Fig 2: TRPM7 siRNA downregulated its mRNA expression and TRPM7-like current in TCMK-1 cells. Compared to the cells treated by the NC siRNA, the expression of TRPM7 mRNA was reduced by TRPM7 siRNA sequence 3239 and 1793 in TCMK-1 cells (P < 0.05 (a). However, TRPM7 siRNA 5326 and 1667 did not statistically decrease TRPM7 mRNA expression. The representative whole cell TRPM7-like currents invoked by the voltage ramp protocol were recorded in TCMK-1 cells transfected with NC siRNA and TRPM7 siRNA 1793 (b). The outward TRPM7-like currents were inhibited by the sequence 1793 of TRPM7 siRNA (c).

Fig 3: CHBP inhibited TRPM7-like currents in TCMK-1 cells. The voltage ramp protocol invoking TRPM7 currents and the representative TRPM7 currents recorded in TCMK-1 cells (a). Time-dependent running up of TRPM7-like currents after break-in when dialyzed with Mg2+ free internal solution by whole cell patch clamp (b). Blockade of TRPM7-like current (at +100 mV) by CHBP (100 nmol/l) (c). The periods of exposure to the normal extracellular solutions and CHBP were indicated by horizontal bars. Outward TRPM7-like currents were inhibited by CHBP, but reversibly changed by CHBP washout. (n = 6, (d)).

Fig 4: CNNMs have TRPM7-dependent and independent activities.(A) A 25Mg uptake assay was employed to assay TRPM7 channel function and its dependence on CNNMs. Moreover, 293-TRPM7 cells have significantly higher 25Mg uptake than the negative control T-REx-293 cell line (293), which expresses the Tet Repressor protein. Compared to 293-TRPM7 cells, 293-M7-?CNNM3/4 exhibited significantly lower 25Mg uptake, which was increased by reexpression of CNNM4. * = p < 0.05 and *** = p < 0.001, 1-way ANOVA plus Tukey post hoc. (B) Similar levels of free Mg2+ were detected in HEK-293T (293T(WT)) and HEK-293T TRPM7 KO (293T(?M7)) cells using the Mag-Fluo4 Mg2+ indicator. White scale bar = 100 µM. Overexpression of CNNM2 or CNNM4 substantially reduced cytosolic Mg2+ in both cell lines to similar degrees. (C) Quantitation of the results from (B). n = 50. *** indicates a p-value of less than 0.001. (D) Mag-Fura-2 was also employed to compare the Mg2+ levels between 293T(WT), 293T(?M7), and 293T(?M7) transfected with CNNM2 or CNNM4. Images of the cells were taken using a 510-nm filter from excitation at 340 nm versus 380 nm on an inverted Olympus IX70 fluorescence microscope, and the ratio of fluorescence intensity of the cells from excitation at the 340 nm versus 380 nm wavelengths (30 cells) was quantified. *** indicates a p-value of less than 0.001. (E) 293T(WT), 293T(WT) transfected with CNNM4, 293T(WT) transfected with CNNM4 and treated with the TRPM7 channel inhibitor NS85903 (10 µM), and 293T(?M7) cells transfected with CNNM4 were loaded with Mag-Fluo4. The fluorescence intensity of the cells (mean of 50 cells) were measured for 1 minute before the extracellular solution bathing the cells was replaced with a Na+ free solution (replacing NaCl with NMDG-Cl) to inhibit Na+-dependent Mg2+ extrusion by CNNM4. Replacement of the buffer occurred between 50 and 100 seconds after imaging began as indicated. Overexpression of CNNM4 increased the Mg2+ levels in 293T(WT) but not 293T(?M7) cells, and the increase in Mg2+ in 293T(WT) cells caused by CNNM4 overexpression could be blocked by NS8593, which was continually present in the imaging medium and was initially added just prior to imaging. The underlying data for this figure can be found in S1 Data. WT, wild-type.

Fig 5: Comparison of the expression of TRPM6 and TRPM7 in left ventricular cardiomyocytes incubated for 2 h vs. 12 h in extracellular solutions with (A,B) and without (C,D) divalent cations (DV and DVF, respectively). (A,C) The cardiomyocytes were fixed after 2 h (filled columns) or 12 h (unfilled columns) of cell isolation: Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. (B,D) Quantification of the intensity of the fluorescence expressed in arbitrary units (a.u.). # p < 0.001 expression after 12 h vs. 2 h of cardiomyocyte incubation.