Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

描述(申请人提供)：细胞的定向运动是生物体发育所必需的，具有适当的极性，如背腹对称、前后对称和左右对称。我们在非洲爪哇发现，第一个被发现具有自己的激活区的离子通道TRPM7的缺失会导致胚胎出现严重的原肠形成和神经折叠闭合缺陷，使TRPM7成为第一个被证明对脊椎动物早期发育有显著影响的离子通道。这一效应的一个可能解释是我们最近报道的发现，TRPM7控制钙依赖的蛋白酶m-calain的活性，以调节细胞黏附。尽管TRPM7的S在细胞运动中的作用已经有了令人信服的画面，但重要的细节仍然缺乏，即TRPM7的S通道被激活的机制，该激酶的调节，以及对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影响原肠形成的具体方面(S)以及它的激酶和通道在这些事件中所起的作用还没有确定。我们提出了两个特定的目标来阐明TRPM7的S功能及其在细胞水平和体内发育过程中的调控。在第一个具体目标中，我们将采用电生理学的方法来研究血小板衍生生长因子受体激活TRPM7‘S通道依赖于PIP2合成的假说。细胞表面生物素化实验将被用来测试PDGF介导的TRPM7的激活是否依赖于从细胞内位置到质膜的通道的募集。此外，我们还建立了TRPM7基因敲除的成纤维细胞系，以研究TRPM7‘S激酶的调节及其磷酸化和PDGFR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-calain和局部粘连翻转。在第二个特定目标中，我们将使用我们在功能丧失/功能获得相结合的方法中创建的通道和激酶死亡突变体，以确定TRPM7的S通道和激酶在非洲爪哇早期图案形成中的作用。这些研究将包括检测TRPM7的S对收敛伸展运动以及胚泡和神经折叠关闭的影响。总之，所提出的实验将极大地促进我们对TRPM7在体内的S功能的理解。对这一双功能通道的研究可以加深我们对许多生理过程的理解，包括神经脊细胞的迁移，并可能导致治疗依赖细胞运动的病理疾病的新策略，如心脏病期间的炎症、癌细胞转移和脊髓损伤。

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.