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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淋巴细胞的活化和增殖是人类适应性免疫应答所必需的。公钙和镁信号通路在这一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。几位参与钙和镁的球员已经在分子水平上鉴定了内流。其中之一，TRPM 7，一种具有双离子通道的蛋白质，丝氨酸/苏氨酸激酶功能对钙和镁都是可渗透的。TRPM 7是T细胞所必需的巨噬细胞的发育和活化。到目前为止，对TRPM 7的通道功能进行了初步的研究膜片钳电生理学。详细了解TRPM 7参与免疫细胞由于缺乏该通道的特异性药物调节剂，信号传导受到阻碍。为了发展为此目的的有效的高通量药物筛选测定，需要完整的细胞。该建议是一个完整的细胞荧光为基础的Na+概念验证研究用于测量TRPM 7通道活性及其对高通量形式的适应的流入测定。测定利用了TRPM 7通道的生物物理特性，特别是细胞外二价阳离子（Ca 2+或Mg 2+）浓度变化时电流-电压关系的急剧变化。通道活性将使用细胞内Na+浓度作为读数进行评价。TRPM 7传导和门控突变体电生理学鉴定，将用于Na+内流试验验证。Na+通量分析将使我们能够比较响应TRPM 7过表达和突变的细胞Na+变化。结束时通过这些研究，我们将开发出新的工具，用于高通量的基于细胞的TRPM 7检测，用于药物发现。