Function and regulation of TRPM7 Mg2+-inhibited cation channels

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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.