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.

项目摘要 要开发具有适当极性的生物（例如dorso-）需要定向细胞运动 腹侧，​​前后和左右对称性。我们在Xenopus laevis中发现了 TRPM7是第一个离子通道，发现具有自己的激酶域，导致胚胎严重 胃结构和神经折叠闭合缺陷，使TRPM7成为表现出戏剧性的第一个离子通道 对脊椎动物发育过程中关键过程的影响。令人惊讶的是，我们的研究表明 将胚胎与过量镁或镁转运蛋白的表达孵育，逆转这一点 表型，提供了第一个证据，表明镁在这一基本发展中起着至关重要的作用 过程。最近据报道，TRPM7最接近的同源性TRPM6的损失最近也引起神经 管闭合缺陷。众所周知，trpm7和trpm6在异源表达时众所周知 组织培养细胞，但报告说TRPM6是否作为体内通道独立起作用。 初步研究表明，TRPM7在非规范WNT途径中起作用以调节 胃结构和神经褶皱闭合。虽然TRPM7的mRNA表达在早期保持恒定 从合子到t阶段的发育，TRPM6的mRNA表达在胃肠道期间上调 和神经期间的峰值。我们提出了两个具体目标，以阐明这些功能和法规 早期开发过程中的两个渠道。由两个通道的删除引起的早期胚胎致死性 小鼠代表了理解这些渠道在体内功能的实质性障碍。在第一个特定 目的，我们将采用Xenopus系统，在该系统中，功能丧失和功能获得实验是 通过使用反义莫诺利诺技术滴定蛋白质的滴定水平允许，以定义TRPM6的作用 及其在早期开发过程中的渠道和激酶域，以确定两个渠道是否是 共同发挥作用，并确定这两个通道对镁稳态的影响 开发胚胎。在第二个特定目标中，我们将研究WNT途径如何调节TRPM7 并将研究我们鉴定出的80k-H，TRPM7和TRPM6相互作用蛋白的潜在作用。 在此过程中。随着不同人群的发病率不同，神经管闭合缺陷 以每1000个出生1的平均率发生，并且是先天性之后的第二大普遍畸形 人类怀孕中的心脏缺陷。总的来说，拟议的实验应该大大推动我们的 了解这些独特的双功能渠道在体内如何运作，这可能导致新 防止神经管封闭缺陷的策略，并为其他病理提供新的见解 与这些通道相关的条件，包括中风和癌症。