Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 批准号: 8148617
• 负责人: INDU S. AMBUDKAR
• 金额: $ 194.39万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148617
• 关键词: Molecular; Salivary Glands; release of sequestered calcium ion into cytoplasm

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. In addition other calcium channels, including TRP channels, are involved in regulating various other cellular functions such as cell growth, development. Some channels are critical mediators of cellular dysfunction. Our long term goal is to define the components that mediate and regulate Ca2+ 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 as well as dysfunction. Our previous findings suggested that TRP proteins are molecular components of SOCE (TRPC1) and volume regulated Ca2+ channels (TRPV4) in salivary gland cells. We also 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. Further, we had reported that Orai1 and STIM1 are required for TRPC1 function and that functional Orai1 was required for TRPC1-SOCE. 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. We have now further assessed the molecular mechanisms involved in regulating TRPC1. Our major findings are as follows: 1.It is now well established that store-operated Ca2+ entry (SOCE) is activated by redistribution of the calcium binding protein, STIM1, from relatively diffused localization in the endoplasmic reticulum into puncta in discrete domains near the cell periphery where it interacts with and activates SOCE channels The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. We had earlier investigated the nature of the plasma membrane domains that determine the sites of STIM1 aggregation and reported that 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 Ca2+ signaling proteins from both the ER and plasma membrane and that intact LRD are required for activation of TRPC1-mediated SOCE. Thus, our findings demonstrate that STIM1-dependent activation of TRPC1 occurs within LRD. We now report that the cholesterol-binding LRD protein Caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland cells (HSG) decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of STIM mutants with disrupted interaction with TRPC1 (ERM-STIM1 or STIM1-KK/EE) prevented dissociation of TRPC1-Cav1 as well activation of TRPC1-SOCE. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1. These important data reveal the intricate processes that regulate store-operated calcium entry. 2. We have now reported a novel relationship between cellular volume change and store-operated calcium influx. Since changes in cell volume are intricately associated with fluid secretion in salivary gland acini, we believe our studies are potentially very important and identify novel effectors of prolonged volume stress. We have shown that when cells undergo swelling due to exposure to hypotonic conditions, there is a disruption in the architecture of the ER-plasma membrane junctional region. Since this spatial positioning is critical for functioning of SOCE, we investigated the effects of hypotonic cell swelling on SOCE. Our data demonstrate that as the cell undergoes swelling, the ER recedes from the plasma membrane. This prevents the positioning of STIM1 within the optimal distance required for its interaction with the plasma membrane channels involved in SOCE. Thus SOCE is not activated under these conditions. We further reported that the reversibility of the loss in SOCE depends on the extent of hypotonic stress. At lower levels of stress, SOCE is recovered when the stress is removed the cell volume returns to normal or even after the cell undergoes normal regulatory volume decrease. We propose that factors regulated by SOCE-dependent signaling might play a role in the survival of cell to long term hypotonic stress. 3. Vesicular trafficking is a key mechanism for controlling the surface expression of TRP channels in the plasma membrane, where they perform their function. We have previously reported that TRPC3 is dynamically trafficked to the plasma membranbe in response to stimuli that lead to PIP2 hydrolysis. TRP channels in vivo are often composed of heteromeric subunits. Experiments using total internal fluorescence reflection microscopy and biotin surface labeling show that Ca(2+) store depletion enhanced TRPV4-C1 translocation into the plasma membrane in human embryonic kidney 293 cells that were coexpressed with TRPV4 and TRPC1. The translocation required STIM1. TRPV4-C1 heteromeric channels were more favorably translocated to the plasma membrane than TRPC1 or TRPV4 homomeric channels. Similar results were obtained in native vascular endothelial cells. Thus, Ca(2+) store depletion stimulates the insertion of TRPV4-C1 heteromeric channels into the plasma membrane, resulting in an augmented Ca(2+) influx in response to flow in the human embryonic kidney cell overexpression system and native endothelial cells. Since we have previously shown that TRPV4 is required for regulatory volume decrease in salivary gland cells and is trafficked during the process, we believe these new findings are also very relevant to salivary gland function. Our studies provide novel understanding of the complex regulatory mechanisms and intricate cross talk between various calcium signaling proteins and pocesses. Thus, our studies have made significant advancement in our understanding of the molecular components and molecular mechanism(s) that are involved in regulation of store operated calcium channels as well as the interaction of the SOCE system with other physiologically relevant processes that are critically involved in salivary gland fluid secretion.

