Molecular determinants of Mg2+ and Ca2+ permeability and pH sensitivity in TRPM6 and TRPM7

Abstract

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg2+ and Ca2+ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg2+ and Ca2+ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than TRPM7, respectively. The binding affinity of Ca2+ and Mg2+ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca2+ and Mg2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca2+ and Mg2+ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca2+ and Mg2+ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels.

FIGURE 1. Alignment of TRPM7 pore region with other TRPM channels

A, schematic structure of the TRPM7 and positions of substituted amino acid residues in the TRPM7 channel. B, alignment of pore region of TRPM channels. Amino acids in boxes are the ones that were selected for analysis. The GenBank^™ accession numbers of the mouse TRPM7, human TRPM1– 6, and TRPM8 are AF376052, AAC8000, AAI12343; NP_060106, NP_055370, Q9BX84; and NP_076985, respectively.

FIGURE 2. Current-voltage relationships of WT TRPM7 and its mutants

A–G, representative currents elicited by ramp protocols ranging from −120 to +100 mV in WT TRPM7 and its mutants. Note the significant changes in the I–V relation of E1047Q (E) and E1052Q (F). H, normalized I–V curves of WT TRPM7 and its mutants. The I–V curves of D1035N, H1039E, H1039M, and D1054A show outward rectification, and are superimposed with that of WT TRPM7; whereas E1047Q exhibits double-rectification with significantly increased inward currents and markedly decreased outward currents. Inward current of E1052Q is substantially larger than that of WT TRPM7.

FIGURE 3. Average current amplitudes of WT TRPM7 and its mutants

A, mean outward(top)and inward (bottom)current amplitudes measured at +100 and −120 mV, respectively(n =8).B, ratios of inward versus outward current amplitude (n =8).

FIGURE 4. Effects of acidic pH on WT TRPM7 and its mutants

A₁–G₁, changes in the inward currents by acidification of external solutions. Current amplitudes were measured at −120 mV at the indicated pH and normalized to the amplitude obtained at pH 7.4. Note that E1047Q currents were inhibited by acidic pH solutions, with maximal inhibition of ~30% at pH 4.0. A₂–G₂, representative recordings of WT TRPM7 and its mutants obtained in the external solutions at pH 7.4 and 4.0. A₃–G₃, concentration-dependent effects of protons on WT TRPM7 and its mutants. The changes in current amplitude at the indicated pH were normalized to the maximal change in current amplitude. Average data were fitted with the Hill equation with average parameters obtained from best fits to individual cells. The 50% potentiation pH (pH_1/2) and Hill coefficient were pH_1/2 = 4.5 ± 0.5 (n_H = 1.2, n = 7) for WT TRPM7; pH_1/2 = 4.1 ±0.2 (n_H = 0.8, n =5) for D1035N; pH_1/2 = 4.5 ± 0.4 (n_H = 0.9, n = 5) for E1052; pH_1/2 = 4.7 ± 0.8 (n_H = 2.1, n = 6) for D1054A; pH_1/2 = 4.2 ± 0.4 (n_H = 1.1, n = 9) for H1039E; and pH_1/2 = 4.6 ±0.6 (n_H = 1.4, n =8) for H1039M; respectively. E1047Q inward currents were blocked by low pH, with the 50% inhibition pH of pH_1/2 = 5.4 ±0.7 (n_H = 0.9, n = 5).

FIGURE 5. Changes in affinity of Ca²⁺ and Mg²⁺ in TRPM7 mutants

A–C, representative recordings of WT TRPM7, E1052Q, and E1047Q obtained in the external solutions containing 1 nM (DVF), 1, 10, 200, and 500 μM Ca²⁺, and in 2 mM Ca²⁺ Tyrode solutions, respectively. D–F, typical currents of WT TRPM7, E1052Q, and E1047Q recorded in 1 nM, 1, 10, 200, or 500 μM Mg²⁺ containing solutions, and in 2 mM Ca²⁺ Tyrode solution, respectively. G and H, dose-response curves of Ca²⁺ and Mg²⁺ for WT TRPM7 and its mutants. The IC₅₀ values obtained by best fit with the Hill equation for the Ca²⁺ block were (μM): 4.1 ± 0.2 (n_H =0.96, n = 10) for WT TRPM7, 3.3 ± 0.1 (n_H = 1.7, n = 7) for D1035N, 593.6 ± 69.9 (n_H = 0.8, n = 5) for E1047Q, 202.2 ± 14.3 (n_H = 0.9, n = 7) for E1052Q, and 2.9 ± 1.3 (n_H =1.5, n =8) for D1054A, respectively. The IC₅₀ values for Mg²⁺ block were (μM): 3.6±0.4 (n_H = 0.7, n = 8) for TRPM7, 7.4± 0.8(n_H = 0.8, n= 7) for D1035N, 442.9 ± 53.6 (n_H = 0.6, n = 6) for E1047Q, 154.7 ± 23.4 (n_H = 0.6, n = 6) for E1052Q, and 3.6 ± 0.2 (n_H = 0.7, n = 8) for D1054A, respectively.

