Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

了解唾液腺细胞中激动剂刺激的钙进入通道的生理功能需要鉴定其分子成分并确定其调控。TRPC（瞬时受体电位规范）蛋白被认为是储存操作钙进入（SOCE）通道的分子候选者。SOCE普遍存在于所有细胞中，并调节多种细胞功能，包括唾液腺液体分泌和炎症。我们的长期目标是确定介导和调节Ca-2+进入唾液腺细胞的成分。为了实现这一目标，我们的研究确定了参与SOCE激活和失活的细胞机制，并确定了TRP通道在唾液腺功能中的作用。这些并非相互排斥，因为确定机制将有助于确定渠道，反之亦然。

A major step towards understanding the physiological function of agonist-stimulated calcium entry channels in salivary gland cells requires identification of their molecular components and defining their regulation. TRPC (transient receptor potential canonical) proteins have been suggested as molecular candidates for store-operated calcium entry (SOCE) channels. SOCE is ubiquitously present in all cells and regulates a variety of cellular functions including salivary gland fluid secretion and inflammation. Our long term goal is to define the components that mediate and regulate Ca-2+ entry into salivary gland cells. Towards this goal, our studies determine cellular mechanisms which are involved in the activation and inactivation of SOCE and define the role of TRP channels in salivary gland function. These are not mutually exclusive as identification of the mechanism will facilitate identification of the channel and vice-versa. Our previous findings suggested that TRP proteins are molecular components of SOCE (TRPC1) and volume regulated Ca-2+ channels (TRPV4) in salivary gland cells. Further, we had reported that Orai1 and STIM1 are required for TRPC1 function. We have now provided evidence using TRPC1(-/-) mouse that TRPC1 accounts for more than 90% of the SOCE in SMG acini and ducts and is required for pilocarpine-stimulated saliva flow. We have also reported that Orai1 is a key component of TRPC1 channel and that Orai1 function is required for TRPC1-SOCE. Additionally, identification of the TRPC3 proteome from rat brain has been fully completed and a manuscript describing these data was published in early 2008. Thus our studies have made significant advancement in our understanding of the molecular components, their assembly, and mechanism(s) of regulation of SOCE channels in salivary gland cells. Together our findings are of wide impact in the field of Ca2+ signaling where this is now a point of major focus. Major findings are summarized below: 1. Physiological function of TRPC1 in salivary glands. A major study accomplished this fiscal year assessed the role of TRPC1 in salivary gland function by using targeted disruption of TRPC1 expression in mice. We reported that neurotransmitter-regulated salivary gland fluid secretion in TRPC1(-/-) mice was severely decreased (by 70%). Further, agonist and thapsigargin stimulated SOC channel activity was significantly reduced in salivary gland acinar and ductal cells isolated from TRPC1(-/-) mice. Deletion of TRPC1 also eliminated sustained KCa activity which is dependent on Ca-2+ entry and required for fluid secretion and volume regulation induced by CCH. Expression of key proteins involved in fluid secretion and Ca-2+ signaling, including STIM1 and other TRPC channels, was not altered. Together these data demonstrate that reduced store-operated calcium entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(-/-) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and essential for salivary gland fluid secretion. We suggest that TRPC1 is a potentially useful target molecule for treatment of salivary gland dysfunction. 2. We have previously reported that TRPC1 is an essential component of the SOC channels in salivary gland cells. We had reported earlier that TRPC1, Orai1 and STIM1 are recruited into a complex following stimulation and that Orai1 and STIM1 were required for TRPC1 function. We hypothesized that STIM1 is a regulator for TRPC1 and that Orai1 is either a scaffold for TRPC1 or contributes more directly to TRPC1 channel. To resolve this we generated functional TRPC1 channels in HEK-293 cells by overexpressing TRPC1+STIM1 and then assessed the role of Orai1 in TRPC1-function. Stimulation of cells expressing Orai1+STIM1 increased Ca2+ entry and activated typical ICRAC current while stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, ISOC. Knockdown of Orai1 decreased SOCE in cells expressing TRPC1 alone or TRPC1+STIM1. Expression of R91WOrai1 (SCID nonfunctional Orai mutant) or E106QOrai1 (dominant-negative Orai1) induced similar attenuation of TRPC1+STIM1-dependent SOCE and ISOC while expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1. Together these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE. We had predicted that if Orai1 was serving as a scaffold, then its channel function would not be critical for TRPC1. Our data do not support this suggestion. Instead they suggest that either Orai1 forms a separate channel (our data do not support this possibility, but we cannot conclusively ruled it out) or that the two proteins somehow contribute to a single channel pore. 3.We have now investigated the nature of the plasma membrane domains that determine the sites of STIM1 aggregation. Plasma membrane lipid rafts domains (LRD) function as centers for the assembly of signaling complexes. We have reported earlier that TRPC1 is assembled in a signaling complex with key Ca-2+ signaling proteins from both the ER and plasma membrane and that intact LRD are required for activation of TRPC1-mediated SOCE. We now show that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). Store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within LRD and this association was dynamically regulated by the status of the ER Ca-2+ store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1D76A which is peripherally localized and constitutively activates SOCE in unstimulated cells displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared to STIM1. Disruption of membrane rafts decreased peripheral STIM1D76A puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane domains. 4. Presence of various protein-interacting domains in TRPC channels have lead to the suggestion that they associate with proteins that are involved in their function and regulation. We have made significant progress in identifying proteins that are associated with TRPC channels. We have used two approaches. (i) TRPC1 and TRPC3 N and C terminals were used as baits to screen rat brain and HEK-cell libraries using a yeast 2-hybrid system. (ii) immunoprecipitation and mass spectrometry were used to analyse the proteins associated with TRPC3 and TRPC1. We have now identified the proteins associated with native TRPC3 from rat brain using a shotgun proteomic approach. 64 specific TRPC3-associated proteins were identified which were grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we reported novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth.