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TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions

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Abstract

The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg2+ homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other’s cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg2+-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg2+, but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect—a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular trafficking and TRPM7-dependent cell growth. All these effects are dependent upon the presence of an active TRPM6 kinase domain. Dysregulated Mg2+-homeostasis causes or exacerbates many pathologies. As TRPM6 and TRPM7 are expressed simultaneously in numerous cell types, understanding how their relationship impacts regulation of Mg2+-uptake is thus important knowledge.

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Abbreviations

TRPM6 and TRPM7:

Transient receptor potential cation channel, subfamily melastatin, member 6 and 7

HSH:

Hypomagnesemia with secondary hypocalcemia

XMEN:

X-linked immunodeficiency with magnesium defect, chronic Epstein–Barr virus infections and neoplasia

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Acknowledgments

This paper was supported by NIH training Grant 5 T32 A1007405 (NIAID) to K.B., by NIH Grant 5R01GM068801 (NIGMS) to A.L.P. and by NIH Grants 5R21AI088421 (NIAID) and 5R01GM090123 (NIGMS & Office of Dietary Supplements) to C.S. We thank Dr. Fabienne Gally for carefully reviewing the manuscript.

Author information

Authors and Affiliations

  1. Integrated Department of Immunology, University of Colorado School of Medicine, 1400 Jackson Street, Denver, CO, 80206, USA

    Katherine Brandao, Francina Deason-Towne, Xiaoyun Zhao, Anne-Laure Perraud & Carsten Schmitz

  2. National Jewish Health, 1400 Jackson Street, Denver, CO, 80206, USA

    Xiaoyun Zhao & Anne-Laure Perraud

  3. Department of Biology, Regis University, 3333 Regis Boulevard, Denver, CO, 80221, USA

    Francina Deason-Towne

Authors
  1. Katherine Brandao
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  2. Francina Deason-Towne
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  3. Xiaoyun Zhao
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  4. Anne-Laure Perraud
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  5. Carsten Schmitz
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Corresponding authors

Correspondence to Anne-Laure Perraud or Carsten Schmitz.

Additional information

A.-L. Perraud and C. Schmitz contributed equally to this work.

Electronic supplementary material

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18_2014_1647_MOESM1_ESM.tif

Figure S1: Complementation approach of TRPM7 deficient DT40 cells with human TRPM7 WT co-expressed with human TRPM6 WT or human TRPM6 kinase dead (TRPM6 KR) mutants. A) Schematic representation of the DT40 complementation system. TRPM7 deficient chicken DT40 B cell complemented with human TRPM7 WT (cWT M7) alone, co-expressed with human TRPM6 WT (cWT +M6 WT) or with a human TRPM6 kinase dead mutant (cWT M7 + M6 K1804R). TRPM6 is Flag- and TRPM7 is HA-tagged. B) FLAG-hTRPM6 K1804R (kinase dead) has been cloned and stably transfected into TRPM7 deficient DT40 cells with stable expression of the Tet repressor and complemented with human TRPM7 WT channels. Cell clones were selected for stable, doxycycline (Dox)-inducible protein expression (+, induced; -, non-induced). Flag-tagged TRPM6 was immunoprecipitated (IP) and subsequently analyzed by immunoblotting using an anti-FLAG antibody. C) Analysis of TRPM6 protein expression levels under high and low Mg2+ concentrations. Co-expression of human TRPM7 WT and TRPM6 WT or a TRPM6 kinase dead mutant (KR) in TRPM7 deficient DT40 cells was induced by adding doxycycline to chemically defined media. 24 h after induced protein overexpression cells have been cultured in 10 or 0 mM Mg2+ for another 24 h. Equal numbers of cells of the hTRPM6/hTRPM7 coexpressing DT40 TRPM7−/− cell line were analyzed by immunoprecipitation (IP), followed by Western immunoblotting (WB) with anti-FLAG or anti-HA antibodies. TRPM7 DT40 deficient cells have been used as negative control (M7 KO). These data are representative of at least two separate experiments. (TIFF 579 kb)

18_2014_1647_MOESM2_ESM.tif

Figure S2: Analysis of TRPM6 channel-kinase regulation of TRPM7 subcellular trafficking in varying Mg2+ growth conditions. Charts show mean % of cells ± SEM. A/C) TRPM7 subcellular distribution was measured using a 1 pixel histogram across each cell. Cells displaying high levels of peripheral TRPM7 trafficking were identified by steep increases in fluorescent intensity near the membrane while cells expressing low levels of peripheral trafficking contained low levels of immunoreactivity near the membrane and shallow increases in fluorescent intensity. 15-30 cells were measured per cell type and growth condition. A/B) TRPM7 WT (0 mM n = 3, 1 mM: n = 4) TRPM7 WT + M6 WT (0 mM n = 5, 1 mM: n = 4) TRPM7 WT + M6 KR (0 mM n = 5, 1 mM: n = 4). A) The percentage of TRPM7 WT cells containing defined peaks of TRPM7 immunoreactivity increased in 0 mM growth conditions compared to the 1 mM growth conditions. The presence of TRPM6 inhibited significant change in TRPM7 transport towards the membrane in hypomagnesic conditions. B) TRPM7 clusters were defined as saturating regions of TRPM7 greater than 0.5 mm in length. The presence of a functional TRPM6 kinase domain corresponded to a significant increase in TRPM7 clusters in both Mg2+ growth conditions. C/D) cWT M7 (0 mM n = 3, 1 mM: n = 5) cWT + M6 WT (0 mM n = 3, 1 mM: n = 5) cWT M7 + M6 KR (0 mM n = 3, 1 mM: n = 4). C) The percentage of cells producing histograms showing sharp TRPM7 peaks near the periphery of the cell instead of diffuse TRPM7 immunoreactivity across the cell increased significantly in cWT M7 grown in 0 mM Mg2+ compared to 1 mM Mg2+. No significant change was measured in cWT M7 + M6 WT or cWT M7 + M6 KR cells in 0 or 1 mM Mg2+ growth conditions, but each cell type displayed a significantly lower percentage of cells containing TRPM7 peripheral trafficking as compared to cWT M7 cells in 0 mM Mg2+. D) TRPM7 expression, measured as the percentage of cells displaying TRPM7 immunoreactivity 3-SD above the mean background levels, increased only in cWT M7 + M6 WT cells grown in a hypomagnesic environment. (TIFF 123 kb)

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Brandao, K., Deason-Towne, F., Zhao, X. et al. TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions. Cell. Mol. Life Sci. 71, 4853–4867 (2014). https://doi.org/10.1007/s00018-014-1647-7

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