Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

描述（由申请人提供）：神经褶皱闭合缺陷（NTD）是人类最常见的出生缺陷之一，平均发生率为1/1000妊娠。母体Mg 2+摄入量减少与NTD风险增加相关，表明Mg 2+渗透离子通道在发育的这一重要阶段发挥关键作用。我们对非洲爪蟾的研究揭示了TRPM 6和TRPM 7离子通道在胚胎发育过程中原肠胚形成和神经折叠闭合中的重要作用。由非洲爪蟾胚胎TRPM 7耗竭引起的神经折叠闭合缺陷可以通过补充Mg 2+或通过表达Mg 2+转运蛋白来预防，这支持了Mg 2+和传导这种重要阳离子的离子通道在这一基本胚胎过程中起关键作用的假设。已知TRPM 7和TRPM 6在组织培养细胞中异源表达时异源寡聚化，但关于TRPM 6是否自身在体内作为通道起作用的报道各不相同。初步研究表明，TRPM 6 mRNA表达在原肠胚形成过程中上调，在神经胚形成过程中达到峰值，支持这两个通道共同发挥作用以调节神经折叠闭合的假设。我们提出了三个具体的目标，以澄清这两个渠道的功能和监管在早期发展。在第一个具体的目标，我们将采用在非洲爪蟾的功能丧失和功能获得的实验，以确定TRPM 6在发展过程中的作用及其连接到非经典的Wnt通路，这已被证明是调节收敛的延伸运动原肠胚形成和神经折叠关闭。在具体目标2中，我们将研究在非洲爪蟾中TRPM 6和TRPM 7及其各个结构域如何共同发挥作用以调节神经折叠闭合，以及这些通道如何影响发育中胚胎的Mg 2+稳态。我们的研究还将集中在TRPM 7对Mg 2+稳态的控制如何影响细胞的迁移行为。在具体目标3中，我们将研究80 K-H的作用，80 K-H是一种TRPM 6和TRPM 7相互作用的蛋白质，在原肠胚形成和神经折叠闭合期间与TRPM 7协同作用，在调节这些通道的蛋白质水平中具有作用，并确定Wnt途径如何影响这种调节。总的来说，拟议的实验应该大大推进我们对这些独特的双功能通道如何在体内发挥作用的理解，这可能会导致预防神经管闭合缺陷的新策略，以及对抗与这些通道相关的其他病理条件的新见解，包括中风和癌症。

DESCRIPTION (provided by applicant): Neural fold closure defect (NTD) is one of the most common birth defects in humans, occurring at an average rate of 1 per 1000 pregnancies. Decreased maternal Mg2+ intake has been associated with an increased risk for NTD, suggesting a key role for Mg2+-permeant ion channels in this essential stage of development. Our research in Xenopus laevis has uncovered important roles for the TRPM6 and TRPM7 ion channels in gastrulation and neural fold closure during embryogenesis. Neural fold closure defects caused by depletion of TRPM7 from Xenopus laevis embryos can be prevented by Mg2+ supplementation or by expression of a Mg2+ transporter, supporting the hypothesis that Mg2+ and the ion channels that conduct this important cation play a critical role during this essential embryonic process. TRPM7 and TRPM6 are known to hetero-oligomerize when heterologously expressed in tissue culture cells, but reports vary as to whether TRPM6 functions by itself as a channel in vivo. Preliminary studies indicate that TRPM6 mRNA expression is upregulated during gastrulation and peaks during neurulation, supporting the hypothesis that the two channels are functioning together to regulate neural fold closure. We propose three specific aims to clarify the function and regulation of these two channels during early development. In the first specific aim, we will employ loss-of-function and gain-of-function experiments in Xenopus laevis to define the role of TRPM6 during development and its connection to the non-canonical Wnt pathway, which has been shown to regulate convergent extension movements during gastrulation and neural fold closure. In specific aim 2 we will examine in Xenopus how TRPM6 and TRPM7 and its individual domains may be functioning together to regulate neural fold closure and how these channels may be impacting Mg2+ homeostasis in the developing embryo. Our research will also focus on how TRPM7's control of Mg2+ homeostasis is affecting the migratory behavior of cells. In specific aim 3 we will investigate the role of 80K-H, a TRPM6- and TRPM7-interacting protein that functions synergistically with TRPM7 during gastrulation and neural fold closure, has in regulating these channels' protein levels, and determine how the Wnt pathway may be impacting this regulation. Collectively, the proposed experiments should greatly advance our understanding how these unique bifunctional channels are functioning in vivo, which could lead to new strategies for preventing neural tube closure defects as well as to new insights for combating the other pathological conditions for which these channels have been associated, including stroke and cancer.