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一个重要步骤需要识别其分子成分并定义其调节。 TRPC（瞬时受体电位规范）蛋白已被建议作为钙池操纵钙进入（SOCE）通道的分子候选者。 SOCE 普遍存在于所有细胞中，调节多种细胞功能，包括唾液腺液分泌和炎症。此外，其他钙通道，包括TRP通道，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质。我们的长期目标是确定介导和调节 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 通道在唾液腺功能和功能障碍中的作用。我们之前的研究结果表明，TRP 蛋白是唾液腺细胞中 SOCE (TRPC1) 和容量调节 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 腺泡和导管中 90% 以上的 SOCE，并且是毛果芸香碱刺激唾液流所必需的。此外，我们报道了TRPC1功能需要Orai1和STIM1，并且TRPC1-SOCE需要功能性Orai1。 因此，我们的研究在我们对唾液腺细胞中 SOCE 通道的分子成分、组装和调节机制的理解方面取得了重大进展。我们现在进一步评估了调节 TRPC1 所涉及的分子机制。 我们的主要发现如下： 1. 现在已经明确，钙库操纵的 Ca2+ 内流 (SOCE) 是通过钙结合蛋白 STIM1 的重新分布而激活的，从内质网中相对分散的定位到细胞外周附近离散域中的斑点，在那里它与 SOCE 通道相互作用并激活 SOCE 通道。参与通道精确靶向及其在这些特定微域中保留的因素尚未确定。我们之前研究了决定 STIM1 聚集位点的质膜结构域的性质，并报道了脂筏结构域 (LRD) 作为信号复合物组装中心的功能。我们之前报道过，TRPC1 与来自内质网和质膜的关键 Ca2+ 信号蛋白组装在信号复合物中，并且完整的 LRD 是激活 TRPC1 介导的 SOCE 所必需的。因此，我们的研究结果表明 TRPC1 的 STIM1 依赖性激活发生在 LRD 内。我们现在报道，胆固醇结合 LRD 蛋白 Caveolin-1 (Cav1) 是一种关键的质膜支架，可将 TRPC1 保留在 STIM1 点在储存耗尽后定位的区域内。这使得 TRPC1 与 STIM1 能够相互作用，这是激活 TRPC1-SOCE 所需的。沉默人颌下腺细胞 (HSG) 中的 Cav1 可减少 TRPC1、TRPC1-STIM1 簇的质膜保留，从而减少 TRPC1-SOCE，而不改变 STIM1 斑点。重要的是，TRPC1-SOCE 的激活与 TRPC1-STIM1 的增加和 TRPC1-Cav1 聚类的减少相关。 与此一致的是，Cav1 的过表达降低了 TRPC1-STIM1 聚类和 SOCE，当 STIM1 相对于 Cav1 以更高水平表达时，这两者都得到恢复。沉默 STIM1 或与 TRPC1 相互作用被破坏的 STIM 突变体（ERM-STIM1 或 STIM1-KK/EE）的表达可防止 TRPC1-Cav1 解离以及 TRPC1-SOCE 激活。 此外，促进 TRPC1-STIM1 聚类和 TRPC1-SOCE 的条件引起 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 是非活性 TRPC1 的关键质膜支架。我们认为 STIM1 激活 TRPC1-SOC 介导 Cav1 通道的释放。这些重要数据揭示了调节商店经营的钙进入的复杂过程。 2. 我们现在报道了细胞体积变化和钙库操纵的钙流入之间的新关系。由于细胞体积的变化与唾液腺腺泡的液体分泌密切相关，我们相信我们的研究可能非常重要，并确定了长期体积应激的新效应器。我们已经证明，当细胞由于暴露于低渗条件而发生肿胀时，内质网-质膜连接区域的结构会受到破坏。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，随着细胞肿胀，内质网从质膜上退缩。这阻止了 STIM1 定位在其与 SOCE 涉及的质膜通道相互作用所需的最佳距离内。 因此 SOCE 在这些条件下不会被激活。我们进一步报告说，SOCE 损失的可逆性取决于低渗应激的程度。在较低水平的压力下，当压力消除、细胞体积恢复正常或什至在细胞经历正常的调节体积减少之后，SOCE 就会恢复。我们认为 SOCE 依赖性信号传导调节的因素可能在细胞对长期低渗应激的存活中发挥作用。 3. 囊泡运输是控制质膜中 TRP 通道表面表达的关键机制，TRP 通道在质膜中发挥其功能。我们之前曾报道过，TRPC3 响应导致 PIP2 水解的刺激而动态运输至质膜。体内TRP通道通常由异聚亚基组成。使用全内荧光反射显微镜和生物素表面标记的实验表明，在与TRPV4和TRPC1共表达的人胚胎肾293细胞中，Ca(2+)存储耗尽增强了TRPV4-C1易位到质膜中。易位需要 STIM1。 TRPV4-C1 异聚通道比 TRPC1 或 TRPV4 同聚通道更容易易位到质膜。在天然血管内皮细胞中也获得了类似的结果。因此，Ca(2+) 储存耗尽刺激 TRPV4-C1 异聚通道插入质膜，导致人胚胎肾细胞过表达系统和天然内皮细胞中的 Ca(2+) 流入增加。由于我们之前已经证明 TRPV4 是唾液腺细胞调节体积减少所必需的，并且在此过程中被贩运，因此我们相信这些新发现也与唾液腺功能非常相关。我们的研究为复杂的调节机制以及各种钙信号蛋白和过程之间错综复杂的串扰提供了新的理解。 因此，我们的研究在我们对参与调节钙通道钙通道的分子成分和分子机制以及 SOCE 系统与其他与唾液腺液分泌密切相关的生理相关过程的相互作用的理解方面取得了重大进展。