Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 批准号: 7593360
• 负责人: INDU S. AMBUDKAR
• 金额: $ 178.34万
• 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/7593360
• 关键词: Acinus organ component; Adenoviruses; Agonist; Attenuated; Binding; Binding Proteins; Calcium; Calcium Channel; Calcium Signaling; Cations; Cell Death; Cell Line; Cell Volumes; Cell membrane; Cell physiology; Cells; Complex; Condition; Coupled; Data; Development; Disease; Duct (organ) structure; EF-Hand Domain; Elevation; Endoplasmic Reticulum; Ensure; Event; Family; Fluids and Secretions; Functional disorder; GTP-Binding Proteins; Generations; Goals; Growth and Development function; Hematopoietic; Human; Hydrolysis; Inflammation; Injury; Ion Channel; Knowledge; Laboratories; Lead; Link; Localized; Location; Lymphocyte; Mediating; Membrane; Molecular; Mutation; Neurotransmitters; Pathogenesis; Pathway interactions; Peptides; Peripheral; Permeability; Personal Satisfaction; Phospholipase; Physiological; Physiology; Pilocarpine; Protein Biosynthesis; Protein Overexpression; Protein Region; Proteins; Range; Rate; Rattus; Regulation; Reporting; Research; Role; STIM1 gene; Salivary; Salivary Glands; Signal Transduction; Signaling Protein; Site; Sjogren' s Syndrome; Stress; Submandibular gland; Syndrome; TRP channel; Thapsigargin; Time; Transmembrane Domain; Work; base; cell growth; cytokine; free radical oxygen; genetic regulatory protein; human STIM1 protein; inhibitor/antagonist; irradiation; member; novel; prevent; protein degradation; protein folding; radiation effect; receptor; release of sequestered calcium ion into cytoplasm; response; saliva secretion; salivary cell; therapeutic target; tool; trafficking; uptake

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 one or more presently known TRP proteins, including TRPC1, are molecular components of Ca-2+ entry channels in salivary gland cells. Further we showed that trafficking and assembly of store-operated and non-store-operated Ca2+ channels are key determinants of their function and regulation and that Ca-2+ entry occurs in specific microdomains where it is regulated via physical and functional association with structural and signaling proteins. In the past year we have continued to work along the same major directions. Our work has provided new information on the role and molecular composition of Ca-2+ entry channels in salivary gland cells. We have identified the translocon complex in the ER as a possible site for passive internal Ca-2+ release. In an important study we have functionally localized for the first time, the intracellular calcium stores that are coupled to store-operated calcium entry. Our studies have also revealed a novel mechanism of regulation of TRPC1-dependent store-operated calcium entry by the newly identified proteins STIM1 and Orai1. 1. Store-operated Ca-2+ entry (SOCE) is activated in response to depletion of intracellular Ca-2+ from the endoplasmic reticulum (ER). A variety of agonists stimulate SOCE via IP-3-dependent Ca-2+ depletion. SOCE is also activated by thapsigargin, an inhibitor of Ca2+ re-uptake into the ER that induces a net Ca-2+ loss from the ER by unmasking a Ca-2+ leak pathway. The molecular identity of this Ca-2+ leak channel and the physiological conditions under which such agonist-independent Ca-2+ depletion might occur remain poorly characterized. Here we report the presence of an agonist-independent mechanism for internal Ca-2+ store depletion and activation of SOCE in salivary gland cells, HSG (human submandibular gland cell line). These significant findings reveal an agonist-independent mechanism for depletion of internal Ca-2+ stores and activation of SOCE. We show