Modulation of TRPM6 and Na(+)/Mg(2+) exchange in mammary epithelial cells in response to variations of magnesium availability.

Mammary epithelial cells (HC11) chronically adapted to grow in a low-magnesium (0.05 mM vs. 0.5 mM) or in a high-magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non-physiological magnesium availability. Magnesium influx was higher in low-magnesium cells compared to control or high-magnesium cells, whereas magnesium efflux was higher in high-magnesium cells compared to control and low-magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na(+)/Mg(2+) exchange. Using a monoclonal antibody detecting a approximately 70 kDa protein associated with Na(+)/Mg(2+) exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high-magnesium cells > control cells > low-magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low-magnesium cells re-added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na(+)/Mg(2+) exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney.