears to be a mediated via store-operated Ca-2+ entry (SOCE) that is ubiquitously present in many other non-excitable cells. The molecular mechanism(s) of this influx has not yet been determined in any cell type. Recently, the transient receptor potential (TRPC) family of ion channel proteins have been proposed as molecular components of the store-operated Ca-2+ influx channel (SOCC). However, the physiological function(s) of the presently identified TRPCs has not yet been fully established. By expressing TRPC1 in vivo in rat SMG by using an adenovirus encoding hTrp1 (AdHA-hTrp1) and in salivary gland cell lines, we had previously reported that TRPC1 is involved in the regulation of store-operated calcium influx in salivary gland cells. In the past year our major effort has continued to be towards characterizing SOCE and identifying the role of TRPC1 in the SOCE mechanism of salivary gland cells. Consistent with our previous studies, we have now reported that TRPC1 is an integral component of SOCC. More importantly, our studies demonstrate that it is involved in the Ca-2+ dependent feedback inhibition of SOCC. We have shown that CaM mediates this feedback inhibition by binding to a domain, aa758-793, in the C-terminus of TrpC1. This study demonstrated for the first time a possible mechanism for the Ca-2+ dependent feedback inhibition of SOCE. Although TRPC1 appears to be required for store-operated calcium channel (SOCC) function, its exact role in SOCC is not known. Based on the available data it is possible that TRPC1 could function either as a regulator of SOCC or as a pore-forming subunit. Towards resolving this we have used a mutagenesis approach to demonstrate that TRPC1 directly contributes to SOCC activity and that acidic amino acid residues in the TRPC1 putative pore domain (between the 5th and 6th TM regions) are involved in SOCC function. Ongoing studies in the lab aim to identify the specific amino acid residue(s) in TRPC1 that determine the calcium permeability of SOCC. These data demonstrate for the first time that a TRPC protein is a pore-forming subunit of SOCC. Thus, these studies represent a major advancement in our understanding of the mechanism of SOCE in salivary gland cells. SOCC has been proposed to consist of TRPC dimers or multimers. In this reporting period we have shown that epitope-tagged TRPC1 proteins co-imunoprecipitate. Further, by using yeast-two hybrid screen and GST-pull down assays, we have determined that TRPC1 monomers interact via their N-terminus, likely at the first ankyrin-repeat domain. Importantly, expression of the TRPC1 N-terminus exerts a dominant negative effect on SOCC activity and disrupts the interaction of TRPC monomers. Thus, we propose that multimerization of TRPC1 monomers is required for SOCC function and that this is achieved via interaction of the first N-terminal ankyrin repeats. In the coming year we will examine heteromeric interaction of TRPC1 with other TRPC proteins. We have reported earlier that TRPC1 and 3 are assembled in multimolecular signaling complexes associated with caveolin-1. We have now identified the binding site for caveolin-1 in the TRPC1 N-terminus and shown that caveolin-1 is required for the plasma membrane localization of TRPC1 and TRPC3. Further, yeast-two hybrid analysis demonstrated that TRPC1 interacts with the SNARE proteins VAMP2 and SNAP. Further studies showed that VAMP2 is involved in the trafficking of TRPC3 and caveolin to the plasma membrane. Disruption of VAMP2 with tetanus toxin altered the plasma membrane localization of TRPC3 and caveolin. We propose that TRPC1 and 3 are delivered to the plasma membrane via an exocytotic pathway. Currently, we are examining whether the assembly into caveolar signaling complexes occurs at the plasma membrane or intracellularly. We will also determine whether retention of TRPC1 signaling complexes in the plasma membrane requires caveolin-1. We propose that the regulation of SOCE occurs via protein-protein interactions in the TRP-associated signaling complex. Thus, it is essential to identify its protein components. Towards this, we have initiated two approaches: a yeast-two hybrid screen and a proteomics-based screen. In the latter we have immunopurified TRPC3 and are examining the proteins that are associated with it by using 2D-MS-MS analysis. Both methods have yielded novel as well as known proteins. In the coming year we will continue these studies and also confirm the initial observations with more detailed experiments.