Function and regulation of TRPM7 Mg2+-inhibited cation channels

描述（由申请方提供）：T淋巴细胞活化伴随钙离子（Ca 2+）动员、碱化、母细胞转化（增大）和膜电位变化。当抗原识别启动生化级联反应时，细胞中的Ca 2+升高，导致该离子从细胞内储存中释放，并随后Ca 2+通过质膜中的离子通道流入。 钙进入过程对于有效的T细胞基因表达和克隆扩增是至关重要的，这是适应性免疫应答的必要步骤。尽管T淋巴细胞中钙库操纵的钙离子进入机制受到了最多的关注，但T细胞中还存在其他钙离子渗透性离子通道，这些通道可以提供转录所需的持续一小时的钙离子升高。一个这样的通道是TRPM 7。它在T细胞中高度表达，并通过细胞内部的碱化被激活，也被Mg 2+抑制。这项研究的中心假设是TRPM 7通道在T细胞活化和增殖过程中起着Ca 2+进入途径的作用。 验证这一假设的具体目的是：1）表征质膜PI（3，4，5）磷脂对天然T细胞和重组TRPM 7的调节; 2）表征细胞内pH对天然T细胞和重组TRPM 7的调节; 3）确定TRPM 7是否可以在T细胞活化期间提供持续Ca 2+内流的途径。4)建立一个新的系统，用于研究哺乳动物TRPM 7通道的隔离，采用果蝇细胞系，缺乏天然TRPM 7同源物。单细胞Ca 2+和pH成像和电生理学将用于检测特定离子通道活性。利用重组DNA技术在非免疫细胞和果蝇细胞中异源表达重组TRPM 7通道，详细研究其特性和调控。这项研究的意义在于，对T细胞活化过程中Ca 2+进入组分的详细了解可能会为免疫调节确定新的药物靶点。 公共卫生相关性：构成人类适应性免疫系统的细胞需要细胞内钙离子浓度的长期升高。钙处理的遗传紊乱导致免疫缺陷。相反，免疫抑制广泛用于器官移植和自身免疫性疾病。目前的提案旨在阐明免疫细胞中钙处理的分子机制，可能为免疫抑制和免疫调节提供新的治疗靶点。

DESCRIPTION (provided by applicant): T-lymphocyte activation is accompanied by calcium ion (Ca2+) mobilization, alkalinization, blast transformation (enlargement) and membrane potential changes. Ca2+ in the cell is elevated when antigen recognition initiates a biochemical cascade, leading to the release of this ion from intracellular stores and ensuing influx of Ca2+ through ion channels in the plasma membrane. The calcium entry process is critical for efficient T- cell gene expression and clonal expansion, necessary steps for the adaptive immune response. Even though store-operated Ca2+ entry mechanism in T lymphocytes has received most attention, other Ca2+ -permeable ion channels are present in the T cell, which can provide sustained, hour-long Ca2+ elevations required for transcription. One such channel is TRPM7. It is highly expressed in T cells and is activated by alkalinization of the cell interior and also inhibited by Mg2+. The central hypothesis of the proposed research is that TRPM7 channels function as a Ca2+ entry pathway during T-cell activation and proliferation. The specific aims to test this hypothesis are: 1) to characterize regulation of native T cell and recombinant TRPM7 by plasma membrane PI(3,4,5) phospholipid 2) to characterize regulation of native T cell and recombinant TRPM7 by intracellular pH. 3) to determine if TRPM7 can provide a pathway for a sustained Ca2+ influx during T cell activation. 4) to establish a novel system for studying mammalian TRPM7 channels in isolation that employs Drosophila melanogaster cell line, which lack native TRPM7 homologs. Single-cell Ca2+ and pH imaging and electrophysiology will be employed for the detection of specific ion channel activity. Recombinant DNA technology will be used for heterologous expression of recombinant TRPM7 channels in non-immune and Drosophila cells for detailed study of their characteristics and regulation. The significance of the proposed research is that detailed understanding of the Ca2+ entry components in T cell activation process will likely identify novel drug targets for immunomodulation. PUBLIC HEALTH RELEVANCE: Cells comprising the human adaptive immune system require prolonged elevations of intracellular calcium ion concentration. Genetic disturbances in calcium handling result in immunodeficiency. Conversely, immunosuppression is widely used in organ transplantation and autoimmune conditions. The current proposal aims to elucidate the molecular mechanisms of calcium handling in immune cells, potentially providing new therapeutic targets for immunosuppression and immunomodulation.