Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

双功能离子通道和激酶 TRPM7 的功能分析

• 批准号: 8713072
• 负责人: LOREN W RUNNELS
• 金额: $ 15.89万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8713072
• 关键词: Affect; Anterior; Biochemical; Biological; Biological Assay; Biological Models; Birth; Brain Ischemia; Cell Proliferation; Cells; Conflict (Psychology); Congenital Abnormality; Congenital Heart Defects; Defect; Development; Developmental Process; Embryo; Embryonic Development; Event; Funding; Heart; Homeostasis; Homologous Gene; Human; Incidence; Incubated; Intake; Ion Channel; Lead; Left; Life; Link; Magnesium; Malignant Neoplasms; Medical; Modeling; Mus; Nervous system structure; Neural Fold; Neural Tube Closure; Neural Tube Defects; Neural tube; Organism; Pathway interactions; Phenotype; Phosphotransferases; Play; Population; Pregnancy; Prevention strategy; Process; Proteins; Regulation; Reporting; Research; Risk; Role; Signal Transduction; Spinal Dysraphism; Staging; Stroke; System; Tadpoles; Technology; Tertiary Protein Structure; Time; Tissues; United States; Xenopus; Xenopus laevis; base; cancer cell; cell behavior; cell motility; cost; design; directional cell; gain of function; gastrulation; human disease; in vivo; innovation; insight; loss of function; mRNA Expression; malformation; mutant; neuron loss; novel; offspring; prevent; protein expression; research study; tissue/cell culture; xenopus development; zygote

PROJECT SUMMARY Directional cell motility is required for the development of an organism with proper polarity such as dorso- ventral, anterior-posterior, and left-right symmetry. We have found in Xenopus Laevis that depletion of TRPM7, the first ion channel discovered to have its own kinase domain, results in embryos with severe gastrulation and neural fold closure defects, making TRPM7 the first ion channel shown to have a dramatic effect on this pivotal process during vertebrate development. Surprisingly, our research revealed that incubation of the embryos with excess magnesium or expression of a magnesium transporter reverses this phenotype, giving the first evidence that magnesium plays a critical role in this essential developmental process. Loss of TRPM7's closest homologue TRPM6 in mice has recently been reported to also cause neural tube closure defects. TRPM7 and TRPM6 are known to heterooligomerize when heterologously expressed in tissue culture cells, but reports vary as to whether TRPM6 functions independently as a channel in vivo. Preliminary studies indicate that TRPM7 functions within the non-canonical Wnt pathway to regulate gastrulation and neural fold closure. While TRPM7's mRNA expression remains constant during early development from the zygote to tadpole stage, TRPM6's mRNA expression is upregulated during gastrulation and peaks during neurulation. We propose two specific aims to clarify the functions and regulations of these two channels during early development. The early embryonic lethality caused by deletion of either channel in mice represents a substantial barrier to understanding these channels' functions in vivo. In the first specific aim, we will employ the Xenopus system, in which loss-of-function and gain-of-function experiments are permitted by titrating levels of the protein using antisense morpholino technology, to define the role of TRPM6 and its channel and kinase domains during early development, to determine whether the two channels are functioning in concert, and to determine the impact of these two channels on magnesium homeostasis in the developing embryo. In the second specific aim, we will investigate how the Wnt pathway is regulating TRPM7 and will examine the potential role that 80K-H, a TRPM7- and TRPM6-interacting protein we identified, may have in this process. With their incidence varying among different populations, neural tube closure defects occur at an average rate of 1 per 1000 births and are the second most prevalent malformation, after congenital heart defects, among human pregnancies. Collectively, the proposed experiments should greatly advance our understanding of how these unique bifunctional channels are functioning in vivo, which could lead to new strategies for preventing neural tube closure defects as well as provide new insight into other pathological conditions with which these channels have been associated, including stroke and cancer.

项目概要 定向细胞运动对于具有适当极性的生物体（例如背侧）的发育是必需的 腹侧、前后、左右对称。我们在非洲爪蟾中发现， TRPM7 是第一个被发现具有自己的激酶结构域的离子通道，它会导致胚胎出现严重的症状。 原肠胚形成和神经折叠闭合缺陷，使 TRPM7 成为第一个被证明具有显着性的离子通道 对脊椎动物发育过程中这一关键过程的影响。令人惊讶的是，我们的研究表明 用过量的镁孵化胚胎或表达镁转运蛋白可以逆转这种情况 表型，首次证明镁在这一重要的发育过程中发挥着关键作用 过程。最近有报道称，小鼠体内 TRPM7 最接近的同源物 TRPM6 的缺失也会导致神经 管封闭缺陷。已知 TRPM7 和 TRPM6 在异源表达时会发生异源寡聚化。 组织培养细胞，但关于 TRPM6 是否作为体内通道独立发挥作用的报道各不相同。 初步研究表明 TRPM7 在非经典 Wnt 通路中发挥作用来调节 原肠胚形成和神经皱襞闭合。虽然 TRPM7 的 mRNA 表达在早期保持恒定 从受精卵到蝌蚪阶段，TRPM6的mRNA表达在原肠胚形成过程中上调 并在神经形成期间达到峰值。我们提出两个具体目标来明确这些机构的职能和规定 早期开发期间有两个渠道。缺失任一通道导致的早期胚胎致死率 小鼠是理解这些通道体内功能的一个重大障碍。在第一个具体 目标，我们将采用 Xenopus 系统，其中功能丧失和功能获得实验 通过使用反义吗啉技术滴定蛋白质水平，可以确定 TRPM6 的作用 及其早期发育过程中的通道和激酶结构域，以确定这两个通道是否 协同运作，并确定这两个通道对镁稳态的影响 发育中的胚胎。在第二个具体目标中，我们将研究Wnt通路如何调节TRPM7 并将研究我们确定的 TRPM7 和 TRPM6 相互作用蛋白 80K-H 的潜在作用 在这个过程中有。由于不同人群的发病率不同，神经管闭合缺陷 平均每 1000 名新生儿中就有 1 人患有这种畸形，是仅次于先天性畸形的第二大常见畸形 人类怀孕期间的心脏缺陷。总的来说，所提出的实验应该极大地推进我们的研究 了解这些独特的双功能通道如何在体内发挥作用，这可能会导致新的 预防神经管闭合缺陷的策略，并为其他病理学提供新的见解 与这些通道相关的疾病，包括中风和癌症。