Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

描述（由申请方提供）：定向细胞运动是生物体发育所需的适当极性，如背腹、前后和左右对称。我们已经在非洲爪蟾中发现，TRPM 7（第一个被发现具有其自身激酶结构域的离子通道）的耗尽导致胚胎具有严重的原肠胚形成和神经折叠闭合缺陷，使得TRPM 7成为第一个显示出对早期脊椎动物发育具有显著影响的离子通道。这种效应的一个可能的解释是我们最近报道的发现，TRPM 7控制钙依赖性蛋白酶m-钙蛋白酶的活性，以调节细胞粘附。尽管TRPM 7在细胞运动中的作用正在出现令人信服的画面，但仍然缺少重要的细节，即TRPM 7通道被激活的机制，激酶的调节，以及对TRPM 7如何以及在何种条件下控制细胞运动的充分理解。最后，受TRPM 7影响的原肠胚形成的具体方面及其激酶和通道在这些事件中所起的作用尚未确定。我们提出了两个具体的目标，以澄清TRPM 7的功能和调节的细胞水平和在体内非洲爪蟾的发展。在第一个具体目标中，我们将采取电生理学方法来研究TRPM 7通道的PDGF受体激活依赖于PIP 2合成的假设。细胞表面生物素化实验将用于测试PDGF介导的TRPM 7活化是否依赖于从细胞内位点向质膜募集通道。此外，我们建立了TRPM 7敲低的成纤维细胞系，以研究PDGF受体对TRPM 7激酶及其磷酸化和肌球蛋白II的调节。最后，我们将测试PDGF受体是否同时利用TRPM 7和ERK信号通路来调节m-钙蛋白酶和粘着斑周转。在第二个具体的目标，我们将采用通道和激酶死亡的突变体，我们已经创建了一个组合的功能丧失/功能获得的方法来定义TRPM 7的通道和激酶在非洲爪蟾的早期模式形成的作用。这些研究将包括TRPM 7对会聚延伸运动和囊胚孔和神经折叠闭合的影响的检查。总的来说，拟议的实验应该大大提高我们对TRPM 7在体内功能的理解。对这种双功能通道的研究可以加深我们对许多生理过程的理解，包括神经嵴细胞迁移，并可能导致治疗依赖于细胞运动的病理条件的新策略，如心脏病，癌细胞转移和脊髓损伤期间的炎症。

DESCRIPTION (provided by applicant): 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 early vertebrate development. A possible explanation for this effect is our recently reported discovery that TRPM7 controls the activity of the calcium-dependent protease m-calpain to regulate cell adhesion. Although a compelling picture is emerging of TRPM7's role in cell motility, important details are still missing, namely, the mechanism by which TRPM7's channel is activated, regulation of the kinase, and a full understanding of how and under what conditions TRPM7 controls cell motility. Finally, the specific aspect(s) of gastrulation affected by TRPM7 and the roles played by its kinase and channel in these events have not been defined. We propose two specific aims to clarify TRPM7's function and regulation on the cellular level and in vivo during Xenopus development. In the first specific aim, we will take an electrophysiological approach to investigate the hypothesis that PDGF-receptor activation of TRPM7's channel is dependent upon PIP2 synthesis. Cell surface biotinylation experiments will be used to test whether PDGF-mediated activation of TRPM7 relies upon the recruitment of the channel to the plasma membrane from intracellular sites. In addition, we've created TRPM7-knockdown fibroblast cell lines to investigate the regulation of TRPM7's kinase and its phosphorylation and regulation of myosin II by the PDGF receptor. Finally, we will test whether the PDGF receptor utilizes both TRPM7 and the ERK signaling pathway to regulate m-calpain and focal adhesion turnover. In the second specific aim we will employ channel- and kinase-dead mutants we've created in a combined loss-of-function/gain-of-function approach to define the roles of TRPM7's channel and kinase in early pattern formation in Xenopus laevis. These investigations will include an examination of TRPM7's influence on convergent extension movements and blastopore and neural fold closure. Collectively, the proposed experiments should greatly advance our understanding of TRPM7's function in vivo. Study of this bifunctional channel could deepen our understanding of many physiological processes including neural crest cell migration and could potentially lead to new strategies for treating pathological conditions dependent on cell motility such as inflammation during heart disease, cancer cell metastasis, and spinal cord injuries.