Fig. 4.

TRPM6 kinase activity inhibits TRPM7 mediated cell growth in HEK-293 and DT40 cells growth under hypomagnesic conditions. a Growth curves of HEK-293 expressing human TRPM7 WT (M7 WT) alone, or co-expressing hTRPM7 WT with either human TRPM6 WT (M6/7 WT), or the TRPM6 K1804R kinase dead mutant (M6KR + M7 WT). Cells were cultured in normal HEK-293 media, prior to Mg²⁺ deprivation protein overexpression was induced for 6 h with doxycycline (1 μg/ml). Cells were spun down and equal number of cells transferred for 24 h into fresh, complete, serum-free growth media with 0 or 1 mM Mg²⁺, as indicated. Note: TRPM7 overexpressing HEK-293 cells die after 36–48 h doxycycline induced TRPM7 overexpression. b–d growth curves of TRPM7 deficient DT40 cells complemented with human TRPM7 WT (cWT M7) or cKR M7 (kinase dead) alone, or with hTRPM7 WT co-expressed with either human TRPM6 WT (cWT M7 + M6 WT), or the TRPM6 K1804R kinase dead mutant (cWT M7 + M6 KR). Cells were cultured in complete, chemically defined, Mg²⁺-free media without serum requirement. Cells were grown under various Mg²⁺ concentrations for several days as indicated. Prior to Mg²⁺ deprivation protein overexpression has been induced for 24–48 h with doxycycline (final concentration: 1 μg/ml) in complete growth media with additional 10 mM MgCl₂ (Note: TRPM7^−/− DT40 cells would die without Mg²⁺ supplementation of the growth media). Cells were spun down and equal number of cells (2 × 10⁵) transferred into fresh, complete growth media with different Mg²⁺ concentrations, as indicated. For statistical analysis, a two-tailed t test was performed. Data shown are the mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. Note: Growth data at physiological (1 mM Mg²⁺) and hyerpmagnesic conditions have been published in 2003/2005 and did not show any differences