that clearing of the translocon pore induces release of Ca-2+ from the internal Ca2+ store(s) and activation of SOCE. Under physiological conditions, such clearing of the translocon pore occurs following termination of protein synthesis and release of the nascent peptide. Although it is presently unclear exactly how much Ca-2+ release from the ER is required for activation of SOCE, several studies show that SOCE can be activated by submaximal depletion of internal Ca-2+ stores. Thus, depending upon the rate of protein turnover, sufficient Ca-2+ could be lost from the ER during termination of protein synthesis to activate SOCE. This link between SOCE and the protein synthesis provides a mechanism whereby the Ca-2+ in the ER can be maintained at levels optimal for protein folding and maturation in the ER. This will also be critical for protecting the cells against ER stress and activation of the unfolded protein response that result from loss of ER-Ca-2+. While it has been well established that SOCE-dependent refilling internal Ca-2+ stores provides ER-Ca-2+ for regulation of cellular functions that are mediated by agonist stimulation of PIP-2 hydrolysis, the present data demonstrate a novel agonist-independent, but physiologically critical, cellular function that is associated with ER-Ca-2+ and SOCE. 2. One of the proposed mechanisms for activation of SOCE predicts that interactions between ER and plasma membrane channel transduces the Ca-2+ status of the ER to the channel, achieving either activation or inactivation. It has been suggested that ER localized in close proximity to the plasma membrane (PM) regulates SOCE. However there are no data to directly demonstrate the location of this local ER store and whether its depletion is correlated with activation of SOCE. The recently identified SOCE-regulatory protein STIM1 is an ER-Ca2+ binding protein which relocalized to the sub-plasma membrane region when internal Ca-2+ store is depleted. This relocalization is suggested to be causal in activation of SOCE. However, it is unclear whether changes in the local Ca2+ store, i.e. in the vicinity where regulation of SOCE seems to be taking place, regulate relocation of STIM1 and activation of SOCE or whether signals from more interior regions of the cell are required. We have examined whether STIM1 regulation of SOCE is dependent on the Ca2+ in subplasma membrane ER or in more internal ER. Data obtained in this study demonstrate that mobilization of STIM1 and activation of SOCE are associated with the decrease of Ca2+ in ER which is localized within the subplasma region of the cell. Thus activation of SOCE by STIM1 is determined by the Ca2+ in the peripheral Ca2+ store where STIM1 is also localized. As long as Ca2+ in this store is below the threshold for binding to the STIM1-EF hand domain, STIM1 will be localized in punctae and SOCE will remain activated. Further studies are required to determine the exact molecular interactions involved in the generation of STIM1-containing punctae and how exactly these punctae regulate gating of the SOCE channel. 3. Despite intense focus on SOCE over the past two decades neither the mechanism(s) by which the status of Ca2+ in the ER is transmitted to the PM, to activate or inactivate SOC channels, nor the molecular components of the channels have yet been conclusively established. Recently two new proteins have emerged as candidate components of SOCE, STIM1 and Orai1. As noted above, STIM1 an EF-hand domain ER protein is shown to be essential for SOCE. The second protein, Orai1, is a four transmembrane domain protein that appears to constitute the pore-forming unit of CRAC channels which are a specific type of SOCE