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.

2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism

“…The pH value of 6.91 is expected to be sufficient to reduce TRPM7 channel current magnitude, since its IC 50 of proton inhibition is pH 6.3. 26 It should be noted however that measurements shown in Figure 3 describe an average value of pH over the whole cytoplasm, whereas TRPM7 channels likely sense the pH near the plasma membrane. 27 In Figure 3D application of 300 μM and 900 μM 2-APB to Jurkat T cells produced increased acidification with increased concentration of the compound.…”

“…29 These findings suggest that 2-APB inhibits TRPM7 channels by acidifying the cytosol, which would be expected to reduce TRPM7 channel activity. 26 We therefore tested if external ammonium would relieve channel inhibition by 2-APB. In Figure 1D, we applied 100 μM 2-APB after TRPM7 channels were fully activated.…”

“…15,18,24,25 Jurkat T-cell TRPM7 channels are sensitive to cytoplasmic Mg 2+ , described by a biphasic inhibition dose-response curve with IC 50 values of ~10 μM and ~165 μM in whole-cell recordings. 26 These channels are also sensitive to intracellular pH with a single IC 50 of pH 6.3. 26,27 We recently found that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit TRPM7 channels in Jurkat T 2-APB is a widely used compound in ion channel research.…”

“…26 These channels are also sensitive to intracellular pH with a single IC 50 of pH 6.3. 26,27 We recently found that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit TRPM7 channels in Jurkat T 2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them.…”

“…Note that in this cell TRPM7 current was pre-activated at break-in. 26 (D) Inhibition of TRPM7 channels by 100 μM 2-APB is relieved by ammonium. One hundred micromolars of 2-APB was applied as indicated by the horizontal bar and elicited pronounced inhibition of TRPM7 current.…”