Molecular Mechanisms Regulating Calcium Flux In Salivary

本项目旨在了解唾液腺细胞中钙信号的调节机制。神经递质对唾液腺液体分泌的刺激是通过胞浆[Ca-2+]的双相升高来调节的；最初的一过性增加是由于内部释放，而后者的持续增加是由于Ca-2+的内流。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，我们的工作集中在唾液腺细胞的钙内流机制上，这种机制似乎是通过广泛存在于许多其他非兴奋性细胞中的商店操作的钙内流(SOCE)来调节的。这种流入的分子机制(S)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被认为是钙离子内流通道(SOCC)的分子成分。然而，目前已鉴定的TRPC的生理功能(S)尚未完全确定。通过利用携带hTrp1基因的腺病毒载体(Adha-hTrp1)在大鼠SMG和涎腺细胞系中表达TRPC1，我们已报道TRPC1参与调节唾液腺细胞内钙内流。在过去的一年里，我们的主要工作继续是确定唾液腺细胞SOCE的特征和确定TRPC1在SOCE机制中的作用。与我们之前的研究一致，我们现在已经报道了TRPC1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的钙依赖反馈抑制。我们已经证明，CaM通过与TrpC1 C末端的一个结构域aa758-793结合来介导这种反馈抑制。本研究首次证实了SOCE的钙依赖反馈抑制的可能机制。 尽管TRPC1似乎是钙离子通道(SOCC)功能所必需的，但它在SOCC中的确切作用尚不清楚。根据现有的数据，TRPC1可能既可以作为SOCC的调节因子，也可能作为成孔亚基发挥作用。为了解决这个问题，我们用突变的方法证明了TRPC1直接参与SOCC的活性，并且TRPC1推测的孔区(在第5和第6 TM区域之间)的酸性氨基酸残基参与了SOCC的功能。实验室正在进行的研究旨在确定TRPC1中决定SOCC钙通透性的特定氨基酸残基(S)。这些数据首次证明TRPC蛋白是SOCC的一个成孔亚基。因此，这些研究代表了我们对唾液腺细胞SOCE机制的理解的重大进步。SOCC已被提议由TRPC二聚体或多聚体组成。在本报告期间，我们已经证明了表位标记的TRPC1蛋白共不共沉淀。此外，通过酵母双杂交筛选和GST下拉试验，我们已经确定TRPC1单体通过其N-末端相互作用，可能在第一个Ankyrin-Repeat结构域。重要的是，TRPC1N末端的表达对SOCC活性具有显性的负面影响，并破坏了TRPC单体之间的相互作用。因此，我们认为TRPC1单体的多聚化是SOCC功能所必需的，这是通过第一个N-末端锚定蛋白重复序列的相互作用实现的。在未来的一年里，我们将研究TRPC1与其他TRPC蛋白的异构体相互作用。 我们早些时候已经报道，TRPC1和3组装在与小窝蛋白-1相关的多分子信号复合体中。我们现在已经确定了小窝蛋白-1在TRPC1N-末端的结合位置，并表明小窝蛋白-1是TRPC1和TRPC3质膜定位所必需的。酵母双杂交分析表明，TRPC1与SNARE蛋白VAMP2和SNAP相互作用。进一步的研究表明，VAMP2参与了TRPC3和小窝蛋白向质膜的运输。破伤风毒素破坏VAMP2改变了TRPC3和小窝蛋白的质膜定位。我们认为TRPC1和TRPC3是通过胞吐途径进入质膜的。目前，我们正在研究空泡信号复合体的组装是发生在质膜还是发生在细胞内。我们还将确定TRPC1信号复合体是否需要小窝蛋白-1才能保留在质膜上。我们认为，SOCE的调节是通过色氨酸相关信号复合体中的蛋白质-蛋白质相互作用发生的。因此，鉴定其蛋白质组分是至关重要的。为此，我们启动了两种方法：酵母双杂交筛选和基于蛋白质组学的筛选。在后者中，我们有免疫纯化的TRPC3，并正在使用2D-MS-MS分析与其相关的蛋白质。这两种方法都产生了新的和已知的蛋白质。在接下来的一年里，我们将继续这些研究，并用更详细的实验来证实初步的观察结果。

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.