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对SOCE的特点和确定的作用TRPC通道激动剂刺激的钙进入唾液腺细胞。与我们以前的研究一致，我们现在已经报道了小窝蛋白通过调节TRPC 1通道的质膜组装在SOCE中具有关键作用。TRPC 1的小窝蛋白结合结构域的突变破坏了其质膜定位，并对SOCE产生显性负效应。此外，我们已经报道了TRPC 3与SNARE蛋白相互作用，并且这种相互作用涉及TRPC 3的膜运输。我们已经表明，TRPC 3经历的组成和管制贩运机制。重要的是，激动剂刺激的PIP 2水解增加TRPC 3胞吐插入质膜，这有助于在受刺激的激动剂细胞中观察到的钙进入的增加。我们以前的研究主要是用HSG细胞进行的，我们已经最终证明TRPC 1是主要的SOCE组分。为了表征其他唾液腺细胞中的SOCE组分，我们现在研究了HSY细胞中的SOCE。我们的研究结果表明，不同的存储操作的钙通道存在于不同的细胞。在HSG细胞中，SOCE与相对Ca 2+选择性电流相关，而HSY细胞显示非选择性阳离子通道。这两个通道都不同于RBL细胞中的CRAC通道，其组分目前是未知的。虽然这些不同的SOCE通道的生理相关性目前还不清楚，我们现在已经检查了HSY细胞中通道的分子组成。有趣的是，虽然HSG通道似乎主要依赖于TRPC 1，但HSY通道似乎是由TRPC 1和TRPC 3的共组装形成的。此外，TRPC 1-TRPC 3相互作用通过其N-末端结构域介导。NTRPC 1的表达破坏了HSY细胞中的SOCE。因此，我们建议，TRCs可以组装成同聚体或异聚体，形成SOC和通道特性所定义的特定TRPC组件所涉及的。 为了评估TRPC通道的生理相关性，我们研究了它们在极化上皮细胞中的路由。我们已经在MDCK和唾液腺上皮（SMIE）细胞中建立了稳定的TRPC表达，这两种细胞在Transwell过滤器上培养时均形成高抗性单层。我们观察到TRPC具有明显的细胞定位。TRPC 3位于顶端，TRPC 1、TRPC 5和TRPC 2位于基底，而TRPC 6存在于细胞的顶端和基底区域。此外，内源性TRPC 3、TRPC 1和TRPC 6被发现在相同的位置。我们还研究了这些细胞区域中TRP通道的调节。与以前的报告一致，Ca 2+信号蛋白也主要定位在这些细胞的顶端区域。此外，TRPC 3与TRPC 6组装在复合物中，而不是TRPC 1，以及关键的Ca 2+信号蛋白如IP 3R，G蛋白和PLC。重要的是，我们发现TRPC 3/TRPC 6通道可以介导极化上皮细胞的顶端钙摄取和跨上皮钙转运。这些数据证明了TRPC 3/TRPC 6通道的新作用;即激动剂刺激的顶端钙摄取。与此一致，我们检测到TRPC 3和TRPC 6在唾液腺和肾导管的顶端区域的定位。研究正在进行中，以确定这些顶端定位的通道是如何调节的，以及它们的生理功能是什么。 在下一个财政年度，我们将继续沿着这些方向进行沿着研究。一个主要的焦点将指向确定新的TRPC相互作用蛋白，以帮助我们了解他们的功能和调节。我们还将继续研究TRPC通道的贩运及其组装和多聚化所涉及的机制。研究还将针对识别由不同TRPC组合产生的特定SOC电流。

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 TRPC channels in agonist-stimulated calcium entry in salivary gland cells. Consistent with our previous studies, we have now reported that caveolin has a critical role in SOCE by regulating the plasma membrane assembly of TRPC1 channels. Mutation in the caveolin-binding domain of TRPC1 disrupted its plasma membrane localization and exerted a dominant negative effect on SOCE. Further, we have reported that TRPC3 interacts with SNARE proteins and that this interaction is involved in membrane trafficking of TRPC3. We have shown that TRPC3 undergoes constitutive and regulated trafficking mechanisms. Importantly, agonist-stimulated PIP2 hydrolysis, increases exocytotic insertion of TRPC3 into the plasma membrane and this contributes towards the increase in calcium entry seen in stimulated agonist-cells. Our previous studies have been largely carried out with HSG cells in which we have demonstrated conclusively that TRPC1 is the primary SOCE component. Towards characterizing SOCE components in other salivary gland cells, we have now studied SOCE in HSY cells. Our results demonstrate that distinct store-operated Ca2+ channels are present in different cells. In HSG cells, SOCE is associated with a relatively Ca2+ selective current whereas HSY cells display a non-selective cation channel. Both of these channels are distinct from the CRAC channel in RBL cells, the components of which are presently unknown. Although the physiological relevance of these different SOCE channels is not presently clear, we have now examined the molecular components of the channel in HSY cells. Interestingly, while the HSG channel appears to primarily depend on TRPC1, the HSY channel appears to be formed by the coassembly of TRPC1 and TRPC3. Further, TRPC1-TRPC3 interactions are mediated via their N-terminal domains. Expression of the NTRPC1 disrupts SOCE in HSY cells. Thus, we propose that TRCs can assemble as homomers or heteromers to form SOC and that the channel properties are defined by the specific TRPC components that are involved. To assess the physiological relevance of TRPC channels we have examined their routing in polarized epithelial cells. We have established stable TRPC expression in MDCK and salivary epithelial (SMIE) cells, both of which form high resistance monolayers when cultured on Transwell filters. We have obsevred that TRPCs have distinct cellular localization. TRPC3 is apically localized, TRPC1, TRPC5, and TRPC2 are basaly localized while TRPC6 is found in both apical and basal regions of the cell. Further, endogenous TRPC3, TRPC1, and TRPC6 were found at the same locale. We have also studied the regulation of the TRP channels in these cellular regions. Consistent with previous reports, Ca2+ signaling proteins were also predominantly localized in the apical region of these cells. Further, TRPC3 was assembled in a complex with TRPC6, but not TRPC1, and key Ca2+ signaling proteins like IP3R, G-proteins, and PLC. Importantly, we showed that TRPC3/TRPC6 channels can mediate apical calcium uptake and transepithelial calcium transport in polarized epithelial cells. These data demonstrate a novel role for TRPC3/TRPC6 channels; i.e. agonist-stimulated apical calcium uptake. Consistent with this, we detected localization of TRPC3 and TRPC6 in the apical regions of salivary gland and kidney ducts. Studies are ongoing to determine how these apically localized channels are regulated and what is their physiological function. In the coming fiscal year we will continue our studies along these directions. A major focus will directed towards determining novel TRPC interacting proteins to help us to understand their function and regulation. We will also continue to study the trafficking of TRPC channels and the mechanisms involved in thier assembly and multimerization. Stduies will also be directed identifying specific SOC currents generated by different TRPC combinations.