了解唾液腺细胞中激动剂刺激的钙进入通道的生理功能的主要步骤，需要鉴定其分子成分并定义其调节。 TRPC（瞬态受体潜在规范）蛋白已被认为是用于存储经营钙进入（SOCE）通道的分子候选。 SOCE在所有细胞中都存在普遍存在，并调节多种细胞功能，包括唾液腺液体分泌和炎症。此外，其他钙通道（包括TRP通道）也参与调节各种其他细胞功能，例如细胞生长，发育。一些通道是细胞功能障碍的关键介体。我们的长期目标是定义介导和调节Ca2+进入唾液腺细胞的成分。为了实现这一目标，我们的研究确定了与SOCE激活和失活有关的细胞机制，并定义了TRP通道在唾液腺功能和功能障碍中的作用。我们以前的发现表明，TRP蛋白是唾液腺细胞中SOCE（TRPC1）和体积调控Ca2+通道（TRPV4）的分子成分。 我们还使用TRPC1（ - / - ）小鼠提供了证据，表明TRPC1占SMG ACINI和管道中SOCE的90％以上，并且是毛carpine刺激的唾液流量所必需的。此外，我们报道了TRPC1函数需要ORAI1和STIM1，并且TRPC1-SOCE需要该功能性ORAI1。 因此，我们的研究在我们对唾液腺细胞中SOCE通道调节的调控的分子成分，组装和机制方面取得了显着进步。现在，我们进一步评估了调节TRPC1的分子机制。 我们的主要发现如下： 1.It is now well established that store-operated Ca2+ entry (SOCE) is activated by redistribution of the calcium binding protein, STIM1, from relatively diffused localization in the endoplasmic reticulum into puncta in discrete domains near the cell periphery where it interacts with and activates SOCE channels The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined.我们早些时候已经研究了确定刺激位点位点的质膜结构域的性质，并报告说脂质筏域（LRD）功能是信号复合物组装的中心。我们早些时候报道说，TRPC1与来自ER和质膜的密钥Ca2+信号蛋白组装在信号复合物中，并且该完整的LRD是激活TRPC1介导的SOCE所必需的。因此，我们的发现表明，trpc1的im1依赖性激活发生在LRD内。现在，我们报告说，胆固醇结合的LRD蛋白小窝蛋白-1（CAV1）是一个关键的质膜支架，在商店耗竭后STIM1点是位置的区域内保留TRPC1。这使TRPC1与trpc1-SOCE激活所必需的STIM1相互作用。沉默的人下腺细胞（HSG）中的cav1降低了TRPC1的质膜保留，TRPC1-Stim1聚类，因此降低了TRPC1-SECE，而不会改变STIM1点。重要的是，TRPC1-SECE的激活与TRPC1-Stim1的增加有关，而TRPC1-CAV1聚类的降低。 与此相一致，CAV1的过表达降低了TRPC1-stim1聚类和SOCE，当STIM1以较高水平相对于CAV1表达时，这两者均已恢复。沉默的刺激或与TRPC1相互作用（ERM-stim1或stim1-kk/ee）相互作用的刺激突变体的表达阻止了TRPC1-CAV1的解离以及TRPC1-SECE的激活。 此外，促进TRPC1-StIM1聚类和TRPC1-SECE的条件引起了SOCE依赖性NFKB激活和细胞增殖的相应变化。这些数据共同表明，CAV1是无效TRPC1的关键质膜支架。我们建议通过STIM1激活TRPC1-SOC会介导该通道从CAV1释放。这些重要的数据揭示了调节储存量的钙进入的复杂过程。 2。我们现在报道了细胞体积变化与储存钙的钙流入之间的新关系。由于细胞体积的变化与唾液腺acini中的液体分泌相关，因此我们认为我们的研究可能非常重要，并确定了长时间应力的新型效应因子。我们已经表明，当细胞因暴露于低渗条件而发生肿胀时，ER - 铂膜连接区域的结构会造成破坏。由于这种空间定位对于SOCE的功能至关重要，因此我们研究了低渗细胞肿胀对SOCE的影响。我们的数据表明，随着细胞的发生肿胀，ER从质膜退缩。这样可以防止STIM1在其与SOCE涉及的质膜通道相互作用所需的最佳距离内的定位。 因此，在这些条件下没有激活SOCE。我们进一步报道，SOCE中损失的可逆性取决于低渗应力的程度。在较低的压力水平下，当应力消除时，SOCE被回收，即使细胞发生正常调节体积减小，细胞体积也会恢复正常。我们提出，由SOCE依赖性信号调节的因素可能在细胞到长期低音应激的存活中起作用。 3。囊泡运输是控制质膜中TRP通道表面表达的关键机制，它们在其中执行其功能。我们先前已经报道说，TRPC3是针对导致PIP2水解的刺激的响应质膜动态运输到质膜的。体内的TRP通道通常由异质亚基组成。使用总内荧光反射显微镜和生物素表面标记的实验表明，Ca（2+）储存耗竭增强了TRPV4-C1的易位到人类胚胎肾脏293个细胞中的质膜，这些肾脏与TRPV4和TRPC1共表达。易位需要STIM1。与TRPC1或TRPV4同源通道相比，TRPV4-C1异构通道更受欢迎地转移到质膜上。在天然血管内皮细胞中获得了类似的结果。因此，Ca（2+）存储耗竭刺激TRPV4-C1异源通道插入质膜，从而导致人类胚胎肾细胞过表达系统和天然内皮细胞的流动响应于流动的增强Ca（2+）涌入。由于我们以前已经表明，唾液腺细胞的调节体积减少需要TRPV4，并且在此过程中被贩运，因此我们认为这些新发现也与唾液腺功能非常相关。我们的研究提供了对各种钙信号蛋白和杂质之间复杂调节机制和复杂的交叉谈话的新了解。 因此，我们的研究在我们对参与储存钙通道调节的分子成分和分子机制的理解方面取得了重大进步，以及SOCE系统与其他生理相关的过程的相互作用，这些过程与唾液腺体流体分泌涉及唾液腺体流体分泌。