TRPM7 channel is regulated by magnesium nucleotides via its kinase domain

Abstract

TRPM7 is a Ca(2+)- and Mg(2+)-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg(2+), the functional roles of intracellular levels of Mg.ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg.ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg(2+). Mg.ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg(2+) and Ca(2+) overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP > or = GTP > or = UTP > ITP approximately free Mg(2+) alone. These nucleotides also enhanced TRPM7 inhibition by free Mg(2+), suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg(2+)-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg.NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg(2+) binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg(2+) binding site extrinsic to that domain.

Figure 1.

Inhibition of TRPM7 by ATP, ADP and free Mg²⁺, but not by AMP. Whole cell currents were recorded in HEK-293 cells induced to overexpress human TRPM7. Cs-BAPTA was used as intracellular calcium buffer. (A) Cells were perfused with 6 mM ATP and no Mg²⁺ (n = 4) or 6 mM Mg·ATP and 790 μM free Mg²⁺ (n = 7). (B) Cells were perfused with 6 mM ADP and no Mg (n = 5) or 6 mM Mg·ADP and 780 μM free Mg²⁺ (n = 6). (C) Cells were perfused with 6 mM AMP and no Mg (n = 9) or 6 mM AMP and 750 μM free Mg²⁺ (n = 5). (D) Representative current–voltage (I/V) relationships for TRPM7, in the conditions described above (A–C). All I/V relationships were taken at 200 s and were derived from a high-resolution current record in response to a voltage ramp of 50 ms duration that ranged from −100 mV to +100 mV. (E) Inhibition of TRPM7 currents by 6 mM Mg·ATP and increasing free Mg²⁺ concentrations (n = 4–8). (F) Dose–response curve of TRPM7 current inhibition by 6 mM Mg·ATP and increasing free Mg²⁺ concentrations (0, 70, 120, 160, 210, 420, 790, 1,600, and 3,200 μM) at 200 s (n = 4–8). The best fit was obtained with a Hill coefficient close to 2 and was therefore fixed at that value.

Figure 2.

Inhibition of TRPM7 by Mg·ATP and free Mg²⁺. Whole-cell currents were recorded in HEK-293 cells induced to overexpress human TRPM7. Cs-BAPTA was used as buffer. (A) Cells were perfused with increasing concentrations of free Mg²⁺ (n = 6–10). (B) Dose–response curve for the inhibition of TRPM7 current by increasing free Mg²⁺ concentrations (0, 70, 210, 780, 1,270, 1,600, and 3,200 μM). Data points correspond to average and normalized current amplitudes measured at + 80 mV after 200 s of whole-cell recording, plotted as a function of free Mg²⁺ concentration (n = 6–10). The best fit was obtained by a Hill coefficient close to 2 and was therefore fixed at that value. The calculated IC₅₀ value for free Mg²⁺ concentration inhibition of TRPM7 current is 720 ± 79 μM. (C) Cells were perfused with increasing concentrations of Mg·ATP (in this example, the free Mg²⁺ concentration was fixed to around 780 μM; n = 4–7). (D) Dose–response curve for the inhibition of TRPM7 current by increasing Mg·ATP concentrations (0, 1, 2, 4, and 6 mM Mg·ATP, free Mg²⁺ concentrations were fixed in this example around 790 μM). Data points correspond to average and normalized current amplitudes measured at +80 mV after 200 s of whole-cell recording, plotted as a function of Mg·ATP concentration (n = 4–7). The best fit was obtained by a Hill coefficient close to 2 and was therefore fixed at that value. The calculated IC₅₀ value for ATP concentration inhibition of TRPM7 current under physiological free Mg²⁺ (790 μM) is 2 ± 0.7 mM. (E) Comparison of dose–response curves as a function of Mg·ATP concentrations at different fixed free Mg²⁺ concentrations (210, 790, and 1,600 μM). Data points from the 790 μM dose–response curve were omitted for clarity as they are presented in B. Each dose–response was calculated as in B and normalized to 1 by dividing with Y_max (n = 4–12). (F) Comparison of dose–response curves as a function of free Mg²⁺ concentrations at different fixed Mg·ATP concentrations (0, 1, 2, 4, and 6 mM). Each dose–response curve was calculated as in Fig. 1 F and normalized to 1 by dividing with Y_max (n = 4–8). All the Hill coefficients of the dose–response curves in C and D were fixed to 2.

Figure 3.

Mg²⁺ and Mg·ATP inhibition of TRPM7 currents is independent of the buffer. Whole-cell currents were recorded in HEK-293 cells induced to overexpress human TRPM7. The currents were normalized by the capacitance of the cells recorded at break-in. (A) Comparison of HEDTA and BAPTA on TRPM7 current inhibition by Mg²⁺. Cells were perfused with 10 mM of either buffer without added Mg²⁺ or with 3.2 mM free Mg²⁺ (n = 4–8). (B) Mg·ATP block of TRPM7 current using the conditions published by Kozak and Cahalan (2003). The cells were perfused with 12 mM EGTA and 0.5 mM MgCl₂ (270 μM free Mg²⁺; n = 5) or with 2.5 mM HEDTA, 3 mM EGTA, 4 mM Mg·ATP, 360 μM free Mg²⁺ (n = 6). Mg·ATP block is clearly shown. Left axis labeling same as in A. (C) Comparison of HEDTA and BAPTA on TRPM7 current inhibition by Mg·ATP. Cells were perfused with 10 mM of either buffer with a fixed concentration of 210 μM free Mg²⁺ with or without 4 mM Mg·ATP (n = 5–8). Left axis labeling same as in A. (D) Typical example of Mg·ATP block of TRPM7 current in the absence of any Mg²⁺ or Ca²⁺ chelator and under physiologically relevant magnesium concentrations. The cells were perfused with a fixed concentration of 650 μM free Mg with or without 4 mM Mg·ATP (n = 6). (E) Mg²⁺ inhibition of TRPM7 current with EGTA buffering. Cells were perfused with 10 mM of EGTA and 2.5 mM Ca²⁺ (≈55 nM free) without added Mg²⁺ or with 0.5 or 1 mM MgCl₂ (370 and 740 μM free Mg²⁺, respectively (n = 7–11).