channels found primarily in lymphocytes and other hematopoietic cells. It has been suggested that Orai1 and STIM1 together are sufficient for the formation of this channel. We have previously reported that TRPC1 is an essential component of the SOC channels in salivary gland cells. Our data demonstrated that knockdown of TRPC1 decreases SOCE, overexpression increases the activity, while expression of TRPC1 with mutations in the proposed pore region of this protein alters the Ca2+ permeability of the channel. We have now investigated the possible role of STIM1 and Orai1 in TRPC1-dependent SOCE. This recent study reveals that all three proteins are essential for generation of TRPC1-SOC channels. Our findings show that STIM1 regulation of TRPC1-SOC is similar to its regulation of ICRAC. Based on these findings we have suggested that dynamic assembly of a TRPC1/STIM1/Orai1 complex is involved in activation of Ca2+ entry. Thus, our studies suggest a common molecular basis for channels in salivary gland cells and hematopoietic cells. Ongoing studies in our laboratory are aimed towards identifying the molecular interactions that regulate the function of this complex.

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。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。这些并不相互排斥，因为机制的识别将有助于通道的识别，反之亦然。 我们之前的研究结果表明，一种或多种目前已知的 TRP 蛋白（包括 TRPC1）是唾液腺细胞中 Ca-2+ 进入通道的分子成分。此外，我们还表明，钙池操作和非钙池操作的 Ca2+ 通道的运输和组装是其功能和调节的关键决定因素，并且 Ca-2+ 进入发生在特定的微域中，在该微域中，它通过与结构和功能相关的物理和功能关联进行调节。信号蛋白。在过去的一年里，我们继续沿着相同的主要方向努力。我们的工作提供了有关唾液腺细胞中 Ca-2+ 进入通道的作用和分子组成的新信息。我们已经确定内质网中的易位子复合体是被动内部 Ca-2+ 释放的可能位点。在一项重要的研究中，我们首次在功能上定位了与钙库操作的钙进入耦合的细胞内钙库。我们的研究还揭示了新发现的蛋白质 STIM1 和 Orai1 调节 TRPC1 依赖性钙池操作的钙进入的新机制。 1. 内质网 (ER) 细胞内 Ca-2+ 耗尽后，钙库操纵的 Ca-2+ 内流 (SOCE) 被激活。多种激动剂通过 IP-3 依赖性 Ca-2+ 消耗来刺激 SOCE。 SOCE 还可被毒胡萝卜素激活，毒胡萝卜素是一种 ER 中 Ca2+ 再摄取的抑制剂，通过揭示 Ca-2+ 泄漏途径，诱导 ER 中的 Ca-2+ 净损失。这种 Ca-2+ 泄漏通道的分子特性以及可能发生这种不依赖于激动剂的 Ca-2+ 耗竭的生理条件仍然知之甚少。在这里，我们报告了唾液腺细胞 HSG（人颌下腺细胞系）中内部 Ca-2+ 储存耗尽和 SOCE 激活的激动剂独立机制的存在。这些重要的发现揭示了内部 Ca-2+ 储存耗尽和 SOCE 激活的不依赖激动剂的机制。我们发现，易位孔的清除会诱导内部 Ca2+ 库释放 Ca-2+ 并激活 SOCE。在生理条件下，这种易位子孔的清除发生在蛋白质合成终止和新生肽释放之后。尽管目前尚不清楚激活 SOCE 需要从 ER 释放多少 Ca-2+，但一些研究表明 SOCE 可以通过内部 Ca-2+ 储备的次最大消耗来激活。因此，根据蛋白质周转率，在蛋白质合成终止以激活 SOCE 期间，足够的 Ca-2+ 可能会从 ER 中丢失。 SOCE 和蛋白质合成之间的这种联系提供了一种机制，使 ER 中的 Ca-2+ 可以维持在 ER 中蛋白质折叠和成熟的最佳水平。这对于保护细胞免受 ER 应激和激活因 ER-Ca-2+ 丢失而导致的未折叠蛋白反应也至关重要。虽然已经明确 SOCE 依赖性再填充内部 Ca-2+ 储存提供 ER-Ca-2+ 来调节由 PIP-2 水解的激动剂刺激介导的细胞功能，但目前的数据证明了一种新的不依赖激动剂的方法，但生理上至关重要的细胞功能与 ER-Ca-2+ 和 SOCE 相关。 2. 所提出的 SOCE 激活机制之一预测 ER 和质膜通道之间的相互作用将 ER 的 Ca-2+ 状态转变成通道，从而实现激活或失活。有人认为，位于质膜 (PM) 附近的 ER 调节 SOCE。然而，没有数据可以直接证明该本地 ER 存储的位置以及其消耗是否与 SOCE 的激活相关。最近发现的 SOCE 调节蛋白 STIM1 是一种 ER-Ca2+ 结合蛋白，当内部 Ca-2+ 储存耗尽时，它会重新定位到质膜下区域。这种重新定位被认为是 SOCE 激活的因果关系。然而，尚不清楚局部 Ca2+ 存储（即 SOCE 调节似乎发生的附近区域）的变化是否会调节 STIM1 的重新定位和 SOCE 的激活，或者是否需要来自细胞更内部区域的信号。我们研究了 STIM1 对 SOCE 的调节是否依赖于质膜下 ER 或更内部 ER 中的 Ca2+。本研究中获得的数据表明，STIM1 的动员和 SOCE 的激活与位于细胞亚浆区域内的 ER 中 Ca2+ 的减少相关。因此，STIM1 对 SOCE 的激活是由外周 Ca2+ 储存中的 Ca2+ 决定的，STIM1 也位于其中。只要该库中的 Ca2+ 低于与 STIM1-EF 手结构域结合的阈值，STIM1 将定位在点中，并且 SOCE 将保持激活状态。需要进一步的研究来确定包含 STIM1 的点的生成所涉及的确切分子相互作用，以及这些点如何准确地调节 SOCE 通道的门控。 3. 尽管在过去的二十年里人们对 SOCE 给予了极大的关注，但对于 ER 中 Ca2+ 的状态传输到 PM 来激活或失活 SOC 通道的机制以及通道的分子成分都还没有研究清楚。最终成立。最近，两种新蛋白质 STIM1 和 Orai1 成为 SOCE 的候选成分。如上所述，STIM1（一种 EF 手结构域 ER 蛋白）被证明对于 SOCE 至关重要。第二种蛋白 Orai1 是一种四跨膜结构域蛋白，似乎构成 CRAC 通道的成孔单元，CRAC 通道是主要存在于淋巴细胞和其他造血细胞中的一种特定类型的 SOCE 通道。有人认为 Orai1 和 STIM1 一起足以形成该通道。我们之前报道过 TRPC1 是唾液腺细胞 SOC 通道的重要组成部分。我们的数据表明，TRPC1 的敲低会降低 SOCE，过度表达会增加活性，而在该蛋白的拟议孔区域中突变的 TRPC1 表达会改变通道的 Ca2+ 通透性。我们现在已经研究了 STIM1 和 Orai1 在 TRPC1 依赖性 SOCE 中的可能作用。最近的这项研究表明，所有三种蛋白质对于 TRPC1-SOC 通道的生成都是必需的。我们的研究结果表明 STIM1 对 TRPC1-SOC 的调节与其对 ICRAC 的调节相似。基于这些发现，我们认为 TRPC1/STIM1/Orai1 复合物的动态组装参与了 Ca2+ 内流的激活。因此，我们的研究表明唾液腺细胞和造血细胞的通道具有共同的分子基础。我们实验室正在进行的研究旨在确定调节该复合物功能的分子相互作用。