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

基本信息

批准号：
10171764
负责人：
Juliusz Ashot Kozak
金额：
$ 18.75万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10171764
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

项目摘要

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淋巴细胞激活和增殖对于人类适应性免疫反应至关重要。很好确定钙和镁信号通路在此过程中起着至关重要的作用损伤导致各种形式的免疫缺陷。几个参与钙和镁的玩家流入已在分子水平上鉴定出来。其中之一，TRPM7，一种带有双离子通道的蛋白质，丝氨酸/苏氨酸激酶功能均可渗透到钙和镁中。 T-Cell需要TRPM7 巨噬细胞的发展和激活。到目前为止，已经研究了TRPM7通道功能贴片钳电生理学。详细了解TRPM7参与免疫细胞该通道的特定药物调节剂缺乏特定的药物调节剂阻碍了信号传导。为了发展有效的高通量药物筛查测定法，以检测通道活动的新型方法完整的细胞是必要的。该建议是对基于细胞荧光的NA+的概念验证研究用于测量TRPM7通道活性及其对高吞吐量格式的涌入测定。测定法利用TRPM7通道的生物物理特性，具体来说，在细胞外偏度阳离子（Ca2+或Mg2+）浓度变化时，电流关系的急剧变化。通道活动将使用细胞内Na+浓度作为读数进行评估。 TRPM7传导和门控突变体从电生理上鉴定出来，将用于NA+涌入测定验证。 NA+通量测定将使我们能够比较细胞Na+对TRPM7的过表达和诱变的响应的变化。结束我们将开发的拟议研究为高通量细胞的TRPM7开发新工具用于药物发现。