MOLECULAR MECHANISMS REGULATING CALCIUM FLUX IN SALIVARY GLANDS

唾液腺液体分泌的神经递质刺激是通过细胞质[Ca-2+]的双期升高介导的；最初是由于内部释放引起的短暂性增加，后来是由于Ca-2+内流引起的持续增加。持续的液体分泌被认为直接依赖于[Ca-2+]的持续升高，从而依赖于Ca-2+内流。本项目旨在了解唾液腺细胞中Ca-2+信号的介导和调节机制。最近，我们的工作集中在介导和调节Ca-2+流入唾液腺细胞的机制上。唾液细胞中的钙-2+内流是存在于许多其他不可兴奋细胞中的储存操作的钙-2+内流机制的原型。这种内流的分子机制尚未在任何细胞类型中确定。最近，瞬态受体电位（Trp）家族离子通道蛋白被认为是储存操作的Ca-2+内流通道的分子组成部分。我们之前已经报道了大鼠Trp1的克隆以及在大鼠和人唾液腺细胞中内源性Trp1蛋白的鉴定和定位。在本报告期间，我们进一步研究了Trp1在商店操作的Ca-2+内流机制中的作用。我们的研究表明，Trp1是唾液腺细胞Ca-2+内流通道的强有力候选。此外，我们已经报道了Trp1定位于脂筏区，在那里它与其他Ca-2+信号分子（如IP3R和g蛋白）一起组装成一个更大的信号plex。唾液腺和人类唾液腺细胞系。我们提出，在该信号通路内促进的蛋白质-蛋白质相互作用调节了储存操作的Ca-2+内流。在我们的功能研究中，我们使用膜片钳方法检查了由商店操作的Ca-2+通道介导的Ca-2+电流，结果也与这些发现一致。我们未来的研究将继续关注储存操作的Ca-2+内流途径及其在唾液腺液体分泌中的调节和作用。

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 suggested to directly dependent upon the sustained elevation of [Ca-2+]and thus on Ca-2+ influx. This project is aimed towards understanding the mechanisms which mediate and regulate Ca-2+ signaling in salivary gland cells. Recently, our efforts have been focused on the mechanisms involved in mediating and regulating Ca-2+ influx into salivary gland cells. Ca-2+ influx in salivary cells is prototypical of the store-operated Ca-2+ influx mechanism 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 (Trp)family of ion channel proteins have been proposed as molecular components of the store-operated Ca-2+ influx channel. We have previously reported cloning of rat Trp1 and identification and localization of the endogenous Trp1 protein in rat and human salivary gland cells. In this reporting period we have further studied the role of Trp1 in the store-operated Ca-2+ influx mechanism. Our studies suggest that Trp1 is a strong candidate for the Ca-2+ influx channel in salivary gland cells. Further, we have reported that Trp1 is localized in a lipid raft region where it is assembled in a larger signaplex along with other Ca-2+ signaling molecules, such as the IP3R and G-proteins. salivary glands and human salivary gland cell lines. We propose that protein-protein interactions facilitated within this signaplex regulate store-operated Ca-2+ influx. Results from our functional studies where we have examined the Ca-2+ current mediated by the store-operated Ca-2+ channel by using patch clamp methodology, are also consistent with these findings. Our future studies will continue to focus on the store-operated Ca-2+ influx pathway, its regulation and role in salivary gland fluid secretion.