Detailed examination of Mg2+ and pH sensitivity of human TRPM7 channels

Abstract

TRPM7 channel kinase is a protein highly expressed in cells of hematopoietic lineage, such as lymphocytes. Studies performed in native and heterologous expression systems have shown that TRPM7 forms nonselective cation channels functional in the plasma membrane and activated on depletion of cellular Mg(2+). In addition to internal Mg(2+), cytosolic pH and the phospholipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] are potent physiological regulators of this channel: protons inhibit, while PI(4,5)P(2) is required for TRPM7 channel activity. These channels are also inhibited from inside by other metal cations and polyamines. While the regulation of TRPM7 channels by internal metal ions, acidic pH, and PI(4,5)P(2) is voltage independent, extracellular metal cations and polyamines block voltage dependently at micromolar concentrations and appear to occupy a distinct blocking site. In the present study we investigated intracellular Mg(2+) and pH dependence of native TRPM7 currents using whole cell patch-clamp electrophysiology in human Jurkat T lymphocytes and HEK293 cells. Our main findings are 1) Mg(2+) inhibition involves not one but two separate sites of high (∼10 μM) and low (∼165 μM) affinity; and 2) while sharing certain characteristics with Mg(2+) inhibition, protons most likely inhibit through one inhibitory site, corresponding to the low-affinity Mg(2+) site, with an estimated IC(50) of pH 6.3. Additionally, we present data on amplitude distribution of preactivated TRPM7 currents in Jurkat T lymphocytes in the absence of prior Mg(2+) or proton depletion.

Fig. 1.

RT-PCR with with Jurkat and HEK293 cell total RNA. RNA isolated from Jurkat T lymphocytes grown in suspension (left) and HEK293 cells grown in a monolayer (right) were used for RT-PCR reactions with melastatin-like transient receptor potential (TRPM) 6-, TRPM7-, and GAPDH-specific primer sets (40 cycles). Predicted sizes of the respective products are 284, 734, and 555 bp (NCBI Primer-BLAST). Amplified fragments were separated by electrophoresis in a 0.8% agarose gel with added ethidium bromide. Left lane: DNA molecular weight (MW) marker (Hi-Lo DNA marker, Minnesota Molecular). All 6 reactions were electrophoresed on the same gel; the vertical line is added for visual aid. The contrast of MW lane was enhanced separately to improve resolution.

Fig. 2.

Mg²⁺ dose-response relation for TRPM7 channels of Jurkat T lymphocytes. A: current-voltage (I-V) relations for TRPM7 channels from 3 cells containing 106 nM, 16.4 μM, and 303 μM free Mg²⁺. I-V relations were taken at the time point when current magnitude reached maximum for each cell. Current amplitudes were obtained from the 120th ramp. B: time course of development of TRPM7 current from the same cells as in panel A, measured at +85 mV. Note that 303 μM Mg²⁺ inhibits preactivated TRPM7 current (triangles) whereas 16.4 μM Mg²⁺ does not. C: maximum current density measurements from Jurkat T cells perfused with various Mg²⁺-containing solutions indicated on the x-axis. Number of cells for each concentration is shown at top of bar. Here and in Figs. 3 and 4, significant differences between pairs of mean currents are indicated by * (Tukey method) and ** (contrast analysis). Not all pairwise comparisons are shown in the graph. D: dose-response relation for internal Mg² on a logarithmic scale. The fit is a biphasic dose-response curve provided by Origin software fitting subroutine (adjusted R-square = 0.99), giving IC₅₀ values of 10.2 μM (SE = 0.55) and 165.0 μM (SE = 3.38) with Hill coefficients of 1.92 and 7.62, respectively. Here and in the other figures, data points represent means and SE.

Fig. 3.

pH dose-response relation for TRPM7 channels in HEK293 cells obtained with EGTA-containing solutions. A: maximally activated I-V relations from 3 different HEK293 cells perfused internally with pH 8.3, 7.4, and 6.1, and 12 mM EGTA. B: time courses of TRPM7 current development in the same cells as in panel A, measured at +85 mV. Note the development and rundown of the proton-activated current at intracellular pH (pH_i) of 6.1. C: bar graphs showing maximal TRPM7 current densities for internal solutions containing 12 mM EGTA at pH values of 8.3 to 5.1. Not all pairwise comparisons are shown in the graph. D: dose-response relation resulting from data in panel B normalized to maximum current amplitude is fitted with biphasic dose-response curve (adjusted R-square = 0.99), giving IC₅₀ values of pH 7.57 (SE = 0.02) and 5.93 (SE = 0.04). Significant differences between pairs of mean currents are indicated by * (Tukey method) and ** (contrast analysis).

Fig. 4.

Dose-response relation for pH dependence of TRPM7 channels in HEK293 cells obtained with N-(2-hydroxyethyl)ethylenediaminetriacetate (HEDTA)-containing solutions. A: mean TRPM7 current densities at various pH_i values for HEDTA-containing internal solution (measured at ramp 250). Not all pairwise comparisons are shown. B: concentration-response relation derived from data in panel A plotted against proton concentration on x-axis and fitted with standard monophasic dose-response curve (adjusted R-square = 0.97) giving H⁺ concentration ([H⁺]) IC₅₀ value of 478 nM (SE = 89.8 nM) (corresponds to pH 6.32). C: superimposed concentration-response relations from Fig. 3D (black circles) and 3B (open squares) fitted with biphasic dose-response curves. Note that the first deflection seen at pH values of 8.3–7.4 is abolished and the decline at pH values of 6.7 and lower is accelerated for HEDTA-containing solutions. Significant differences between pairs of mean currents are indicated by * (Tukey method).

Fig. 5.

Amplitude histograms of preactivated TRPM7 currents and current densities in Jurkat T lymphocytes. Current amplitudes were measured at +85 mV for internal solutions containing 106, 266, and 399.5 nM free Mg²⁺. In panel B, the same measurements were normalized to cell capacitance and plotted as in panel A. The y-axis is the number of cells expressing current amplitudes/densities indicated on x-axis. No leak subtraction was performed (A, B). Histograms in panels A and B were fitted with monoexponential decay functions.