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

用荧光指示剂furaptra测定细胞内游离Mg^<2 +>浓度（[Mg^<2+>]_i），研究Mg^<2+>从细胞中排出的机制。我们建立了一种小鼠肾皮质小管（MCT）细胞的突变株，该突变株可以在极高Mg^2+浓度的培养基中生长（[Mg^2+] o >100 mM：Mg耐受细胞）。耐镁细胞的[Mg^<2+>] i水平低于野生型细胞。当[Mg^<2+>]_o从51 mM降低到1 mM时，耐镁细胞中细胞外[Mg^<2+>]_i的Na依赖性降低明显加快，表明耐镁细胞中Na^+-Mg^<2 +>交换体对Mg^<2+>的挤出增强。DNA微阵列分析表明，耐镁细胞中许多基因的表达增强。在克隆Mg ^2+转运蛋白的同时，我们还检测了心肌细胞Na^+-Mg^2+交换的功能特征。大鼠心室肌细胞在Mg^<2+>负荷后，加入细胞外Na^+诱导Mg^<2+>外流，用furaptra测定[Mg^<2+>] i的下降率，并进行分析。[Mg^2+] i的小幅升高可显著诱导Mg^2+的外排，在1.9 mM时达到最大激活的一半。另一方面，细胞外Mg^2+可抑制转运，在10 mM时抑制率为50%。细胞外Na^+可加速Mg^2+的外排转运，在55 mM时达到最大激活的一半。在40 mM浓度下，细胞内Na^+可使Mg ^2+流出速率减慢50%。细胞内外Ca^2+浓度和细胞外K^+浓度的变化均不显著影响Mg^2+流出速率。这些结果有力地支持了Na^+-Mg^<2+>交换假说，并有助于详细描述迁移过程。

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.