Cellular regulation of magnesium homeostasis

描述（由申请人提供）：尽管镁离子在所有生命形式中都有丰富和普遍的使用，但这种阳离子在细胞生理学中的关键作用以及其作为信使离子的潜在作用已被充分记录，但对调控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，TRPM7是新发现的trpm -阳离子通道亚家族的成员，似乎在脊椎动物的Mg 2+生理中起着重要作用：我们发现，在DT40鸡b细胞系统中，TRPM7纯合缺失引起的致死表型可以通过补充高浓度的Mg 2+完全恢复。此外，最近的研究表明，患有遗传性低镁血症的患者携带TRPM6突变，TRPM7的近亲。TRPM6和TRPM7在所有已知的离子通道中都是独特的，因为它们在c端具有激酶结构域。我们已经证明TRPM7可渗透并受Mg 2+的调节，并且TRPM7激酶结构域的磷酸转移酶活性对调节通道门控的相同浓度范围内的Mg 2+敏感。我们的初步结果表明，虽然激酶结构域不是通道激活所必需的，但通道和激酶在功能上偶联，因此激酶改变了通道的Mg2+敏感性，并且激酶结构域的外部结构负责观察到的TRPM7的Mg2+敏感性。基于这些结果，我们提出了三个关于TRPM7调控和功能的重要问题。具体目标1：结构-功能关系研究。在这个目标中，我们计划使用三种实验方法来表征不同的TRPM7激酶突变体以及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缺陷的DT40细胞中使用互补方法分析选定突变对细胞生理和Mg 2+稳态的影响。具体目标2：确定TRPM7 C端结构域磷酸化位点的作用：我们将首先进行磷酸肽定位分析，并设计一系列C端区域的截断和点突变体来定位潜在的磷酸化位点。随后在TRPM7全长通道中选定的位置发生突变，并使用Aim 1中描述的方法分析这些突变。具体目标3：确定TRPM7激酶结构域在调节基因表达和蛋白质合成中的作用：利用cDNA微阵列技术监测基因表达模式的变化，我们计划研究不同条件下DT40细胞中TRPM7存在或相对缺失时一般细胞过程的潜在变化。另一组实验将分析TRPM7调节蛋白质合成的潜在作用。

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.