TRPM7 Permeant Divalent Metal Cytotoxicity During Magnesium Depletion and Loading

Abstract

TRPM7 channels, highly expressed in immune cells, are believed to conduct divalent metal cations such as Mg2+, Zn2+, Cd2+ and Mn2+. Normally, TRPM7 channels are inhibited by cytosolic Mg2+, polyamines and protons, resulting in very small basal current magnitudes. In patch-clamp, these channels are traditionally activated by removal of intracellular Mg2+. Here, we have investigated the consequences of long-term reductions in extracellular [Mg2+] on TRPM7 channel activity. Jurkat T lymphocytes were maintained at external Mg2+ concentrations of 400 nM to 1.4 mM [Mg2+]o for 1-2 days. Incubating cells in 400 nM or 8 uM [Mg2+] resulted in almost complete current activation at break-in (t=0). Conversely, 1.4 mM external [Mg2+] was sufficient to eliminate basal currents. These findings suggested that long-term culture in low [Mg2+] results in cytosolic Mg2+ depletion and TRPM7 current activation. Presence or absence of external Ca2+, did not influence Mg2+ depletion or loading. Next, we investigated if entry and cytotoxicity of divalent metals is influenced by TRPM7 channel activity. We exposed the cells to 0.4 mM Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ba2+, Sr2+ for 1-2 days and quantified cell viability by trypan blue exclusion. In cells grown in 8 uM and 0.4 mM [Mg2+] the cytotoxicity sequence was: Cd2+ > Zn2+ > Co2+ > Ni2+ >Mn2+ with Ba2+ and Sr2+ virtually nontoxic. Maintaining cells in 0.4 or 1.4 mM Mg2+ had a ≤20% protective effect over 8 uM Mg2+ for the tested metal ions except Cd2+. Blockade of TRPM7 channels with NS8593 or La3+ also had a modest protective effect. In TRPM7 CRISPR knockdown HAP1 cells, metal cytotoxicity persisted and was modestly reduced for Co2+, Ni2+, Mn2+ but not Cd2+, Zn2+. Our experiments suggest that only a fraction of divalent metal cation entry depends on TRPM7 channels.