Cellular regulation of magnesium homeostasis

镁稳态的细胞调节

• 批准号: 6884661
• 负责人: ANNE-LAURE PERRAUD
• 金额: $ 27.06万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6884661
• 关键词: active sites; binding sites; cell biology; cell line; chimeric proteins; gene expression; homeostasis; magnesium; membrane channels; microarray technology; phosphorylation; point mutation; protein biosynthesis; protein kinase; protein structure function; translation factor; voltage /patch clamp

DESCRIPTION (provided by applicant): Despite the abundance and ubiquitous use of Mg 2+ in all forms of life, the well documented, crucial role of this cation in cellular physiology, as well as its potential role as a messenger ion, extremely little is known about molecular components and mechanisms acting to regulate Mg2+-homeostasis, especially in vertebrates. Our preliminary data demonstrates that TRPM7, a member of the newly discovered TRPM-subfamily of cation channels, appears to play an essential role in Mg 2+physiology in vertebrates: We showed that the lethal phenotype caused by the homozygous deletion of TRPM7 in the DT40 chicken B-cell system can be totally reverted by supplementing the growth medium with high concentrations of Mg 2+. Furthermore, it was recently shown that patients suffering from an inherited form of hypomagnesemia bear mutations in TRPM6, the closest relative of TRPM7. Both TRPM6 and TRPM7 are unique among all known ion-channels in that they possess a kinase domain at their C-terminus. We have shown that TRPM7 is permeable to and regulated by Mg 2+ and that the phosphotransferase activity of the TRPM7 kinase domain is sensitive to Mg 2+ in the same concentration range that regulates channel gating. Our preliminary results demonstrate that while the kinase domain is not essential for channel activation, channel and kinase are functionally coupled such that the kinase alters the channel's Mg2+-sensitivity and that a structure extrinsic to the kinase domain is responsible for the observed Mg2+-sensitivity of TRPM7. Based on these results, we propose to address three important questions about the regulation and function of TRPM7. Specific aim 1: Structure-function relationships studies. In this aim, we plan to characterize different TRPM7 kinase mutants as well as TRPM7/TRPM6 chimeric molecules using three experimental approaches: Biochemical analysis of channel phosphorylation, electrophysiological characterization of channel function, and analysis of the effects of selected mutations on cellular physiology and Mg 2+ homeostasis using a complementation approach in the TRPM7-deficient DT40 cells. Specific aim 2: Determining the role of phosphorylation sites in the C-terminal domains of TRPM7: We will first conduct phosphopeptide mapping analyses and design a series of truncations and point mutants of the C terminal regions to localize potential sites of phosphorylation. Selected positions will subsequently be mutated in the TRPM7 full-length channel, and these mutants analyzed using the approaches describeed in Aim 1. Specific Aim 3: Defining the role of the TRPM7 kinase domain in regulating gene expression and protein synthesis: Using cDNA microarray techniques to monitor changes in gene expression patterns, we plan to investigate the potential changes in general cellular processes in the presence or relative absence of TRPM7 in the DT40 cells under various conditions. Another set of experiments will analyze the potential role of TRPM7 regulating protein synthesis.

描述(申请人提供)：尽管镁离子在所有生命形式中的丰富和普遍使用，这种阳离子在细胞生理中的关键作用，以及它作为信使离子的潜在作用，人们对调节镁离子稳态的分子成分和机制知之甚少，特别是在脊椎动物中。我们的初步数据表明，新发现的TRPM-阳离子通道亚家族成员TRPM7似乎在脊椎动物的镁生理中起着重要的作用：我们的研究表明，在DT40鸡B细胞系统中，由于TRPM7纯合缺失而导致的致死表型可以通过添加高浓度的镁离子而完全逆转。此外，最近有研究表明，患有遗传性低镁血症的患者携带TRPM6的突变，TRPM7是TRPM7的近亲。在所有已知的离子通道中，TRPM6和TRPM7都是独一无二的，因为它们在C端都有一个激动域。结果表明，TRPM7对镁离子具有通透性，并受镁离子的调节，在与调节通道门控相同的浓度范围内，TRPM7蛋白的磷酸化转移酶活性对镁离子敏感。我们的初步结果表明，虽然激活域对通道激活不是必不可少的，但通道和激酶在功能上是偶联的，从而使激酶改变了通道对镁离子的敏感性，并且激酶结构域外部的结构导致了观察到的TRPM7对镁离子的敏感性。基于这些结果，我们提出了关于TRPM7的调控和功能的三个重要问题。具体目标1：结构-功能关系研究。为此，我们计划使用三种实验方法来表征不同的TRPM7激酶突变体和TRPM7/TRPM6嵌合分子：通道磷酸化的生化分析，通道功能的电生理表征，以及在TRPM7缺失的DT40细胞中利用互补方法分析所选突变对细胞生理和镁离子稳态的影响。具体目标2：确定TRPM7 C-末端结构域的磷酸化位点的作用：我们将首先进行磷酸化多肽图谱分析，并设计一系列C末端区域的截短和点突变来定位潜在的磷酸化位点。所选择的位置随后将在TRPM7全长通道中发生突变，并使用目标1中描述的方法对这些突变进行分析。具体目标3：确定TRPM7激酶结构域在调控基因表达和蛋白质合成中的作用：利用基因表达谱芯片技术监测基因表达模式的变化，我们计划研究在不同条件下DT40细胞中存在或相对不存在TRPM7的情况下一般细胞过程中的潜在变化。另一组实验将分析TRPM7调节蛋白质合成的潜在作用。