Functional Analysis of the Bifunctional Ion Channel and Kinase TRPM7

点击翻译按钮获取中文摘要

PROJECT SUMMARY 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 this pivotal process during vertebrate development. Surprisingly, our research revealed that incubation of the embryos with excess magnesium or expression of a magnesium transporter reverses this phenotype, giving the first evidence that magnesium plays a critical role in this essential developmental process. Loss of TRPM7's closest homologue TRPM6 in mice has recently been reported to also cause neural tube closure defects. TRPM7 and TRPM6 are known to heterooligomerize when heterologously expressed in tissue culture cells, but reports vary as to whether TRPM6 functions independently as a channel in vivo. Preliminary studies indicate that TRPM7 functions within the non-canonical Wnt pathway to regulate gastrulation and neural fold closure. While TRPM7's mRNA expression remains constant during early development from the zygote to tadpole stage, TRPM6's mRNA expression is upregulated during gastrulation and peaks during neurulation. We propose two specific aims to clarify the functions and regulations of these two channels during early development. The early embryonic lethality caused by deletion of either channel in mice represents a substantial barrier to understanding these channels' functions in vivo. In the first specific aim, we will employ the Xenopus system, in which loss-of-function and gain-of-function experiments are permitted by titrating levels of the protein using antisense morpholino technology, to define the role of TRPM6 and its channel and kinase domains during early development, to determine whether the two channels are functioning in concert, and to determine the impact of these two channels on magnesium homeostasis in the developing embryo. In the second specific aim, we will investigate how the Wnt pathway is regulating TRPM7 and will examine the potential role that 80K-H, a TRPM7- and TRPM6-interacting protein we identified, may have in this process. With their incidence varying among different populations, neural tube closure defects occur at an average rate of 1 per 1000 births and are the second most prevalent malformation, after congenital heart defects, among human pregnancies. Collectively, the proposed experiments should greatly advance our understanding of how these unique bifunctional channels are functioning in vivo, which could lead to new strategies for preventing neural tube closure defects as well as provide new insight into other pathological conditions with which these channels have been associated, including stroke and cancer.