Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

描述（由申请人提供）：定向细胞运动是发育具有适当极性的生物体所必需的，例如背-腹、前后和左右对称。我们在非洲爪蟾中发现，第一个被发现具有自身激酶结构域的离子通道TRPM7的耗尽会导致胚胎出现严重的原肠胚形成和神经折叠闭合缺陷，这使得TRPM7成为第一个被证明对早期脊椎动物发育有显著影响的离子通道。对这种效应的一个可能解释是我们最近报道的TRPM7控制钙依赖性蛋白酶m-calpain的活性来调节细胞粘附。尽管TRPM7在细胞运动中的作用令人信服，但重要的细节仍然缺失，即TRPM7通道被激活的机制，激酶的调节，以及TRPM7如何以及在什么条件下控制细胞运动的充分理解。最后，TRPM7影响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明确。我们提出了两个具体的目标来阐明TRPM7在爪蟾发育过程中在细胞水平和体内的功能和调控。在第一个具体目标中，我们将采用电生理学方法来研究TRPM7通道的pdgf受体激活依赖于PIP2合成的假设。细胞表面生物素化实验将用于测试pdgf介导的TRPM7激活是否依赖于从细胞内部位向质膜募集通道。此外，我们创建了TRPM7敲低的成纤维细胞系，以研究PDGF受体对TRPM7激酶及其磷酸化的调节以及对肌球蛋白II的调节。最后，我们将测试PDGF受体是否同时利用TRPM7和ERK信号通路来调节m-calpain和局灶黏附转换。在第二个特定目标中，我们将使用我们在功能丧失/功能获得的组合方法中创建的通道和激酶死亡突变体来定义TRPM7通道和激酶在非洲爪蟾早期模式形成中的作用。这些研究将包括检查TRPM7对会聚伸展运动、胚孔和神经襞闭合的影响。总的来说，这些实验将极大地促进我们对TRPM7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包括神经嵴细胞迁移在内的许多生理过程的理解，并可能为治疗依赖于细胞运动的病理状况（如心脏病、癌细胞转移和脊髓损伤期间的炎症）提供新的策略。

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.