(A) Average time course of outward (at +80 mV) current densities from HEK293 cells transfected with TRPM6 in control conditions (open circles) and after EGF treatment (30 minutes, 10 nM, filled circles) in comparison with mock-transfected cells without EGF treatment (triangles) and after EGF stimulation (filled triangles). n = 17–20. (B) Dose-response curve of EGF-induced current in TRPM6-transfected HEK293 cells indicating half maximal effective concentration of 1.7 nM. n = 11–14. (C) Histogram presenting the current densities at +80 mV of TRPM6-transfected cells (200 seconds after establishment of the whole-cell configuration) treated with mock (mock), pro-EGF (EGF WT) or pro-EGF–P1070L (EGF-P1070L) supernatant. All treatments were performed for 30 minutes at 37°C. White bars indicate experimental conditions in which HEK293 cells were transfected with mock DNA whereas black bars indicate HEK293 cells transfected with TRPM6. Asterisk indicates significance in comparison with nontreated TRPM6-transfected cells. P = 0.024; n = 6. (D) Supernatants of mock-, pro-EGF–, or pro-EGF–P1070L–transfected HEK293 cells were analyzed by ELISA. Asterisk indicates mock and pro-EGF–P1070L supernatant were significantly different from EGF supernatant. P = 0.006; n = 4. (E) Histogram summarizing the current density (pA/pF) at +80 mV (200 seconds after break-in) of HEK293 cells treated with the apical or basolateral supernatant of MDCK cells stably transfected with either wild-type pro-EGF, pro-EGF–P1070L, or mock DNA (30 minutes, 37°C). White bars indicate experimental conditions in which HEK293 cells were transfected with mock DNA whereas black bars indicate HEK293 cells transfected with TRPM6. Crosses indicate significance in comparison with the mock treatment at the apical side (wild-type pro-EGF apical, P = 0.0001, n = 6; pro-EGF–P1070L apical, P = 0.029, n = 6), and pound symbol represents significance in comparison with the mock treatment at the basolateral side (wild-type pro-EGF basolateral P = 0.031, n = 6). (F) Schematic model illustrating how pro-EGF–P1070L mutation results in IRH. The pro-EGF–P1070L mutation leads to impaired basolateral sorting of pro-EGF, resulting in abrogated stimulation of the EGFR localized at the basolateral membrane. Activation of the EGFR in DCT by EGF is necessary to prevent renal Mg²⁺ wasting by stimulation of the epithelial Mg²⁺ channel TRPM6.