Sensitivity of TRPM7 Channels to Mg2+ Characterized in Cell-Free Patches of Jurkat T Lymphocytes

Transient receptor potential melastatin 7 (TRPM7) channels were originally identified electrophysiologically when depletion of cytosolic Mg ²⁺ resulted in the gradual development of an outwardly rectifying cation current. Conversely, inclusion of millimolar Mg ²⁺ in internal solutions prevented activation of these channels in whole cell patch clamp. We recently demonstrated that the Jurkat T-cell whole cell TRPM7 channels are inhibited by internal Mg ²⁺ in a biphasic manner, displaying high [IC ₅₀₍₁₎ ≈ 10 μM] and low [IC ₅₀₍₂₎ ≈ 165 μM] affinity inhibitor sites. In that study, we had characterized the dependence of the maximum cell current density on intracellular Mg ²⁺ concentration. To characterize Mg ²⁺ inhibition in Jurkat T cells in more detail and compare it to whole cell results, we recorded single TRPM7 channels in cell-free membrane patches and investigated the dependence of their activity on Mg ²⁺ added on the cytoplasmic side. We systematically varied free Mg ²⁺ from 265 nM to 407 μM and evaluated the extent of channel inhibition in inside-out patch for 58 patches. We found that the TRPM7 channel shows two conductance levels of 39.0 pS (γ ₁ ) and 18.6 pS (γ ₂ ) and that both are reversibly inhibited by internal Mg ²⁺ . The 39.0-pS conductance is the dominant state of the channel, observed most frequently in this recording configuration. The dose-response relation in inside-out patches shows a steeper Mg ²⁺ dependence than in whole cell, yielding IC ₅₀₍₁₎ of 25.1 μM and IC ₅₀₍₂₎ of 91.2 μM.. Single-channel analysis shows that the primary effect of Mg ²⁺ in multichannel patches is a reversible reduction of the number of conducting channels (N _o ). Additionally, at high Mg ²⁺ concentrations, we observed a saturating 20% reduction in unitary conductance (γ ₁ ). Thus Mg ²⁺ inhibition in whole cell can be explained by a drop in individual participating channels and a modest reduction in conductance. We also found that TRPM7 channels in some patches were not sensitive to this ion at submaximal Mg ²⁺ concentrations. Interestingly, Mg ²⁺ inhibition showed the property of use dependence: with repeated applications, Mg ²⁺ effect became gradually more potent, which suggests that Mg ²⁺ sensitivity of the channel is a dynamic characteristic that depends on other membrane factors.