The TRPM6 kinase domain determines the Mg·ATP sensitivity of TRPM7/M6 heteromeric ion channels

Abstract

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg(2+)) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg(2+) and therefore unlikely to be active at physiological levels of [Mg(2+)]i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex.

Keywords: Magnesium; Metabolic Regulation; Protein Complexes; Protein Kinases; TRP channels.

FIGURE 1.

Differential modulation of TRPM6, TRPM7, and TRPM7/M6 channels by 2-APB. Whole-cell currents in HEK-293 cells were measured in an external solution in which Mg²⁺ was omitted but 1 mm Ca²⁺ was present to maintain the typical I-V shape. The internal solution was Mg²⁺-free in the presence of 10 mm EDTA. 200 μm 2-APB was applied to the cells as indicated by the black bars. Currents were analyzed at +80 and −80 mV for outward and inward currents, respectively. Representative I-V curves were extracted before application of 2-APB for respective channel type. A and B, whole-cell measurement of outward and inward TRPM6 currents in wild-type HEK-293 cells transiently transfected with TRPM6 cDNA (n = 7). C, whole-cell measurement of co-overexpressed outward and inward TRPM7/M6 currents in HEK-293 cells induced by tetracycline (n = 8). D shows representative I-V curves for control uninduced TRPM7/M6 cells (− tet) and induced TRPM7/M6 cells (+ tet). E, whole-cell measurement of overexpressed outward and inward TRPM7 currents in HEK-293 cells induced with tetracycline (n = 8). F and G, whole-cell measurement of outward TRPM6 currents in tetracycline-inducible HEK-293 cells in the presence (●, induced, n = 8) or absence (○, uninduced, n = 8) of tetracycline. G shows the typical I-V curve for induced currents (+ tet) prior to 2-APB application. H, typical I-V curve for current controls in uninduced TRPM6 cells prior to 2-APB application (− tet) is shown. pF, picofarad.

FIGURE 2.

Magnesium-independent waxenicin A inhibition and sensitivity to osmotic gradients. HEK-TRPM7/M6 cells were induced by tetracycline (A, B, E, and F). TRPM6 currents were measured in HEK-293 cells transiently transfected with TRPM6 (C and D). 10 mm EDTA was used to completely remove internal Mg²⁺ (0 [Mg²⁺]_i, C and E), whereas free Mg²⁺ was clamped by 10 mm EGTA (D and F). A, whole-cell currents in HEK-293 cells were measured in an external solution in which Mg²⁺ was omitted but 1 mm Ca²⁺ was present to maintain the typical I-V shape. The internal solution was clamped to 780 μm [Mg²⁺]_i. Currents were analyzed at +80 and −80 mV for outward and inward currents, respectively. Current development assessed in TRPM7/M6 tetracycline-induced cells was challenged (○ n = 5) or not (● n = 5) with 10 μm waixenicin A as indicated by the black bar. B, current development assessed in TRPM7/M6 tetracycline-induced cells challenged (○ n = 8) or not (● n = 9) with 10 μm waixenicin A in the absence of internal Mg²⁺. C, TRPM6 currents recorded in the absence of internal Mg²⁺. The bar indicates application of solutions with low (210 mosm, n = 9) or high osmolarity (510 mosm, n = 9). D, TRPM6 currents recorded in the presence of 20 μm Mg²⁺. The bar indicates application of isotonic (310 mosm, n = 11), hypotonic (210 mosm, n = 8), or hypertonic solutions (510 mosm, n = 9). E, TRPM7/M6 currents in response to hypotonic (210 mosm, n = 5) or hypertonic challenge (510 mosm, n = 5) in the absence of internal Mg²⁺. F and G, normalized TRPM7/M6 currents measured in the presence of 790 μm Mg²⁺. All currents were normalized to maximum currents at 100 s after break-in. Bars indicate application of various solutions with different osmolarity. The application was started at 150 s but terminated variably depending upon the tested osmolarity. Note that the solid black line is the control for hypotonic solution in which the osmolarity was normalized to 310 mosm with mannitol. Data plots in the dose-response curve represent n = 7, 12, 9, 10, 6 cells for 200, 310, 380, 510, 800 mosm, respectively. The ratios of currents (250 s versus the starting point of application at 150 s) were plotted versus the osmotic gradients (E). Dashed line represents the dose-response curve for the osmosensitivity of TRPM7 channels (reproduced from Ref. 22). pF, picofarad.

