Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

项目摘要 定向细胞运动是具有适当极性的生物体发育所必需的，如背- 腹侧、前后和左右对称。我们在非洲爪蟾身上发现， TRPM 7是第一个被发现具有自身激酶结构域的离子通道，它导致胚胎具有严重的 原肠胚形成和神经折叠关闭缺陷，使TRPM 7成为第一个显示出具有显着的离子通道。 在脊椎动物发育过程中对这一关键过程的影响。令人惊讶的是，我们的研究显示， 用过量的镁孵育胚胎或表达镁转运蛋白可逆转这种情况。 表型，给出了第一个证据表明，镁在这一重要的发育中起着关键作用， 过程最近有报道称，小鼠中TRPM 7最接近的同源物TRPM 6的缺失也会引起神经系统疾病。 管路闭合缺陷。已知TRPM 7和TRPM 6在大肠杆菌中异源表达时异源寡聚化。 组织培养细胞，但关于TRPM 6是否在体内独立地作为通道发挥作用的报道各不相同。 初步研究表明，TRPM 7在非经典Wnt途径中起调节细胞凋亡的作用。 原肠胚形成和神经褶闭合。虽然TRPM 7的mRNA表达在早期阶段保持不变， 从受精卵发育到蝌蚪期，TRPM 6的mRNA表达在原肠胚形成过程中上调 并在神经形成期达到峰值我们提出了两个具体目标，以澄清这些职能和法规， 早期发展的两个渠道。两个通道缺失引起的早期胚胎死亡率均为100%， 小鼠代表了理解这些通道在体内功能的实质性障碍。在第一个具体的 目的，我们将采用非洲爪蟾系统，其中功能丧失和功能获得实验是 通过使用反义吗啉技术滴定蛋白质水平，以确定TRPM 6的作用， 和它的通道和激酶结构域在早期发展，以确定这两个通道是否是 功能一致，并确定这两个通道对镁稳态的影响， 胚胎发育在第二个具体目标中，我们将研究Wnt通路如何调节TRPM 7。 并将研究我们鉴定的TRPM 7和TRPM 6相互作用蛋白80 K-H可能在 在这个过程中。神经管闭合缺陷在不同人群中的发病率不同， 发生率平均为每1000名新生儿1例，是仅次于先天性畸形的第二大常见畸形。 心脏缺陷，在人类怀孕中。总的来说，拟议的实验应该大大推进我们的研究。 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。

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.