Metal ions contribute to fundamental biological processes, such as maintenance of protein structure, enzymeactivity and signal transduction. Transport mechanisms for divalent metal ions are not well-characterized.Only recently a novel ion channel, TRPM7, was found to conduct significant amounts of these ions into cells.TRPM7 is a ubiquitously expressed protein combining a channel and a kinase domain (channel kinase). Itschannel activity is regulated by intracellular magnesium (Mg2*) and nucleotriphosphates (Mg-NTP). Thechannel plays a key role in Mg homeostasis due to its unprecedented permeation preference for divalentions. TRPM7 is highly conserved across species, including zebrafish, emphasizing its critical role in cellphysiology. An additional protein, TRPM6, is structurally similar to TRPM7, but mainly expressed inabsorptive tissues such as kidney and intestine. Dysfunctional TRPM6 activity leads to hypomagnesemia inhumans. We here hypothesize that TRPM7 plays a ubiquitous role in regulating cellular magnesiumhomeostasis and that co-assembly with TRPM6 enhances its function in specialized absorptivetissues. We hypothesize that the alpha kinase domains of both proteins serve a dual function byregulating ion channel sensitivity to intracelLular Mg2* and Mg-ATP and by phosphorylatingsubstrates unrelated to channel function. Hence, in Aim1 we suggest electrophysiological analyses incollaboration with Project 2 (Ryazanov) and Project 4 (Stokes) investigating the functional consequences ofchannel kinase heteromerization on magnesium and metal ion influx. This will be pursued in aco-expressionsystem using HEK293 cells and in a native system from cells isolated from wild-type, heterozygous channelkinase knock-out (KO) and kinase-mute mice models.
In Aim 2 we will identify structural features of the poreregion of TRPM7 and TRPM6 that confer the selectivity filter for divalent ions, including magnesium. Incollaboration with Project 2 (Ryazanov) a series of pore mutants will be tested for their permeation profile inboth TRPM7 and TRPM6/TRPM7 heteromeric channels, leading to a delineation of the molecular basis forchannel selectivity. Together with Project 3 (Scharenberg) we will determine in Aim3 whether the biophysicaland functional properties of TRPM7 and TRPM6/TRPM7 are conserved across species. Based on ourcurrent knowledge of TRPM7 physiology we will focus on the biophysical characterization of zebrafishchannel kinase. Further differential analyses will identify regions of channel kinases responsible for specificphysiological functions. Detailed understanding of the functional consequences of TRPM6/TRPM7interaction and role of their kinase domains will open new possibilities to influence magnesium and metal ionabsorption in health and disease.
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