FIGURE 6. Voltage-dependent effects of Mg²⁺ on monovalent currents of WT TRPM7, E1047Q, and E1052Q

A–C, dose-response curves for the inhibition of inward monovalent currents by Mg²⁺ at the indicated voltages for WT TRPM7 (A), E1047Q (B), and E1052Q (C). D–F, the IC₅₀ values obtained by best fit with the Hill equation at various voltages (n = 8 for TRPM7; n = 6 for E1047Q and E1052Q, respectively). G–I, current ratios (I/I₀) of TRPM7 (G), E1047Q (H), and E1052Q (I) in the presence of various Mg²⁺ concentrations, respectively. Note the relief of Mg²⁺ block at hyperpolarized potentials observed in TRPM7 and E1052Q, but not in E1047Q. Dotted lines represent I/I₀, where I is the current in the presence of Mg²⁺, and I₀ is the current in DVF solution. Solid lines represent the best fit of the current ratio to the Boltzmann functions (see “Experimental Procedures”). In G, for the voltage-dependent block on TRPM7, V_0.5 values for 3 (blue), 10 (red), and 100 (green) μM Mg²⁺ were 0.3, 20.9, and 40.6 mV, respectively; the values of k(depol) were almost identical, with 15.2 mV at 3 μM Mg²⁺, 15.5 mV at 10 μM Mg²⁺, and 15.6 mV at 100 μM Mg²⁺, respectively; for the voltage-dependent relief of block on TRPM7, V_0.5 for 3 and 10 μM Mg²⁺ were −64.9 and −124.2 mV, respectively, and the values of k(hyperpol) were 49.8 and 50.4 mV, respectively. In H, Boltzmann fit of the voltage-dependent block on E1047Q at 500 μM (blue), 10 mM (red), and 20 mM(green) Mg²⁺ generated V_0.5 values of −39.3, −27.5, and −16.3 mV, respectively; and k(depol) values of 36.2, 35.8, and 35.6 mV, respectively. In I, for voltage-dependent block on E1052Q at 150 μM (blue), 1 mM (red), and 5 mM (green) Mg²⁺, the V_0.5 values were 2.5, 15.2, and 50.6 mV, respectively; and k(depol) values were 24.5, 24.3, and 24.4 mV, respectively. For voltage-dependent relief of Mg²⁺ block on E1052Q, the V_0.5 values were −87.7 and −95.4 mV at 150 μM, and 1 mM Mg²⁺, respectively, and k(hyperpol) values were 29.1 and 29.0 mV at 150 μM and 1 mM Mg²⁺, respectively. The fraction of the membrane electrical field δ_out calculated from k(depol) based on k = RT/zδF was 0.84 for TRPM7, 0.36 for E1047Q, and 0.52 for E1052Q, respectively (see supplementary materials Table S2).

FIGURE 7. Changes in divalent permeability of TRPM7 mutants (mean ±S. E.,n =6)

A–E, currents recorded under the indicated conditions were normalized to the current amplitude value obtained in 30 mM Ca²⁺ external solutions. The sequence of monovalent permeability was not changed (K⁺>Cs⁺>Na⁺) in the mutants compared with WT TRPM7; however, the monovalent permeabilities in E1047Q were significantly larger than that of WT TRPM7. F, currents obtained in 30 mM Ca²⁺ or Mg²⁺ were normalized to the current amplitude obtained in Tyrode solution. Note that the ratios of I_Ca/I_Tyrode and I_Mg/I_Tyrode for E1047Q were 0.014 and 0.0096, respectively. The I_Ca(E1047Q)/I_Ca(TRPM7) was 0.023, and I_Mg(E1047Q)/I_Mg(TRPM7) was 0.011, respectively.

FIGURE 8. E1047Q diminishes Ca²⁺ permeation and eliminates Mg²⁺ permeation

A–C, representative currents of TRPM7, E1047Q, and E1052Q elicited by ramp protocols in 2 mM Ca²⁺/Tyrode, NMDG, isotonic Ca²⁺, and isotonic Mg²⁺ solutions, respectively. P2 pipette solution containing reduced Cs⁺ concentration (10 mM) was used. D–F, inward currents measured at −120 mV under different conditions in the representative cells for TRPM7 (D), E1047Q (E), and E1052Q (F). G–I, average current amplitudes (mean ±S.E., n =6) measured at −120 mV under the indicated conditions for TRPM7, E1047Q, and E1052Q. J–L, changes in reversal potentials of TRPM7 (J), E1047Q (K), and E1052Q (L) (mean ± S.E., n =6). ΔE_rev was obtained by subtracting the reversal potential under indicated conditions by the reversal potential in Tyrode solution.

