Molecular Mechanisms Regulating Calcium Flux In Salivary Glands

调节唾液腺钙通量的分子机制

• 批准号: 7967039
• 负责人: INDU S. AMBUDKAR
• 金额: $ 186.27万
• 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/7967039
• 关键词: Accounting; Acinus organ component; Address; Agonist; Attenuated; Binding; Calcium; Calcium Channel; Calcium Signaling; Calcium-Binding Proteins; Carbachol; Cations; Cell Death; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cholesterol; Complex; Data; Diffuse; Disease; Dissociation; Duct (organ) structure; Embryonic Development; Endoplasmic Reticulum; Ensure; Event; Family; Fluids and Secretions; Functional disorder; GTP-Binding Proteins; Goals; Growth and Development function; Human; Hydrolysis; ITPR1 gene; Inflammation; Injury; Knowledge; Lead; Mediating; Mediator of activation protein; Membrane Microdomains; Molecular; Morphogenesis; Mus; Mutation; N-terminal; Nature; Neurotransmitters; Pathogenesis; Pathway interactions; Pattern; Phosphatidylinositol 4,5-Diphosphate; Phospholipase; Physiological; Physiology; Pilocarpine; Process; Protein Biosynthesis; Proteins; Radiation; Reactive Oxygen Species; Regulation; Relative (related person); Reporting; Research; Role; STIM1 gene; Saliva; Salivary; Salivary Glands; Signal Transduction; Signaling Protein; Site; Sjogren' s Syndrome; Submandibular gland; Surface; Syndrome; TRP channel; TRPC3 ion channel; Tertiary Protein Structure; Thapsigargin; Tissues; caveolin 1; cell growth; cytokine; endoplasmic reticulum stress; gland development; irradiation; member; mutant; novel; overexpression; prevent; radiation effect; receptor; receptors for activated C kinase; release of sequestered calcium ion into cytoplasm; salivary cell; scaffold; sulfated glycoprotein 2; therapeutic target

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. Activation of TRPC3 channels is concurrent with IP3R-mediated intracellular Ca2+ release and associated with PIP2 hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with Receptor for Activated C-Kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also interaction of IP3R with RACK1 and IP3-dependent intracellular Ca2+ release. We show that TRPC3 interacts with RACK1 via N-terminal residues E232, D233, E240, and E244. Carbachol (CCh)-stimulation of HEK293 cells expressing wt-TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP3R complex and increased surface expression of TRPC3 and Ca2+ entry. Mutation of the putative RACK1-binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP3R complex as well as increased surface expression of TRPC3 and receptor-operated Ca2+ entry were also attenuated. Importantly, CCh-induced intracellular Ca2+ release was significantly reduced as was RACK1-IP3R association without any change in thapsigargin-stimulated Ca2+ release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP3R association and decreased CCh-stimulated Ca2+ entry. Further, an oscillatory pattern of CCh-stimulated intracellular Ca2+ release was seen in these cells compared to the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca2+ release was seen following CCh-stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP3R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating interaction between IP3R and RACK1.IP3R is a central and critical Ca2+ signaling protein in cells. It is assembled in a complex with both plasma membrane, cytosolic, and ER proteins. IP3R function is tightly controlled and the channel is involved in regulating both physiological and cell death pathways. Studies over the past several years have demonstrated that IP3Rs interact with plasma membrane TRPC channels and more recently, with other TRP channels such as polycystin2. While many of these earlier studies propose a role for IP3R in the regulation of TRPC channel function, to our knowledge the findings we have presented here provide the first demonstration that TRPC channels have a role in intracellular Ca2+ release via IP3R. 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 in salivary gland cells. Additionally, we have now identified a critical role for TRPM7 in salivary gland branching morphogenesis and a potentially important role for TRPM2 in salivary gland damage due to irradiation. Ongoing studies are focused on further resolving the exact functions of Orai1 and TRPc1 in SOCE and to determine how TRPC1 channels are assembled. Further, we are focusing our efforts on understanding the novel functions of TRPM2 and TRPM7 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。此外，其他钙通道，包括TRP通道，参与调节各种其他细胞功能，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）和体积调节Ca2+通道（TRPV4）的分子组分。我们还利用TRPC1（-/-）小鼠提供了证据，表明TRPC1占SMG腺泡和导管中SOCE的90%以上，并且是匹罗卡品刺激的唾液流动所必需的。此外，我们已经报道了TRPC1功能需要Orai1和STIM1， TRPC1- soce需要功能性Orai1。因此，我们的研究在了解唾液腺细胞中SOCE通道的分子组成、组装和调节机制方面取得了重大进展。我们现在进一步评估了参与调节TRPC1的分子机制。我们的主要发现如下：