Modulation of TRPM6 and Na⁺/Mg²⁺ exchange in mammary epithelial cells in response to variations of magnesium availability

Abstract

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.

EGF stimulates Mg2+ influx in mammary epithelial cells

“…The EGF signal transduction pathway in epithelial cells similarly requires PLCc1 activation to trigger Ca 2+ release [17], and our results show that the presence of magnesium is essential for the EGF-induced Ca 2+ signal, which suggests the involvement of the same molecular players. At present we can only speculate as to which of the magnesium channels expressed in HC11 cells [13] takes part in the described process. Available information does not rule out a putative role for any of them.…”

“…In selected experiments, cells were pretreated with 2 lM Tyrphostin AG1478 (Sigma-Aldrich, Milan, Italy) for 90 min. Cell loading was performed as previously described [13].…”

“…We used an in vitro cellular model that has been extensively characterized in our laboratory. We already described HC11 mammary epithelial cells in terms of proliferative behavior, magnesium content and magnesium channel expression in conditions of different magnesium availability [3,[11][12][13]. HC11 cells express the magnesium channels TRPM6, TRPM7 and MagT1, which are responsible for Mg 2+ influx from the extracellular compartment, and the Na + / Mg 2+ exchanger, most likely SLC41A1, which mediates Mg 2+ efflux from the cell [13].…”

A model of secreting murine mammary epithelial HC11 cells comprising endogenous Bcrp/Abcg2 expression and function

“…Furthermore, HC11 cells have been applied to examine the impact of toxic compounds on lactating mammary cells (Öhrvik et al 2010) and also to characterize some transporters belonging to the Solute Carrier (SLC) family and ion channels, which are implicated in the flux of magnesium, zinc, copper, and calcium across the membranes of the lactating mammary epithelium Lönnerdal 2005, 2006;Boyd and Náray-Fejes-Tóth 2007;Öhrvik et al 2010;Wolf et al 2010;McCormick and Kelleher 2012;Ross et al 2013). However, the HC11 cell model has not yet been used to characterize expression and/or function of ATP Binding Cassette (ABC) transporters.…”

The role of Mg2+ in immune cells

“…influx and efflux mechanisms in multiple cellular systems under varying Mg 2? availability [32][33][34].…”