MOLECULAR MECHANISMS REGULATING CALCIUM FLUX IN SALIVARY GLANDS

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+)的内流。本项目旨在了解唾液腺细胞中钙信号的调节机制。最近，我们的工作集中在调节和调节Ca~(2+)进入唾液腺细胞的机制上。唾液细胞中的钙离子内流是许多其他非兴奋性细胞中存在的钙离子内流机制的典型。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(Trp)家族的离子通道蛋白被认为是钙离子内流通道的分子成分。我们之前已经报道了大鼠Trp1的克隆，并在大鼠和人唾液腺细胞中鉴定和定位了内源性Trp1蛋白。在本报告所述期间，我们进一步研究了Trp1在商店操作的钙离子内流机制中的作用。我们的研究表明，Trp1是唾液腺细胞中钙离子内流通道的有力候选者。此外，我们已经报道了Trp1定位于脂筏区域，在那里它与其他钙信号分子，如IP3R和G-蛋白一起组装在一个更大的信号复合体中。唾液腺和人唾液腺细胞株。我们认为，在这个信号复合体中促进的蛋白质-蛋白质相互作用调节商店操作的钙内流。我们的功能研究结果也与这些发现一致，我们用膜片钳方法检测了由商店操作的钙通道介导的钙电流。我们未来的研究将继续集中在存储操作的钙内流途径，它的调节和唾液腺液体分泌中的作用。

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.