Despite the abundance and ubiquitous use of Mg 2+ in all forms of life, the well documented, crucial role of this cation in cellular physiology, as well as its potential role as a messenger ion, extremely little is known about molecular components and mechanisms acting to regulate Mg2+-homeostasis, especially in vertebrates. Our preliminary data demonstrates that TRPM7, a member of the newly discovered TRPM-subfamily of cation channels, appears to play an essential role in Mg 2+physiology in vertebrates: We showed that the lethal phenotype caused by the homozygous deletion of TRPM7 in the DT40 chicken B-cell system can be totally reverted by supplementing the growth medium with high concentrations of Mg 2+. Furthermore, it was recently shown that patients suffering from an inherited form of hypomagnesemia bear mutations in TRPM6, the closest relative of TRPM7. Both TRPM6 and TRPM7 are unique among all known ion-channels in that they possess a kinase domain at their C-terminus. We have shown that TRPM7 is permeable to and regulated by Mg 2+ and that the phosphotransferase activity of the TRPM7 kinase domain is sensitive to Mg 2+ in the same concentration range that regulates channel gating. Our preliminary results demonstrate that while the kinase domain is not essential for channel activation, channel and kinase are functionally coupled such that the kinase alters the channel's Mg2+-sensitivity and that a structure extrinsic to the kinase domain is responsible for the observed Mg2+-sensitivity of TRPM7. Based on these results, we propose to address three important questions about the regulation and function of TRPM7. Specific aim 1: Structure-function relationships studies. In this aim, we plan to characterize different TRPM7 kinase mutants as well as TRPM7/TRPM6 chimeric molecules using three experimental approaches: Biochemical analysis of channel phosphorylation, electrophysiological characterization of channel function, and analysis of the effects of selected mutations on cellular physiology and Mg 2+ homeostasis using a complementation approach in the TRPM7-deficient DT40 cells. Specific aim 2: Determining the role of phosphorylation sites in the C-terminal domains of TRPM7: We will first conduct phosphopeptide mapping analyses and design a series of truncations and point mutants of the C terminal regions to localize potential sites of phosphorylation. Selected positions will subsequently be mutated in the TRPM7 full-length channel, and these mutants analyzed using the approaches describeed in Aim 1. Specific Aim 3: Defining the role of the TRPM7 kinase domain in regulating gene expression and protein synthesis: Using cDNA microarray techniques to monitor changes in gene expression patterns, we plan to investigate the potential changes in general cellular processes in the presence or relative absence of TRPM7 in the DT40 cells under various conditions. Another set of experiments will analyze the potential role of TRPM7 regulating protein synthesis.