Overexpression of Na + /Mg 2+ exchanger SLC41A1 attenuates pro-survival signaling

// Gerhard Sponder 1 , Nasrin Abdulhanan 1, * , Nadine Frohlich 2 , Lucia Mastrototaro 1 , Jorg R. Aschenbach 1 , Monika Rontgen 3 , Ivana Pilchova 4 , Michal Cibulka 5 , Peter Racay 4, 5 and Martin Kolisek 1, 4, * 1 Institute of Veterinary-Physiology, Free University of Berlin, Berlin, Germany 2 PerkinElmer Life and Analytical Sciences GmbH, Rodgau, Germany 3 Leibnitz Institute for Farm Animal Biology, Department of Muscle and Growth Physiology, Dummerstorf, Germany 4 Biomedical Center Martin, Division of Neurosciences, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia 5 Institute of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia * Present/Current address: bess pro GmbH, Berlin, Germany Correspondence to: Martin Kolisek, email: kolisek@jfmed.uniba.sk Keywords: Na + /Mg 2+ exchanger; Mg 2+ homeostasis; Akt/PKB; dynamic mass redistribution; signaling Received: February 17, 2017      Accepted: November 13, 2017      Published: December 22, 2017 ABSTRACT The Na + /Mg 2+ exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg 2+ efflux system, and its overexpression decreases [Mg 2+ ] 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 [Mg 2+ ] 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 [Mg 2+ ] 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 Thr 308 and/or Ser 473 and of Erk1/2 on Thr 202 /Tyr 204 in the presence of 0 or 1 mM (physiological) Mg 2+ in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg 2+ ] i in the presence of [Mg 2+ ] 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 [Mg 2+ ] e (and consequently also [Mg 2+ ] 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.