Identification of magnesium transporter molecules and functional analysis with high temporal/spatial resolution.

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Mechanisms of Mg^<2+> extrusion from the cells were studied by measuring intracellular free concentrations of Mg^<2+> ([Mg^<2+>]_i) with the fluorescent indicator furaptra. We established a mutant strain of mouse renal cortical tubular (MCT) cells that could grow in culture media with very high Mg^<2+> concentrations ([Mg^<2+>]_o >100 mM : Mg-tolerant cells). The [Mg^<2+>]_i levels of the Mg-tolerant cells were kept lower than those in the wild-type cells. When [Mg^<2+>]_o was lowered from 51 mM to 1 mM, the extracellular Na-dependent decrease in [Mg^<2+>]_i was significantly faster in the Mg-tolerant cells, suggesting that Mg^<2+> extrusion by the Na^+-Mg^<2+> exchanger was enhanced in the Mg-tolerant cells. The DNA microarray analysis showed that expression of many genes were enhanced in the Mg-tolerant cells. Screening of these candidate genes for the Mg^<2+> transporter is now on-going.In parallel with molecular cloning of the Mg^<2+> transporter, we also examined functional characteristics of the Na^+-Mg^<2+> exchange in cardiac myocytes. After Mg^<2+> loading of rat ventricular myocytes, Mg^<2+> efflux was induced by addition of extracellular Na^+, and the rates of decrease in [Mg^<2+>]_i were measured with furaptra, and were analyzed. A small elevation of [Mg^<2+>]_i significantly induced Mg^<2+> efflux with half maximal activation at 1.9 mM. On the other hand, extracellular Mg^<2+> inhibited the transport with 50% inhibition at 10 mM. The Mg^<2+> extrusion transport was accelerated by extracellular Na^+ with half maximal activation at 55 mM, and was slowed by intracellular Na^+ with 50% inhibition at 40 mM. Neither changes in intracellular and extracellular concentrations of Ca^<2+> nor extracellular K^+ concentration did not significantly influence the Mg^<2+> efflux rate. These results strongly support the Na^+-Mg^<2+> exchange hypothesis, and help detailed characterization of the transport.