Our previously published TRPM6/7 surface area biotinylation study in HEK-293 cells demonstrated that TRPM6 and TRPM7 have to associate in order for TRPM6 to be detected at the plasma membrane . (also phosphorylated by TRPM6 kinase) , eukaryotic Elongation Factor 2 kinase (eEF2K)  and Phospholipase C gamma 2 (PLC2) . TRPM7s phosphotransferase activity may regulate the activity of its channel domain in accordance to the environmental availability of Mg2+, as the inhibitory phosphorylation of eEF2K via TRPM7 increases under hypomagnesic cell culture conditions . Mutations and deletions of both TRPM6 and TRPM7 cause profound cellular dysfunction and are often lethal, indicative of the important role these channels play in regulating Mg2+ homeostasis. TRPM6 mutations in humans have been linked to an autosomal recessive form of familiar hypomagnesemia with secondary hypocalcemia (HSH). These patients fail to build a functional TRPM6 pore and suffer from neurological symptoms, including seizures and muscle spasms during infancy, and eventually die if not treated by Mg2+ supplementation [4, 5]. Over the last decade numerous studies have demonstrated that TRPM7 plays an important role in cell proliferation (, reviewed in ), cell migration , protein translation , immuno receptor signaling , cytoskeleton building (reviewed in [23, 24], cancer development (reviewed in ) and cancer metastasis . TRPM6 and TRPM7 knock out mice (TRPM6-/- and TRPM7-/-) are both embryonically lethal [7-9, 27]. Mice with inducible, T cell restricted TRPM7 deletion show a block in thymocyte development at the double negative stage and a depletion of thymic medullary cells, but no measureable changes in intracellular Ca2+ or Mg2+ concentrations (, reviewed in [6, 28]). However, in another study the same research group excluded any role for TRPM7 kinase in Fas induced apoptosis in TRPM7-/- T-cells (, reviewed in ). Future studies will need to clarify whether this developmental phenotype is T cell specific, or if TRPM7 Adamts4 is such an essential gene that its absence is causing decreased viability and developmental failures in any cellular context. Homozygous TRPM7 kinase deletion mutants generated by Ryazanova and colleagues  are embryonically lethal as well, whereas the corresponding heterozygote mice are viable, but hypomagnesic, and exhibit reduced intestinal Mg2+ absorption . The same group were able to rescue TRPM7 kinase deficient embryonic stem cells going into growth MEK162 (ARRY-438162, Binimetinib) arrest by additional Mg2+ supplementation . In analogy, TRPM7 deficient chicken DT40 B-cells go into cell-growth arrest and die under physiological levels of Mg2+ (~1mM), but grow normally if the medium is supplemented with 5-10 mM Mg2+. TRPM7-/- DT40 cells can be rescued MEK162 (ARRY-438162, Binimetinib) by overexpression of human TRPM7 WT, TRPM7 kinase dead mutants , and partially by the human Mg2+ transporters MagT1  and SLC41A2 , but not via overexpression of TRPM6 WT alone . Due to some conflicting data in literature, it still remains to be determined whether native TRPM6 homomers can form functional channels at the MEK162 (ARRY-438162, Binimetinib) cell surface that are physiologically relevant, or if TRPM6/TRPM7 heteromers might represent the more common and functionally important configuration of these channels for cellular fate. In order to provide further insights into TRPM6 and TRPM7 function, we have carefully examined the biological effects of TRPM6 and TRPM7 co-expression in two different cellular systems, and demonstrate for the first time that TRPM6 phosphotransferase activity affects TRPM7 subcellular localization and cellular growth. We show that TRPM6 regulates TRPM7 trafficking, and that under hypomagnesic cell culture conditions, TRPM6 has an inhibitory effect on TRPM7-dependent cell growth. 2. Material and Methods 2.1. Cloning, cell culture and generation of cell lines overexpressing proteins of interest A) The generation of HEK-293 T-Rex cells (Invitrogen) with transient or stable, doxycycline (dox)-inducible expression of human TRPM6 and TRPM7 wt and kinase dead or deletion mutants, as well as the cell culture conditions of these cells have MEK162 (ARRY-438162, Binimetinib) been previously described . In order to study the biological effects of Mg2+ deprivation, expression of proteins of interest in HEK-293 cells was induced with 1 g/ml doxycycline for 24-48 hrs prior deprivation. After 12 hrs of induced protein expression regular HEK media was removed and replaced with customized, chemically defined, serum-free media (HyQ.
Our previously published TRPM6/7 surface area biotinylation study in HEK-293 cells demonstrated that TRPM6 and TRPM7 have to associate in order for TRPM6 to be detected at the plasma membrane