Capacitative Calcium Entry

电容式钙输入

• 批准号: 8553755
• 负责人: JAMES W PUTNEY
• 金额: $ 210.06万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/8553755
• 关键词: 1,2-diacylglycerol; Apoptosis; Area; Calcium; Calcium Channel; Categories; Cell membrane; Cell model; Cell physiology; Cells; Chemicals; Chemotaxis; Complementary DNA; Confocal Microscopy; Development; Differentiation and Growth; Diglycerides; Disease; Dominant-Negative Mutation; Electrophysiology (science); Endoplasmic Reticulum; Environment; Environmental Risk Factor; Family; Genes; Goals; Homeostasis; Human; Image; In Vitro; Investigation; Ion Channel; Learning; Lipids; Mammalian Cell; Measurement; Membrane; Microscopy; Molecular; Nature; Pathologic Processes; Pathway interactions; Peptides; Phosphatidylinositols; Phospholipase C; Physiological Processes; Plants; Play; Process; Proteins; Pseudogenes; Pump; Receptor Activation; Regulation; Role; Signal Pathway; Signal Transduction; Skin; Study models; Surface; Thapsigargin; Transfection; Wound Healing; base; bone; cell motility; cell type; computerized data processing; environmental intervention; falls; fluorescence imaging; interest; member; mouse model; patch clamp; receptor; release of sequestered calcium ion into cytoplasm; sensor; tumor

The broad aim of this project is to understand at the cellular and molecular level the mechanisms involved in regulation of calcium entry across the plasma membrane in non-excitable cells. This is a process that is ubiquitous to non-excitable cells, occurs in many excitable cell types and which is known to play an important role in the control of a large variety of cell processes, including secretion, contraction, motility, growth, differentiation and apoptosis. This signaling process is potentially vulnerable to environmental intervention by chemical and physical agents (such as EMF). Capacitative calcium entry is believed to involve a signal generated in the endoplasmic reticulum when Ca2+ is released by IP3 and which then interacts with the plasma membrane to activate calcium channels. The nature of this signal and the nature of the channels involved are prime areas of investigation. Specifically, we are intested in the roles of Stim1 and Stim2, which act as sensors for endoplasmic reticulum calcium, and Orai1, 2 and 3, which function as subunits of the store-operated channels. We utilize as a model for studying this process the actions of a tumor-promoting plant product, thapsigargin. Thapsigargin acts by inhibiting endoplasmic reticulum Ca2+ pumps, thereby depleting intracellular Ca2+ stores in a passive manner. We utilize the tecnhiques of single cell fluorescence imaging, confocal microscopy, total internal reflectance microscopy and patch-clamp electrophysiology. We are currently using mouse models to evaluate the role of this pathway in various physiological processes, including bone homeostasis, skin development, wound healing, cell migration and chemotaxis. We are also interested in other calcium-permeable channels activated as a result of surface receptor activation, specifically the phospholipase C-regulated TRPC channels. There are 7 members of the TRPC family of ion channels identified in mammalian cells. In humans, there are 6, as TRPC2 is a pseudogene. These fall into three categories based on sequence similarity: TRPC1, TRPC3/6/7 and TRPC4/5. We are expressing these channel genes in cell types in which they are normally expressed and in cell types which do not express these channel proteins. We intend to examine the effects of interfering with, or deletion of these proteins by transfection of cells with siRNAs, and cDNAs encoding for potentially dominant negative peptides. We are currently investigating the mechanisms by which lipids regulate this class of channels, specifically, diacylglycerols and phosphatidylinositol 4,5-bisphosphate. We are hopeful that by achieving a better understanding of the molecular and cellular modes of regulation of these important signaling pathways, we can learn how calcium entry is altered by environmental factors and by disease states.

该项目的广泛目的是在细胞和分子水平上了解与不可激发细胞中钙膜跨质膜进行调节有关的机制。这是一个无处不在的细胞的过程，发生在许多令人兴奋的细胞类型中，并且已知在控制各种细胞过程中起着重要作用，包括分泌，收缩，运动，生长，分化和凋亡。这种信号传导过程可能容易受到化学和物理剂（例如EMF）的环境干预的影响。据信，当Ca2+由IP3释放时，电容性钙进入涉及内质网中产生的信号，然后与质膜相互作用以激活钙通道。该信号的性质和所涉及的渠道的性质是主要调查领域。具体而言，我们在STIM1和STIM2的作用中无知，它们充当内质网钙的传感器，而Orai1、2和3，它们充当了商店经营的通道的亚基。我们利用一个模型来研究此过程，促进肿瘤的植物产品Thapsigargin的作用。 thapsigargin通过抑制内质网Ca2+泵起作用，从而以被动方式耗尽细胞内Ca2+储存。我们利用单细胞荧光成像，共聚焦显微镜，总内反射显微镜和斑块钳电生理学的tecnhiques。我们目前正在使用小鼠模型来评估该途径在各种生理过程中的作用，包括骨骼稳态，皮肤发育，伤口愈合，细胞迁移和趋化性。 我们还对由于表面受体激活而激活的其他钙渗透通道感兴趣，特别是磷脂酶C调节的TRPC通道。在哺乳动物细胞中鉴定出的TRPC家族有7个成员。在人类中，有6个，因为TRPC2是假基因。这些基于序列相似性分为三类：TRPC1，TRPC3/6/7和TRPC4/5。我们在通常表达的细胞类型中表达这些通道基因，并且在不表达这些通道蛋白的细胞类型中。我们打算检查通过用siRNA转染细胞来干扰或缺失这些蛋白质的影响，以及编码潜在显性负肽的cDNA。我们目前正在研究脂质调节这类通道的机制，特别是二酰基甘油和磷脂酰肌醇4,5-双磷酸。我们希望，通过更好地了解这些重要信号通路调节的分子和细胞模式，我们可以学习如何通过环境因素和疾病状态改变钙的进入。