Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

描述（由申请人提供）：神经折叠闭合缺陷（NTD）是人类最常见的出生缺陷之一，平均发生率为每1000例妊娠中有1例。母体Mg2+摄入量的减少与NTD风险的增加有关，这表明Mg2+渗透离子通道在这一重要发育阶段起着关键作用。我们对非洲爪蟾的研究揭示了TRPM6和TRPM7离子通道在胚胎发生过程中原肠胚形成和神经襞闭合中的重要作用。由TRPM7缺失引起的非洲爪蟾胚胎神经襞闭合缺陷可以通过Mg2+补充或Mg2+转运体的表达来预防，这支持了Mg2+和传导这一重要阳离子的离子通道在这一重要胚胎过程中发挥关键作用的假设。已知TRPM7和TRPM6在组织培养细胞中异种表达时会发生异齐聚，但关于TRPM6是否在体内单独作为通道起作用的报道各不相同。初步研究表明，TRPM6 mRNA的表达在原肠胚形成期间上调，在神经发育期间达到峰值，支持了这两个通道共同调节神经襞闭合的假设。我们提出了三个具体目标来阐明这两个渠道在早期发育过程中的功能和调控。在第一个具体目标中，我们将利用非洲爪蟾的功能丧失和功能获得实验来确定TRPM6在发育过程中的作用及其与非规范Wnt通路的联系，该通路已被证明在原肠胚形成和神经襞关闭期间调节收敛性伸展运动。在具体目标2中，我们将在爪蟾中研究TRPM6和TRPM7及其单个结构域如何共同调节神经折叠关闭，以及这些通道如何影响发育中的胚胎中的Mg2+稳态。我们的研究还将重点关注TRPM7对Mg2+稳态的控制如何影响细胞的迁移行为。在特定的目的3中，我们将研究80K-H在调节这些通道蛋白水平中的作用，80K-H是一种TRPM6-和TRPM7相互作用的蛋白，在原肠形成和神经折叠关闭期间与TRPM7协同作用，并确定Wnt途径如何影响这种调节。总的来说，所提出的实验将极大地促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.