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It is well known that magnesium deficiency or altered IMH can trigger many pathophysiological conditions, thus a correct functioning of Mg2+ transporters and channels is essential for normal cellular physiology. Mutations in many MRG induce hypomagnesemia which often represents one of the complications of many human ailments.
Based on previous data which have characterized SLC41A1 as a plasma membrane Na+/Mg2+ exchanger (4, 5) and as being overexpressed in preeclamptic women (6), the present thesis aimed at a further characterization of SLC41A1 and Mg2+ efflux in some pathophysiological conditions. It further aimed at the molecular characterization of two others MRGs, CNNM2 and SLC41A3, in order to achieve a better understanding of Mg2+ homeostasis and to link the mechanisms of Mg2+ mobilization across the plasma membrane or between intracellular compartments with mitochondrial dysfunction and disease states (e.g.: Parkinson’s disease (PD), diabetes, etc).
Proceeding from previous results of Kolisek et al (4, 5), describing SLC41A1 as the major Mg2+ efflux system of the cell, the first study examined the complex-forming ability of SLC41A1 in vivo and identified EBP and other members of the SLC superfamily as potential binding partners. Further experiments evaluated the transport activity of the PD-associated SLC41A1 variant pA350V and defined it as a “gain-of-function” mutation enhancing Mg2+ efflux compared with the wild-type protein. A next question was whether SLC41A1 transport activity could be influenced and regulated by insulin in order to explain the molecular basis of hypomagnesaemia often observed in diabetes patients. The present study shows that insulin reduces the SLC41A1-mediated Mg2+ efflux and, most importantly, it seems to have an effect on intracellular Mg2+ stores, since an earlier onset of Mg2+ release from intracellular stores was observed.
In the second part of this thesis, experiments were conducted on stably transfected HEK293 cells overexpressing SLC41A3 in order to uncover the function of SLC41A3 with regard to its ability to transport Mg2+, its mode of Mg2+ transport and its role in cellular Mg2+ homeostasis. To assess the role of SLC41A3 for cellular Mg2+ homeostasis and gain insight into the regulation of transport activity and/or membrane insertion, knowledge about the precise cellular localization and the identification of the binding partners are essential. The present data reveal a specific mitochondrial localization for SLC41A3 and its function as mitochondrial Mg2+ efflux system. They further suggest that the effect of insulin on intracellular Mg2+ stores is most likely mediated via SLC41A3. Given that mitochondria serve as intracellular Mg2+ stores, a mitochondrial dysfunction might affect cellular Mg2+ homeostasis and this could be one reason for intracellular Mg2+ deficiency observed in diseases such as diabetes, PD and hypertension.
The last part of the project focused on the physiological characterization of two isoforms of another Mg2+ responsive gene, CNNM2, because a mutation in this gene has been recently associated with severe familial hypomagnesaemia.
A previous study showed that CNNM2 is overexpressed in diabetic patients (114) but its expression does not correlate with Mg2+ plasma levels. However, the present study shows an overexpression of CNNM2 in Jurkat and JVM-13 cells after Mg2+ starvation. The protein has an extensive localization in the cells, including the mitochondrial membrane, and its putative interactors include proteins involved in the regulation of mitochondrial homeostasis (mitophagy, clearance of ROS). A further question was whether the two isoforms (I1 and I2) of CNNM2 are able to transport Mg2+, but the data presented herein clearly indicate that CNNM2 transports Mg2+ neither in electrogenic nor in electroneutral mode in transgenic HEK293 cells overexpressing I1 or I2. This strongly suggests that CNNM2 might represent the first magnesium homeostatic factor without being a Mg2+ transporter per se. Instead CNNM2 can be postulated to sense the changes in extracellular and/or intracellular Mg2+ concentration and consequently activates other proteins responsible for Mg2+ mobilization in the cell.
From these data a role of CNNM2 in intracellular Mg2+ homeostasis can be assessed and it can be speculated that the two SLC41 proteins act cooperatively with CNNM2-mediated Mg2+ sensing in controlling the cellular magnesium homeostasis.