Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis.
Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis.
复制标题
体外铁螯合对多巴胺转运蛋白的下调是通过改变运输而不是合成来介导的。
DOI：
10.1111/j.1471-4159.2006.04175.x
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发表时间：
2007
影响因子：
4.7
通讯作者：
Beard,JohnL
中科院分区：
文献类型：
--
作者：
Wiesinger,JasonA;Buwen,JamesP;Cifelli,ChristopherJ;Unger,EricaL;Jones,ByronC;Beard,JohnL
生命早期缺铁会对神经发育和多巴胺 (DA) 神经传递功能产生不利影响。缺铁大鼠的尾壳核和伏隔核中细胞外 DA 显着升高，多巴胺转运蛋白 (DAT) 密度降低。为了探索细胞铁浓度控制 DAT 功能的可能机制，使用表达内源性 DAT 的 PC12 细胞来确定铁螯合对 DAT 蛋白和 mRNA 表达模式的影响。此外，我们使用人 DAT (hDAT) 转染 Neuro2a (N2A) 细胞来检查 DAT 降解和运输模式。在 PC12 和 N2A 细胞中，用铁螯合剂去铁敏 (DFO) 以 50 µm 浓度处理 24 小时，以剂量依赖性方式显着降低多巴胺摄取，而 Km 没有明显变化。两种细胞系中 DA 摄取的减少都伴随着浓度和时间依赖性的总 DAT 蛋白水平的降低。暴露于浓度增加的 DFO 并没有显着改变 PC12 或 N2A 细胞中的 DAT mRNA。然而，暴露于 50 µmDFO 24 小时的两种细胞类型中，DAT 降解率增加了三到五倍。 N2A 细胞中的生物素化研究表明膜组分中 DAT 的损失更为显着，而 OptiPrep 分级分离实验表明铁螯合导致溶酶体 DAT 增加。用星形孢菌素抑制蛋白激酶 C 活化可防止铁螯合对 DAT 功能的影响，表明体外铁螯合主要通过影响运输而非合成来影响 DAT。
Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron‐deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT‐expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)‐transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 µmtreatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose‐dependent manner, with no apparent change inKm, in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration‐ and time‐dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three–fivefold in both cell types exposed to 50 µmDFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting thatin vitroiron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.