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TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity.

ABSTRACT: TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg²⁺ and divalent metal cations such as Ca²⁺, Zn²⁺, Cd²⁺, Mn²⁺, and Ni²⁺. TRPM7 channels are inhibited by cytosolic Mg²⁺, rendering them largely inactive in intact cells. The dependence of channel activity on extracellular Mg²⁺ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg²⁺ concentrations varying between 400 nM and 1.4 mM for 1-3 days, obtaining an IC₅₀ value of 54 μM. Maintaining the cells in 400 nM or 8 μM [Mg²⁺]_o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg²⁺ depletion. A total of 1.4 mM [Mg²⁺]_o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg²⁺ depletion but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni²⁺, Zn²⁺, Cd²⁺, Co²⁺, Mn²⁺, Sr²⁺, and Ba²⁺ requires TRPM7 channel activity. Mg²⁺ loading modestly reduced cytotoxicity of Zn²⁺, Co²⁺, Ni²⁺, and Mn²⁺ but not of Cd²⁺. Channel blocker NS8593 reduced Co²⁺ and Mn²⁺ but not Cd²⁺ or Zn²⁺ cytotoxicity and interfered with Mg²⁺ loading as evaluated by TRPM7 channel basal activity. Ba²⁺ and Sr²⁺ were neither detectably toxic nor permeant through the plasma membrane. These results indicate that in Jurkat T cells, entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg²⁺ entry requires TRPM7 channels.

SUBMITTER: Mellott A

PROVIDER: S-EPMC7561021 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:TRPM7 is a Ca(2+)- and Mg(2+)-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg(2+), the functional roles of intracellular levels of Mg.ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg.ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg(2+). Mg.ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg(2+) and Ca(2+) overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP > or = GTP > or = UTP > ITP approximately free Mg(2+) alone. These nucleotides also enhanced TRPM7 inhibition by free Mg(2+), suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg(2+)-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg.NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg(2+) binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg(2+) binding site extrinsic to that domain.

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TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity.

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