FIGURE 3.

Suppression of native TRPM7 and overexpressed TRPM6 and TRPM7/M6 channels by Mg²⁺. Native whole-cell TRPM7 currents were assessed in wild-type HEK-293 cells. Whole-cell TRPM6 currents (transiently overexpressed in wild-type HEK-293 cells) were recorded in Mg²⁺-free external solutions, whereas TRPM7/M6 currents (induced by tetracycline) were measured in normal external solution containing 2 mm Mg²⁺. The chelator EDTA was used to clamp free Mg²⁺ levels for TRPM6 currents. EGTA was the Mg²⁺ chelator for native TRPM7 currents and induced TRPM7/M6 currents. The detailed composition of the pipette solutions for current recording is shown in Tables 1 and 2. A and B, Mg²⁺ sensitivity of native TRPM7 currents assessed in wild-type HEK-293 cells by whole-cell patch clamping. Plots represent n = 7, 8, 10, 11, 4, and 4 cells for 0, 70, 210, 790, 1600, and 3200 μm Mg²⁺, respectively. A dose-response fit rendered an IC₅₀ of 569 μm with a Hill coefficient of 1.27 (peak currents extracted at 434 s). C, RT-PCR analysis of the expression of native TRPM6 (546 bp) and native TRPM7 (519 bp) in HEK-293 wild-type cell line, as well as the housekeeping gene, GAPDH (532 bp) (2nd and 3rd lanes). The gene-specific primers for TRPM6 and TRPM7 amplified the same sized bands from heterologously expressed human TRPM6 and TRPM7 plasmids, respectively (5th and 6th lanes). D, whole-cell patch clamp analysis of Mg²⁺ block of transiently transfected TRPM6. The number of patched cells is 7, 6, 5, 5, 6, 5, and 7 for 0, 10, 30, 100, 300, 900, and 1200 μm Mg²⁺, respectively. E, whole-cell patch clamp analysis of Mg²⁺ block of TRPM7/M6 currents. Plots represent n = 12, 7, 7, 7, 11, 8, 6, and 5 cells for 0, 20, 70, 210, 790, 1200, 1600, and 3200 μm Mg²⁺, respectively. F, dose-response curves of Mg²⁺-mediated suppression of native TRPM7 (long-dashed line taken from B and normalized to 1), overexpressed TRPM7 (small-dashed line reproduced from Ref. and normalized to 1), TRPM6 (extracted at 100 s, closed circles, Hill = 0.69), and TRPM7/M6 currents (extracted at 120 s, open circles, Hill = 0.8). pF, picofarad.

FIGURE 4.

Mg²⁺ sensitivity of TRPM6 phosphotransferase activity-deficient point mutant (TRPM6-K1804R) and TRPM6-Δkinase alone and in channel complex with TRPM7. The detailed composition of the pipette solutions for current recording is shown in Tables 1 and 2. A, whole-cell patch clamp assessment of Mg²⁺ block of TRPM6-K1804R currents. B, whole-cell patch clamp assessment of Mg²⁺ block of TRPM6-Δkinase currents. C, dose-response curves for Mg²⁺ suppression of whole-cell currents carried by various TRPM6 constructs. Data represent peak currents at the indicated Mg²⁺ concentrations normalized to the maximum currents attained at 0 Mg²⁺. Peak currents extracted at 100, 48, and 48 s were averaged for dose-response analysis for TRPM6 WT, TRPM6 K1804R, and TRPM6 Δkinase, respectively. Note that the plots of TRPM6 WT are derived from Fig. 2D. Plots represent n = 7, 6, 5, 5, 6, and 5, experiments (TRPM6 WT, 0, 10, 30, 100, 300, and 900 μm Mg²⁺, respectively), n = 5, 5, 5, 5, 5, and 5, experiments (K1804R, 0, 10, 30, 100, 300, and 900 μm Mg²⁺, respectively), and n = 5, 8, 6 experiments (Δkinase, 0, 30, 100 μm Mg²⁺, respectively). D–F are from heteromeric channels where HEK-TRPM7/M6-K1804R cells and HEK-TRPM7/M6-Δkinase cells were induced by tetracycline to co-overexpress TRPM7/M6-K1804R and TRPM7/M6-Δkinase, respectively. The internal and external solutions for current recordings (D and E) are the same as those for TRPM7/M6 in Fig. 2E. D, whole-cell patch clamp analysis of Mg²⁺ inhibition of TRPM7/M6 K1804R currents. Pipette solution contained the indicated concentrations of free Mg²⁺. Plots represent n = 8, 9, 6, 6, 6, and 5 cells for 0, 20, 210, 790, 1600, and 3200 μm Mg²⁺, respectively. E, whole-cell patch clamp assessment of Mg²⁺ suppression of induced TRPM7/M6 Δkinase currents. Plots represent n = 5, 5, 5, 7, 6, and 5 cells for 0, 20, 210, 790, 1600, and 3200 μm Mg²⁺, respectively. F, dose-response curves for Mg²⁺ suppression of TRPM7/M6 K1804R (peak currents extracted at 76 s, Hill = 1.34) and TRPM7/M6 Δkinase currents (extracted at 136 s Hill = 1.28). pF, picofarad.