FIGURE 9. Changes in Ca²⁺ and Mg²⁺ permeability in TRPM6 mutants E1024Q and E1029Q

A–C, current-voltage relations for TRPM6, E1029Q, and E1024Q elicited by voltage ramps ranging from −120 to +100 mV. D, normalized I–V curves of TRPM6, E1029Q, and E1024Q. E and F, dose-response curves of Ca²⁺ and Mg²⁺ for TRPM6 and its mutants. The IC₅₀ values obtained by best fit with the Hill equation for the Ca²⁺ block (E) were (μM): 4.6± 0.4 (n_H = 0.8, n =10) for TRPM6, 153.5 ± 20.0 (n_H=0.6, n =5) for E1024Q, and 50.5 ±9.0 (n_H =0.5, n =5) for E1029Q, respectively. The IC₅₀ values for Mg²⁺ block were (μM): 3.4 ±0.3 (n_H = 0.7, n =8) for TRPM6, 237.2 ±26.2 (n_H = 0.5, n =6) for E1024Q, and 149.2 ±19.2 (n_H = 0.6, n =6) for E1029Q, respectively. G–I, inward current amplitude of TRPM6, E1024Q, and E1029Q measured at −120 mV under the indicated conditions. J, average current amplitudes of TRPM6, E1024Q, and E1029Q obtained in isotonic Ca²⁺ and Mg²⁺ solutions (120 mM) normalized to the current amplitude obtained in Tyrode solution.

FIGURE 10. Effects of external protons on TRPM6 and its mutants E1024Q and E1029Q

A–C, representative recordings of TRPM6 (A), E1024Q (B), and E1029Q (C) obtained in the external solutions at pH 7.4 and 4.0. Note that E1024Q currents were blocked by low pH (B). D–F, changes in inward current amplitude measured at −120 mV at the indicated pH values. G–I, concentration-dependent effects of protons on TRPM6 (G), E1024Q (H), and E1029Q (I). Average data were fitted with the Hill equation with average parameters obtained from best fits to individual cells. The 50% potentiation pH (pH_1/2) and Hill coefficient were pH_1/2 = 4.3 ±0.4, n_H = 0.7 (n =8) for TRPM6, and pH_1/2 = 3.7 ±0.2, n_H = 1.0 (n =5) for E1029Q, respectively; and the 50% inhibition pH (pH_1/2) and Hill coefficient were pH_1/2 = 5.0 ± 0.7, n_H = 0.5 (n = 5) for E1024Q.

FIGURE 11. Changes in Mg²⁺ and Ca²⁺ permeability and pH sensitivity in the double mutant E1047Q/E1052Q of TRPM7

A, typical recording of the E1047Q/E1052Q current elicited by a voltage ramp protocol ranging from −120 to +100 mV. B, currents of E1047Q/E1052Q recorded in Tyrode, NMDG, isotonic Ca²⁺ and Mg²⁺ (120 mM) solutions. Note the diminished inward currents in isotonic Ca²⁺ and Mg²⁺ solutions. C, inward current amplitude measured at −120 mV under various conditions. D, average current amplitudes at −120 mV in isotonic Ca²⁺ and Mg²⁺ solutions normalized to the current amplitude in the Tyrode solution. The current amplitude of E1047Q/E1052Q in isotonic Ca²⁺ and Mg²⁺ (120 mM) solutions was 0.1 ±0.06 and 0.08 ±0.04 (n = 6) of that in Tyrode solution, respectively. E, concentration-dependent effects of Mg²⁺ on E1047Q/E1052Q at various voltages. The IC₅₀ values of Mg²⁺ were 132.7 ±25.6 μM at −120 mV, 135.2 ±17.3 μM at −80 mV, 138.4 ±41.3 μM at −40 mV, 632.3 ±76.7 μM at +40 mV, and 2.2± 0.4 mM at +80 mV, respectively (n =5). F, current ratio I/I₀ at 100 μM and 2 mM Mg²⁺ plotted as a function of membrane potential. The best fit with the Boltzmann equation for voltage-dependent block of Mg²⁺ at 100 μM and 2 mM yielded the V₅₀ values of −9.6 and 12.3 mV, respectively; and k values of 29.3 and 29 mV, respectively. The fraction of the membrane electrical field δ_out calculated based on k =RT/zδF was 0.44. G, effects of external protons on E1047Q/E1052Q. Inward current amplitude was increased by ~32% at pH 4.0. H, concentration-dependent effects of protons on E1047Q/E1052Q. The best fit with the Hill equation yielded pH_1/2 of 3.5 ±0.1 (n_H = 0.7, n = 5).