TRPM7 and Cellular pH

描述(申请人提供)：T淋巴细胞的激活和增殖是人类适应性免疫反应的基础。钙信号在这一过程中起着至关重要的作用，钙信号的缺失会导致免疫缺陷。淋巴细胞中的钙信号是由参与特定基因转录的周期性细胞内钙离子升高所控制的。钙的两个主要来源是细胞内细胞器，如内质网和质膜中的钙内流途径。在过去的十年中，已经在分子水平上确定了参与钙离子内流的几个主要角色。 TRPM7是一种具有双离子通道和丝氨酸/苏氨酸激酶功能的蛋白质，在人T淋巴细胞和其他造血细胞中高表达。该通道的一个特点是它对细胞质中的镁离子和pH敏感。了解这一调控机制对于理解TRPM7参与T细胞钙信号转导具有重要意义。功能激酶可以自动磷酸化TRPM7，但它在调节该蛋白的离子通道活性中的作用尚不清楚。激酶结构域也被认为介导了镁离子对该通道的抑制，但目前尚不清楚该激酶是否参与了pH调节。我们建议研究TRPM7激酶和磷脂酰肌醇在免疫细胞pH调节中的作用。在目标1中，我们将比较从野生型和激酶死亡突变小鼠分离的白细胞中TRPM7的通道特性，并确定激酶在该通道的pH调节中的作用。在目标2中，我们将测试TRPM7激酶失活是否会影响白细胞的吞噬和增殖等功能。在目标3中，我们将检验T细胞质膜中磷脂酰肌醇的耗竭影响pH敏感性和天然TRPM7通道激活的假设。在这些研究的结论中，我们将阐明控制白细胞中TRPM7通道调节的因素，并开发新的工具来研究ITS激酶结构域的功能。

DESCRIPTION (provided by applicant): T lymphocyte activation and proliferation underlies human adaptive immune response. It is well established that calcium signaling plays a crucial role in this process and its deficiencies result in immunodeficiency. Calcium signaling in lymphocytes is governed by periodic intracellular calcium ion elevations that participate in specific gene transcription. Two main sources of calcium are intracellular organelles such as endoplasmic reticulum and calcium influx pathways in the plasma membrane. In the last decade several of the main players involved in calcium influx have been identified at the molecular level. TRPM7 is a protein with dual ion channel and serine/threonine kinase function and is highly expressed in human T lymphocytes and other hematopoietic cells. A hallmark of this channel is its sensitivity to Mg2+ and pH in the cytoplasm. Understanding the mechanism of this regulation is important for the understanding of TRPM7 involvement in T cell calcium signaling. The functional kinase can autophosphorylate TRPM7 but its function in modulating the ion channel activity of this protein is not well understood. The kinase domain has also been proposed to mediate Mg2+ inhibition of this channel, but it is not known if the kinase participates in pH regulation. We propose to investigate the role of TRPM7 kinase and phosphoinositides in regulation pH in immune cells. In Aim 1 we will compare TRPM7 channel properties in leukocytes isolated from wild type and kinase-dead mutant mice and determine the role of kinase in pH regulation of the channel. In Aim 2 we will test if function of leukocyte such as phagocytosis and proliferation are influenced by inactivation of TRPM7 kinase. In Aim 3 we will test the hypothesis that depletion of phosphoinositides in the plasma membranes of T cells affects pH sensitivity and activation of native TRPM7 channels. At the conclusion of these studies we will have elucidated the factors governing the regulation of TRPM7 channels in white blood cells and developed new tools to study the functions of the its kinase domain.