Sodium Influx Assay for Measurement of TRPM7 Channel Activity in Intact Cells

用于测量完整细胞中 TRPM7 通道活性的钠流入测定

• 批准号: 10057778
• 负责人: Juliusz Ashot Kozak
• 金额: $ 22.1万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057778
• 关键词: Action Potentials; Agonist; Alkalinization; Antigens; Biological Assay; Blood; Calcium; Carrier Proteins; Cations; Cell membrane; Cells; Chemosensitization; Collection; Critical Pathways; Divalent Cations; Drug Targeting; Dyes; Electrophysiology (science); Embryo; Exhibits; Family; Fluorescence; Fluorescence Microscopy; Gene Expression; Genetic; Goals; Human; Immune; Immune response; Immunity; Immunologic Deficiency Syndromes; Immunomodulators; Immunosuppressive Agents; Impairment; Ion Channel; Ion Transport; Ions; Knock-out; Link; Lymphocyte; Lymphocyte Activation; Macrophage Activation; Magnesium; Measurement; Measures; Mediating; Membrane; Membrane Potentials; Methods; Molecular; Monovalent Cations; Mus; Mutagenesis; Mutation; Neurons; Optics; Pathway interactions; Permeability; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Physiology; Play; Potassium Channel; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Reader; Recombinants; Reporter; Research; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Sodium; Specificity; Splenomegaly; Stimulus; Surface; System; T-Cell Activation; T-Cell Development; T-Cell Proliferation; T-Lymphocyte; Testing; Validation; adaptive immune response; base; biophysical properties; divalent metal; driving force; drug discovery; extracellular; gain of function; high throughput screening; high-throughput drug screening; lymphocyte proliferation; macrophage; member; miniaturize; mutant; novel; overexpression; patch clamp; programs; receptor; response; small molecule; sodium ion; sodium-binding benzofuran isophthalate; thymocyte; tool; voltage

T lymphocyte activation and proliferation is essential for the human adaptive immune response. It is well established that calcium and magnesium signaling pathways play a crucial role in this process and their impairments result in various forms of immunodeficiency. Several players involved in calcium and magnesium influx have been identified at the molecular level. One of them, TRPM7, a protein with dual ion channel and serine/threonine kinase function is permeable to both calcium and magnesium. TRPM7 is required for T-cell development and activation of macrophages. Until now TRPM7 channel function has been studied primarily with patch-clamp electrophysiology. Detailed understanding of TRPM7 involvement in immune cellular signaling has been hampered by the lack of specific drug modulators of this channel. In order to develop effective high throughput drug screening assays for this purpose, novel ways of detecting channel activity in intact cells are necessary. This proposal is a proof-of-concept study of an intact cell fluorescence-based Na+ influx assay for measuring TRPM7 channel activity and its adaptation to high throughput format. The assay takes advantage of the biophysical properties of TRPM7 channels, specifically, drastic changes in current-voltage relation upon changes in extracellular divalent cation (Ca2+ or Mg2+) concentrations. Channel activity will be evaluated using intracellular Na+ concentrations as a readout. TRPM7 conduction and gating mutants identified electrophysiologically, will be used for Na+ influx assay validation. The Na+ flux assay will allow us to compare cellular Na+ changes in response to overexpression and mutagenesis of TRPM7. At the conclusion of the proposed studies we will have developed new tools for high throughput cell-based assays of TRPM7 for use in drug discovery.

T淋巴细胞的活化和增殖是人体适应性免疫反应的必要条件。很好