Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

项目总结 具有适当极性的生物体的发育需要定向细胞运动，如 腹侧对称，前后对称，左右对称。我们在非洲爪哇中发现了耗尽的 TRPM7是第一个被发现具有自己的激活域的离子通道，导致严重的胚胎 原肠形成和神经折叠闭合缺陷，使TRPM7成为第一个被证明具有戏剧性 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，我们的研究显示 用过量的镁孵育胚胎或表达镁转运蛋白可以逆转这一点 表型，首次证明镁在这一重要的发育过程中起着关键作用 进程。最近有报道称，在小鼠中丢失TRPM7的S最接近的同源基因TRPM6也会导致神经 管子关闭缺陷。已知TRPM7和TRPM6在异源基因中表达时会异位齐聚 组织培养细胞，但关于TRPM6是否在体内作为通道独立发挥作用的报道各不相同。 初步研究表明，TRPM7在非典型的Wnt途径中发挥调节作用 原肠形成和神经皱折闭合。而TRPM7‘S基因的表达在早期保持不变 从受精卵发育到蝌蚪阶段，TRPM6‘S基因在原肠发育过程中表达上调 并在神经形成过程中达到峰值。我们提出两个具体目标，以澄清这些机构的职能和规则 早期发育中的两个渠道。两个通道中的任何一个的缺失导致的早期胚胎死亡 小鼠是理解这些通道在体内的功能的实质性障碍。在第一个具体的 目的，我们将采用非洲爪哇系统，在该系统中，功能损失和功能获得实验 允许使用反义吗啉技术滴定蛋白质水平，以确定TRPM6的作用 以及它在早期发育过程中的通道和激活域，以确定这两个通道是否 ，并确定这两个通道对体内镁稳态的影响。 发育中的胚胎。在第二个具体目标中，我们将研究Wnt途径是如何调节TRPM7的 并将研究我们发现的一种与TRPM7和TRPM6相互作用的蛋白质80K-H可能发挥的作用 在这个过程中。神经管闭合缺陷在不同人群中的发生率不同 平均每1000名新生儿中就有1人发生，是仅次于先天性的第二大最常见的畸形 心脏缺陷，在人类怀孕中。总的来说，拟议的实验应该会极大地推动我们的 了解这些独特的双功能通道是如何在体内发挥作用的，这可能导致新的 预防神经管闭合缺陷的策略以及为其他病理疾病提供新的见解 与这些渠道相关的条件，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.