As opposed to Ca2+, the role of change in intracellular Mg2+-concentrations, [Mg2+]i, in lymphocyte activation remains unclear. Yet Mg2+ is the most abundant intracellular cation, and an essential cofactor for many cellular enzymes, as well as a critical regulator of cell growth and differentiation. Several studies undertaken in B-lymphocytes document an increase in [Mg2+]i following an increase in [Ca2+]i in response to receptor activation or ionophore treatment. A main difficulty in studying the relevance of [Mg2+]i in B-cell activation has been the very poor understanding of molecular mechanisms underlying Mg2+-homeostasis regulation in vertebrates. The Mg2+-permeable ion channel TRPM7, a member of the newly discovered TRPM-subfamily of cation channels, plays an essential role in Mg2+-homeostasis in DT40 B-lymphocytes in such that TRPM7 deficient DT40 B-cells become Mg2+-deficient, experience growth arrest within 24 hr and eventually die. Both, viability and proliferation of TRPM7 deficient DT40 B-cells are rescued by supplementation of Mg2+ to the growth media. Preliminary data in this grant proposal show furthermore the presence of Mrs2 in DT40 cells, the first mitochondrial Mg2+-transporter in B-cells. It was recently proven that reduced Mg2+-concentrations in Mrs2 knock-out mutant in yeast could be partially restored by complementing with the human form of Mrs2. Furthermore several studies in lymphocytes have shown that Mg2+ from intra-/extracellular sources is regulating phosphoinositide metabolism. For a better understanding of Mg2+-physiology and of Mg2+-dependent signaling events in immune cells, this grant proposes to further investigate the role of Mrs2 and TRPM7 in B-lymphocytes after receptor stimulation, and to analyze their involvement in Mg2+-homeostasis as well as Mg2+-dependent signaling events including PLCgamma activation, phosphoinositide metabolism and diacylglycerol production (DAG), following three lines of investigation: 1) What is the influence of Mrs2 and TRPM7 on intracellular Mg2+- and Ca2+-concentrations after B-cell receptor (BCR) stimulation in DT40 B-lymphocytes? 2) What is the role of Mrs2 and TRPM7 in activation of phosphoinositide metabolism after BCR stimulation? 3) How is gene expression regulated in TRPM7 and Mrs2 deficient DT40 B-cells under different Mg2+-concentrations in the growth media after BCR stimulation?
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