权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

镁转运蛋白分子的鉴定和高时间/空间分辨率的功能分析。

基本信息

批准号：
14370016
负责人：
KONISHI Masato
金额：
$ 5.95万
依托单位：
Tokyo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370016/
关键词：
intracellular Mg concentration Mg transport Mg-tolerant cells fluorescent Mg indicator Na-Mg exchange molecular cloning cardiac myocytes functional analysis of membrane transprot Mg輸送体細胞内Mg 交換輸送 Na 心筋 Mg耐性

项目摘要

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.

用荧光指示剂Furaptra测定细胞内游离镁离子浓度，研究了镁离子从细胞中排出的机理。我们建立了一种突变的小鼠肾皮质小管(MCT)细胞株，该突变株可以在非常高的镁浓度([镁^&lt；2+&gt；]_o&gt；100 mm：镁耐受细胞)的培养液中生长。耐镁细胞的[mg^&lt；2+&gt；]i水平低于野生型细胞。当[mg^&lt；2+&gt；]_o从51 mm降低到1 mm时，耐镁细胞[mg^&lt；2+&gt；]_i的胞外钠依赖性下降速度明显加快，表明Na+-镁离子交换器对细胞外[mg^&lt；2+&gt；]_i的排泄作用增强。DNA芯片分析表明，在耐镁细胞中，许多基因的表达都有所增强。这些候选的镁离子转运蛋白基因的筛选正在进行中。在进行镁离子转运蛋白分子克隆的同时，我们还研究了心肌细胞中钠离子交换的功能特征。大鼠心肌细胞镁离子负荷后，加入细胞外Na+诱导镁离子外流，用Furaptra法测定[mg^&lt；2+&gt；]i下降率，并进行分析。[mg^&lt；2+]_i的少量升高可显著地诱导镁离子外流，在1.9 mM处为激活的一半。胞外Mg~(2+)和Gt~(2+)在10 mM处抑制转运，抑制率为50%。胞外Na~(2+)促进镁离子的排出转运，在55 mM时激活一半，而胞内Na~(++)在40 mM时抑制50%。胞内和胞外Ca~(2+)浓度及胞外K~(++)浓度的变化均不显著影响镁离子外流速率。这些结果有力地支持了Na~(++)-Mg~(2+)&Gt；~(2+)交换假说，并有助于对转运的详细描述。