Detailed Examination of TRPM7 Channel Mg2+ Dependence

Abstract

TRPM7, a member of transient receptor potential superfamily of ion channels, underlies the Mg2+-inhibited cation currents highly expressed in various cell types of hematopoietic lineage. TRPM7 channels are sensitive to intracellular as well as extracellular Mg2+ and other polyvalent cations. The present study was undertaken to characterize the inhibition of TRPM7 channels by intracellular Mg2+ in detail. In order to generate a dose-response curve for Mg2+i, we systematically varied the internal Mg2+ concentration and measured the corresponding maximal TRPM7 current amplitude with whole-cell patch clamp in Jurkat T lymphocytes and HEK293 cells. Mg2+-containing solutions were buffered with HEDTA. We tested free Mg2+ concentrations of 100 nM to 400 μM in n ≥ 70 cells. Unexpectedly, we find that the dose-response curve for Mg2+ is biphasic, yielding IC50 values of ∼8 μM and ∼200 μM. 300 μM free Mg2+ is sufficient for complete inhibition of channel activity. This finding suggests the existence of two inhibitor sites for TRPM7 activity. Since in whole-cell configuration the channel interior is only exposed to one solution throughout the recording, it is impossible to separate Mg2+ effect on the likelihood of channel opening from its effect on an already open channel. Moreover, reversible inhibition cannot be distinguished from time-dependent rundown of activity. In order to examine the nature of Mg2+-mediated inhibition more thoroughly, we recorded TRPM7 channels in inside-out patch configuration. Membrane patches were excised into a Mg2+-free bath solution and TRPM7 single-channel activity recorded. Mg2+ was then added to the solution facing intracellular side of the channel, using rapid application. We observed reversible inhibition by Mg2+ in the 35 - 400 micromolar concentration range and have characterized changes in single-channel characteristics responsible for the inhibition. A comparison of Mg2+ and proton sensitivities is also presented.