Project 3: Mechanism of Mg2+ Homeostatis in Growth and Cell Signaling

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TRPM6 and TRPM7 are unique dual function ion channel/protein kinases. While the individual functions of the channel and kinase domains have been extensively analyzed, and the physiological roles of each protein have been convincingly linked to regulation of Mg2* homeostasis, the molecular mechanisms through which these proteins act to influence Mg2* homeostasis remain poorly understood. We here hypothesize: 1) TRPM7 plays a ubiquitous role In regulating cellular Mg2* homeostasis, and this function is Influenced by coassembly with TRPM6 in specialized absorptive tissues, and 2) the alpha kinase domains of both proteins serve a dual function by regulating ion channel sensitivity to intracellular Mg2* and Mg-ATP, and by phosphorylating substrates unrelated to the channel function. As Mg2* homeostasis must be regulated in concert with cell growth, this proposal will explore three lines of investigation to better define the relationship between TRPM7 function, cellular Mg2* homeostasis, and cellular mechanisms which regulate growth/proliferation. In Specific Aim 1, we will pursue an important implication of TRPMT's role in regulating Mg2+ homeostasis: its function must be integrated with that of other Mg2* transport pathways. In Subaim 1a, we will evaluate the Mg2* transport functions of a novel family of proteins, the SLC41 family, and compare/contrast the function of these proteins with TRPM7's role in Mg2* transport. In Subaim 1b, we will analyze the membrane topology of SLC41 family proteins as an initial step towards understanding the molecular mechanisms which regulate their Mg2* transport function. In Specific Aim 2, the linkage between TRPM7's Mg2* regulatory function and cell growth regulation will be analyzed. These studies will focus on understanding how regulation of Mg2* homeostasis via TRPM7 influences the activity of a central growth regulatory pathway, analysis of the role of TRPM7-mediated Mg2* regulation during lymphocyte stimulation via different mitogens, and identification of potential protein targets of Mg2*- dependent activation of the TRPM7 kinase domain. In aim 3, we will further develop zebrafish TRPM7 nutria mutants, which exhibit defects in growth and cation homeostasis, as a model organism for in vivo studies of TRPM7 function. In subaim 3a, we will analyze TRPM6 and TRPM7 expression in zebrafish, express and characterize zebrafish WT TRPM7, and assess Mg2* homeostasis in WT and existing TRPM7 nutria mutant zebrafish. In subaim 3b, we will isolate temperature sensitive mutants of TRPM7, characterize the phenotype of the mutant fish under various restrictive and permissive conditions, and compare the function of ts TRPM7 alleles to the WT zebrafish TRPM7. Together, the proposed studies will generate a broad foundation of knowledge on the" molecular mechanisms of Mg2*, uptake and their integration with mechanisms which regulate cell growth and proliferation in vertebrates.