TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Abstract

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) >> Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.

Figure 1.

TRPM7 is an influx pathway for Ca²⁺. Simultaneous whole-cell patch-clamp recordings of MagNuM and fura-2 measurements of [Ca²⁺]i in HEK-293 cells overexpressing TRPM7. (A) Average inward and outward MagNuM currents at −80 and 80 mV, respectively, in cells perfused with Cs-glutamate–based internal solution in the absence of Mg·ATP (filled circles, n = 6) and with 3 mM Mg·ATP (open circles, n = 6). Note the different Y-axis scaling. (B) Representative high-resolution current record obtained in response to a 50-ms voltage ramp from −100 to 100 mV from a cell dialyzed with 0 Mg·ATP or 3 mM Mg·ATP, respectively, showing the characteristic signature of MagNuM (strong outward rectification at potentials above 40 mV and suppression by increased Mg·ATP concentrations). Note that data are not leak-corrected which reveals a reversal potential of −33.2 mV ± 2.4 mV (n = 9) under conditions where intracellular calcium is weakly buffered. (C) Average intracellular Ca²⁺ signals recorded from cells patched in A showing a steady rise in [Ca²⁺]i in the absence of Mg·ATP. In contrast, [Ca²⁺]i remains at steady basal levels when TRPM7 is blocked by 3 mM Mg·ATP or in control uninduced HEK-293 cells not overexpressing the channel (dotted line, n = 5).

Figure 2.

Equimolar substitution of 10 mM Ca²⁺ by transition metals. Whole-cell currents were recorded in HEK-293 cells overexpressing TRPM7 kept in a bath containing 10 mM Ca²⁺, without Mg²⁺, and exposed for 60 s to an otherwise identical external solution where 10 mM Ca²⁺ was equimolarly replaced by the test cation. Average inward and outward currents at −80 and 80 mV were scaled so that the inward and outward current amplitudes immediately preceding the solution change were set to 1. (A) Exposure to 10 mM Zn²⁺ (n = 5) and 10 mM Ni²⁺ (n = 9) caused a large increase of the inward current, with a block of the outward current. (B) Left, 10 mM Co²⁺ (n = 3), 10 mM Mg²⁺ (n = 5), and 10 mM Mn²⁺ (n = 5) caused a slight to moderate increase of the inward current, with a block of the outward current. Right, 10 mM Ba²⁺ (n = 6), 10 mM Sr²⁺ (n = 3), and 10 mM Cd²⁺ (n = 3) caused a slight to moderate increase of the inward current, and increase of the outward current. Note that the first 150 s of whole-cell time are not shown. (C) Representative high-resolution current record obtained in response to a 50-ms voltage ramp from −100 to 100 mV from a cell before (200 s whole-cell time, dotted control) and during (250 s whole-cell time, thick continuous line) application of 10 mM Ni²⁺ showing the right shift in reversal potential during application. Note that data are not leak-corrected, which reveals an average control reversal potential of −7.2 mV ± 2.7 mV (n = 39) under conditions where intracellular calcium is buffered to zero. The black open box indicates the area shown enlarged in the inset to the left. (D) The bottom panel shows the rank order of permeation through TRPM7 based on percentage increase (±SEM) of the inward current when carrying the test cation relative to the current magnitude at 10 mM Ca²⁺. The top panel plots effects on the outward current as percent increase or inhibition (±SEM) for each divalent cation.

Figure 3.

Permeation of transition metals in isotonic solutions. Whole-cell currents were recorded in HEK-293 cells overexpressing TRPM7 in standard external solution containing 1 mM Ca²⁺ and 2 mM Mg²⁺, and subsequently exposed to an isotonic solution of the test cation for 60 s. Average inward and outward currents at −80 and 80 mV were scaled so that the inward and outward current amplitudes immediately preceding the solution change were set to 1. (A–D) Exposure to isotonic Ca²⁺ (n = 4), Mg²⁺ (n = 5), Co²⁺ (n = 3), Mn²⁺ (n = 5), Ba²⁺ (n = 3), Sr²⁺ (n = 7), and Ni²⁺ (n = 4) elicits characteristic responses for each cation. Data for Ca²⁺ and Mg²⁺ reprinted in normalized form by permission from Nature [Nadler et al., 2001], copyright 2001 Macmillan Publishers Ltd. (E) Representative high-resolution current records obtained in response to a 50-ms voltage ramp from −100 to 100 mV from a cell before (control at 200-s whole-cell time) and during (230-s whole-cell time) exposure to isotonic Ni²⁺ illustrating the right shift in the reversal potential. Note that data are not leak-corrected which reveals an average control reversal potential of −7.2 ± 2.7 mV (n = 39) under conditions where intracellular calcium is buffered to zero. The black open box indicates the area shown enlarged in the inset to the left.