FIGURE 5.

Mg·ATP does not affect the TRPM7·M6 channel complex and rescues TRPM6 from inactivation. Transiently overexpressed TRPM6 currents were measured in HEK-293 cells and in Mg²⁺-free external solutions (A–D), whereas tetracycline-inducible currents of TRPM7 plus various TRPM6 mutants were recorded in normal external solution containing 2 mm Mg²⁺ and 1 mm Ca²⁺ (F–H). The intracellular free Mg²⁺ for TRPM6 current recording was 25.4 μm (A, C, and D; see Table 3 for composition of pipette solutions) or 20 μm (B; see Table 4 for composition of pipette solutions) and was 753 μm for heteromeric channels of TRPM7 plus TRPM6 (F–H; see Table 5 for composition of pipette solutions). A, HEK-293 cells transiently overexpressing TRPM6 were dialyzed with pipette solutions containing the indicated concentrations of Mg·ATP. Plots represent n = 14, 11, and 10 cells for 0, 3, and 9 mm Mg·ATP, respectively. B, Na·ATP rather than Mg·ATP was tested in TRPM6 WT. Plots were averaged from 10, 10, and 9 cells for 0, 3, and 9 mm Mg·ATP, respectively. C, TRPM6 K1804R mutant was transiently overexpressed. Plots represent n = 7, 7, and 9 cells for 0, 3, and 9 mm Mg·ATP, respectively. D, effects of Mg·ATP on the TRPM6 Δkinase mutant were tested in HEK-293 cells transiently overexpressing the mutant channel. Internal solutions had the same composition as in A and C. Plots represent n = 7, 8, and 9 cells for 0, 3, and 9 mm Mg·ATP, respectively. E, normalized to current peak percent inactivation of transiently expressed TRPM6 WT in HEK-293. Cells were perfused with either no addition of Mg·ATP (control, n = 14, see A), 3 mm (n = 11, A), or 9 mm Mg·ATP (n = 10, A) and 3 mm AMP-PNP (n = 13), 3 mm ADP (n = 9), 3 mm AMP (n = 12), or 300 μm ATPγS (n = 9). F, whole-cell TRPM7/M6 currents measured at 753 μm Mg²⁺ under the indicated conditions. Plots are representative of 14, 19, and 19 cells for 0, 4, and 8 mm Mg·ATP, respectively. G, whole-cell currents of TRPM7/M6 K1804R co-expressed mutant channel complex measured at 753 μm Mg²⁺ under the indicated conditions. Plots represent n = 17, 14, and 16 cells for 0, 4, and 8 mm Mg·ATP, respectively. H, whole-cell recording of tetracycline-induced TRPM7/M6 Δkinase mutant complex currents at 753 μm free Mg²⁺ under the indicated conditions. Note that the experimental conditions in E–G are identical. The number of patched cells is 9, 13, and 9 for 0, 4, and 8 mm Mg·ATP, respectively. pF, picofarad.