Figure 4.

Di- and triphosphate nucleotides inhibit TRPM7 currents. Whole cell currents were recorded in HEK-293 cells induced to overexpress human TRPM7. Cs-BAPTA was used as intracellular calcium buffer. (A) Typical example of TRPM7 current inhibition by NTP. Cells were perfused with 6 mM Mg·GTP and increasing free Mg²⁺ concentrations (n = 5–12). (B) Dose–response curve of TRPM7 current inhibition by 6 mM Mg·GTP and increasing free Mg²⁺ concentrations (0, 160, 420, 790, 1,600, and 3,200 μM). Data points correspond to average and normalized current amplitudes measured at +80 mV after 200 s of whole-cell recording, plotted as a function of free Mg²⁺ concentration (n = 5–12). The calculated IC₅₀ value for free Mg²⁺ concentration in the presence of 6 mM Mg·GTP is 340 ± 95 μM. (C) Comparison of dose–response curves as a function of free Mg concentrations for different NTP. Each dose response was calculated as in B and normalized to 1 by dividing with Y_max. (D) Typical example of TRPM7 current inhibition by ADP. Cells were perfused with 6 mM Mg·ADP and increasing free Mg²⁺ concentrations (n = 6–15). (E) Dose–response curve of TRPM7 current inhibition by 6 mM Mg·ADP and increasing free Mg²⁺ concentrations (0, 160, 210, 780, and 1,600 μM). Data points correspond to average and normalized current amplitudes measured at +80 mV after 200 s of whole-cell recording, plotted as a function of free Mg²⁺ concentration (n = 6–15). The calculated IC₅₀ value for free Mg²⁺ concentration in the presence of 6 mM Mg·ADP is 250 ± 81 μM. (F) Comparison of dose–response curves as a function of free Mg²⁺ concentration for different NDP. Each dose–response was calculated as in E and normalized to 1 by dividing with Y_max. All the Hill coefficients of the dose–response curves were fixed to 2.

Figure 5.

Phosphotransferase-deficient point mutants of TRPM7 affect NTP inhibition. Whole cell currents were recorded in HEK-293 cells induced to overexpress human TRPM7 WT or human TRPM7 mutants. The intracellular calcium buffer was Cs-BAPTA. (A) HEK-293 cells induced to overexpress human TRPM7 WT. Comparison of TRPM7 current inhibition by 6 mM Mg·ATP, Mg·GTP, Mg·ITP, and no nucleotide. In all conditions, free Mg²⁺ concentration was clamped at 210 μM (n = 5–20). (B) Comparison of TRPM7 current inhibition by 6 mM Mg·ATP and Mg·ITP with free Mg²⁺ concentration clamped at 1,600 μM (n = 7–8). (C) HEK-293 cells induced to overexpress human TRPM7 point mutation K1648R. Comparison of TRPM7 current inhibition by 6 mM Mg·ATP, Mg·GTP, Mg·ITP, or no nucleotide. In all conditions, free Mg²⁺ concentration was clamped at 210 μM (n = 6–11). (D) HEK-293 cells induced to overexpress human TRPM7 point mutation G1977D. Comparison of TRPM7 current inhibition by 6 mM Mg·ATP, Mg·GTP, Mg·ITP, or no nucleotide. In all conditions, free Mg²⁺ concentration was clamped at 210 μM (n = 6–12). (E) HEK-293 cells induced to overexpress human TRPM7 Δ-kinase mutation. Comparison of TRPM7 current inhibition by 1 mM Mg·ATP, Mg·GTP, Mg·ITP, or no nucleotide. In all conditions, free Mg²⁺ concentration was clamped at 210 μM (n = 6–17).

Figure 6.

Hypothetical model of Mg²⁺ and Mg-nucleotide regulation of TRPM7. The model depicts the binding of Mg²⁺ and Mg·NTP to the wt TRPM7 channel, the phosphotransferase-deficient point mutant K1648R, and the Δ-kinase truncation mutant. The wt TRPM7 channel provides two distinct sites (M and N sites) that jointly confer intermediate sensitivity to Mg²⁺ and Mg·NTP. The functional kinase domain (highlighted in orange) harbors the N site and awards specificity for various nucleotide species. The phosphotransferase-deficient mutant lacks the functional N site and is primarily regulated by the M site, but the loss of the synergistic N site interaction results in a channel with reduced susceptibility to Mg²⁺ block. Finally, the Δ-kinase truncation mutant completely eliminates the kinase domain and the N site, but exposes the M site, which previously was only accessible to Mg²⁺, to NTP binding. This results in a channel with enhanced sensitivity to both Mg²⁺ and NTP, but does not impart nucleotide specificity, suggesting that the Mg·NTP may simply function as a Mg donor to the M site.