Figure 4.

(A) The rank order of permeation through TRPM7 based on percentage increase (±SEM) of the peak inward current. Note that increases are relative to 1 mM Ca²⁺. (B) Corresponding effects on the outward current as percent inhibition (±SEM). (C) Relative shift of reversal potential (E_rev) to control E_rev (see below) induced by application of the respective isotonic divalent solutions. Data are sorted according to the rank order of inward current (A). The average control reversal potential before application was −7.5 mV ± 5 mV (n = 31) as indicated in Fig. 3 E. (D) Mn²⁺-induced quench of fura-2 fluorescence at 360 nm excitation in HEK-293 cells induced to overexpress TRPM7 (n = 3) and transfected cells that remained uninduced (n = 3). Cells were kept in an extracellular solution supplemented with 3 mM Ca²⁺ and 0 Mg²⁺. The application contained 1 mM Mn²⁺, 1 mM Ca²⁺, and 1 mM Mg²⁺ as divalent ions. (E) Ni²⁺-induced quench of fura-2 fluorescence at 360 nm excitation in HEK-293 cells induced to overexpress TRPM7 (n = 4) and control cells that remained uninduced (n = 3). Cells were kept in an extracellular solution as described in Fig. 4 D. The application contained standard solution with 1 mM Ni²⁺, 1 mM Ca²⁺, and 1 mM Mg²⁺ as divalent ions. (F) Co²⁺-induced quench of fura-2 fluorescence at 360 nm excitation in HEK-293 overexpressing TRPM7 (n = 3) and in uninduced control cells (n = 3). Cells were kept in an extracellular solution as described in Fig. 4 D. The application contained standard solution with 1 mM Co²⁺, 1 mM Ca²⁺, and 1 mM Mg²⁺ as divalent ions.

Figure 5.

Permeation of zinc. (A) Whole-cell currents were recorded in HEK-293 cells overexpressing TRPM7. Closed circles indicate cells that were kept in standard external solution containing 10 mM Ca²⁺ and 0 mM Mg²⁺, and subsequently exposed to a choline-Cl₂ based solution containing 10 mM Zn²⁺ (n = 8). Open circles show cells that were kept in a solution that contained 160 mM choline-Cl, 10 mM TRIS, and 1 mM Ca²⁺ (n = 5). During the time indicated by the black bar, cells were superfused with an otherwise identical solution, except that 1 mM Ca²⁺ was replaced with 1 mM Zn²⁺. Average inward and outward currents at −80 and 80 mV were normalized so that the inward and outward current amplitudes immediately preceding the solution change were set to 1. (B) High-resolution current records from a representative cell showing current-voltage relationships just before (control) and immediately after application of 10 mM Zn²⁺ solution. (C) Whole-cell currents were recorded in WT HEK-293 cells with experimental protocol as in (Fig. 2). Two datasets were generated (a) MagNuM was activated in the absence of Mg·ATP (n = 12) and (b) MagNuM was suppressed by 4 mM Mg²⁺ (n = 5) Datasets were averaged and subtracted to compensate for Zn²⁺ inhibition of background currents (see text). Note the different Y-axis scaling. (D) Average inward and outward currents at −80 and 80 mV, respectively, recorded in HEK-293 cells overexpressing TRPM7 in standard external solution containing 10 mM Ca²⁺ without Mg²⁺. 10 mM Gd31 (n = 3) or La31 (n = 6) were applied as indicated. Note the different Y-axis scaling.