Cellular regulation of magnesium homeostasis

镁稳态的细胞调节

• 批准号: 7057799
• 负责人: ANNE-LAURE PERRAUD
• 金额: $ 26.42万
• 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/7057799
• 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（由申请人提供）：尽管Mg 2+在所有生命形式中的丰度和普遍使用，该阳离子在细胞生理学中的重要作用以及其作为信使离子的潜在作用已得到充分证明，但对调节Mg 2+稳态的分子组分和机制知之甚少，特别是在脊椎动物中。初步研究结果表明，TRPM 7是新近发现的阳离子通道TRPM亚家族的成员之一，在脊椎动物的Mg 2+生理学中起着重要的作用。我们发现，在DT 40鸡B细胞系统中，由TRPM 7纯合缺失引起的致死表型可以通过补充高浓度Mg 2+的生长培养基而完全逆转。此外，最近显示患有遗传形式的低镁血症的患者在TRPM 6中携带突变，TRPM 6是TRPM 7的最近亲属。TRPM 6和TRPM 7在所有已知的离子通道中是独特的，因为它们在其C-末端具有激酶结构域。我们已经表明，TRPM 7是可渗透的镁2+和调节的TRPM 7激酶结构域的磷酸转移酶活性是敏感的镁2+在同一浓度范围内，调节通道门控。我们的初步结果表明，虽然激酶结构域不是必不可少的通道激活，通道和激酶功能耦合，使激酶改变通道的Mg 2+敏感性和结构外在的激酶结构域是负责观察到的Mg 2+敏感性TRPM 7。基于这些结果，我们提出了三个重要的问题，TRPM 7的调节和功能。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TRPM 7激酶突变体以及TRPM 7/TRPM 6嵌合分子：通道磷酸化的生化分析，通道功能的电生理学表征，以及在TRPM 7缺陷的DT 40细胞中使用互补方法来分析所选突变对细胞生理学和Mg 2+稳态的影响。具体目标2：确定磷酸化位点在TRPM 7的C端结构域中的作用：我们将首先进行磷酸肽图谱分析，并设计一系列C端区域的截短和点突变体来定位潜在的磷酸化位点。随后在TRPM 7全长通道中突变所选位置，并使用目标1中描述的方法分析这些突变体。具体目标3：定义TRPM 7激酶结构域在调节基因表达和蛋白质合成中的作用：使用cDNA微阵列技术来监测基因表达模式的变化，我们计划研究在各种条件下DT 40细胞中存在或相对不存在TRPM 7时一般细胞过程的潜在变化。另一组实验将分析TRPM 7调节蛋白质合成的潜在作用。