Fig 4. Inhibition of PIP₂ hydrolysis by Mg²⁺ or Ba²⁺ accumulated into cells through TRPM7.

(A) Schematic diagram. HEK293-TRPM7 cells were transfected with M₁R and PH-YFP and TRPM7 channels were activated by voltage clamp to accumulate divalent. (B) Confocal images of PH-YFP where the fluorescence is coded as yellow. Images are taken before and during application of 1 μM oxotremorine-M (Oxo-M) in control group. Top cell was patched. Region of interest (ROI) used for the PH-YFP translocation analysis is indicated in red circle. Black scale bar indicates 20 μm. (C) The average rate of PIP₂ hydrolysis by PLC was estimated by the monitoring of translocation of PH-YFP into the cytosol upon activation of M₁R with 1 μM Oxo-M. For the accumulation of divalent cations into the cells, external solutions containing 10 mM MgCl₂ (N = 5) or 10 mM BaCl₂ (N = 7) were perfused at the indicated time, and their influx through TRPM7 was triggered by a negative membrane potential (-80 mV). Error bars were omitted for clarity. (D) Comparison of PLC activity before and after the divalent accumulation. Percent increase of cytosolic PH-YFP upon 2nd Oxo-M treatment was divided by that of 1st Oxo-M (see Methods for details). The results are mean ± SEM and representative of two independent experiments. *** P < 0.001 compared to control group (N = 5).