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SOCE的特征和确定TRPC通道在激动剂刺激的唾液腺细胞钙内流中的作用。与我们之前的研究一致，我们现在报道了小窝蛋白通过调节TRPC1通道的质膜组装在SOCE中发挥关键作用。TRPC1的小窝蛋白结合区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TRPC3与SNARE蛋白相互作用，这种相互作用参与了TRPC3的膜转运。我们已经表明，TRPC3经历了结构性和受管制的贩运机制。重要的是，激动剂刺激的PIP2水解，增加TRPC3的胞吐插入质膜，这有助于在刺激的激动剂细胞中看到的钙内流增加。我们之前的研究主要是在HSG细胞上进行的，我们已经确凿地证明TRPC1是SOCE的主要成分。为了表征其他唾液腺细胞中的SOCE成分，我们现在研究了HSY细胞中的SOCE。我们的结果表明，不同的细胞中存在不同的存储操作的钙通道。在HSG细胞中，SOCE与相对钙离子选择性电流有关，而HSY细胞则表现为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，目前尚不清楚CRAC通道的组成。尽管这些不同的SOCE通道的生理相关性目前还不清楚，但我们现在已经研究了HSY细胞中该通道的分子组成。有趣的是，虽然HSG通道似乎主要依赖于TRPC1，但HSY通道似乎是由TRPC1和TRPC3共同组装形成的。此外，TRPC1-TRPC3的相互作用是通过它们的N-末端结构域介导的。NTRPC1的表达破坏HSY细胞的SOCE。因此，我们认为TRPC可以作为同质或异质组装形成SOC，并且通道属性由所涉及的特定TRPC组件来定义。 为了评估TRPC通道的生理学相关性，我们研究了它们在极化上皮细胞中的路径。我们已经在MDCK和唾液上皮(SMIE)细胞中建立了稳定的TRPC表达，当在Transwell过滤器上培养时，这两个细胞都形成了高阻的单层。我们观察到TRPC具有明显的细胞定位。TRPC3定位于顶端，TRPC1、TRPC5和TRPC2基本定位，TRPC6定位于细胞的顶端和基底部。此外，在同一地点还发现了内源TRPC3、TRPC1和TRPC6。我们还研究了这些细胞区色氨酸通道的调节。与以前的报道一致，钙信号蛋白也主要定位于这些细胞的顶端区域。此外，TRPC3与TRPC6，而不是TRPC1，以及关键的钙信号蛋白，如IP3R，G-蛋白和PLC一起组装在一个复合体中。重要的是，我们发现TRPC3/TRPC6通道可以介导极化上皮细胞顶端钙摄取和跨上皮钙转运。这些数据显示了TRPC3/TRPC6通道的一种新作用，即激动剂刺激的心尖部钙摄取。与此一致，我们检测到TRPC3和TRPC6定位于唾液腺和肾管的顶端。目前正在进行研究，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，以帮助我们了解它们的功能和调节。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。研究还将指导确定由不同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.