Overexpression of Na⁺/Mg²⁺ exchanger SLC41A1 attenuates pro-survival signaling

The Na⁺/Mg²⁺ exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg²⁺ efflux system, and its overexpression decreases [Mg²⁺]_{intracellular}. IMH plays an important role in the regulation of many cellular processes, including cellular signaling. However, whether the overexpression of A1 and the consequent drop of [Mg²⁺]_i impact on intracellular signaling is unknown. To examine the latter, we utilized dynamic mass redistribution (DMR) assay, PathScan^® RTK signaling antibody (PRSA) array, confirmatory Western blot (WB) analyses of phosphorylation of kinases selected by PRSA, and mag-fura 2-assisted fast filter spectrometry (FFS). We demonstrate here that the overexpression of A1 quantitatively and qualitatively changes the DMR signal evoked by the application of PAR-1-selective activating peptide and/or by changing [Mg²⁺]_{extracellular} in HEK293 cells. PRSA profiling of the phosphorylation of important signaling nodes followed by confirmatory WB has revealed that, in HEK293 cells, A1 overexpression significantly attenuates the phosphorylation of Akt/PKB on Thr³⁰⁸ and/or Ser⁴⁷³ and of Erk1/2 on Thr²⁰²/Tyr²⁰⁴ in the presence of 0 or 1 mM (physiological) Mg²⁺ in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg²⁺]_i in the presence of [Mg²⁺]_e = 0 or 1 mM. This correlates with the observed attenuation of prosurvival Akt/PKB - Erk1/2 signaling in these cells. Thus, A1 expression status and [Mg²⁺]_e (and consequently also [Mg²⁺]_i) modulate the complex physiological fingerprint of the cell and influence the activity of kinases involved in anti-apoptotic and, hence, pro-survival events in cells.