Sacher A, Cohen A, Nelson N
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
J Exp Biol. 2001 Mar;204(Pt 6):1053-61. doi: 10.1242/jeb.204.6.1053.
Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life. Metal-ion transporters play a major role in maintaining the correct concentrations of the various metal ions in living cells. Little is known about the transport mechanism of metal ions by eukaryotic cells. Some insight has been gained from studies of the mammalian transporter DCT1 and the yeast transporter Smf1p by following the uptake of various metal ions and from electrophysiological experiments using Xenopus laevis oocytes injected with RNA copies (c-RNA) of the genes for these transporters. Both transporters catalyze the proton-dependent uptake of divalent cations accompanied by a 'slippage' phenomenon of different monovalent cations unique to each transporter. Here, we further characterize the transport activity of DCT1 and Smf1p, their substrate specificity and their transport properties. We observed that Zn(2+) is not transported through the membrane of Xenopus laevis oocytes by either transporter, even though it inhibits the transport of the other metal ions and enables protons to 'slip' through the DCT1 transporter. A special construct (Smf1p-s) was made to enhance Smf1p activity in oocytes to enable electrophysiological studies of Smf1p-s-expressing cells. 54Mn(2+) uptake by Smf1p-s was measured at various holding potentials. In the absence of Na(+) and at pH 5.5, metal-ion uptake was not affected by changes in negative holding potentials. Elevating the pH of the medium to 6.5 caused metal-ion uptake to be influenced by the holding potential: ion uptake increased when the potential was lowered. Na(+) inhibited metal-ion uptake in accordance with the elevation of the holding potential. A novel clutch mechanism of ion slippage that operates via continuously variable stoichiometry between the driving-force pathway (H(+)) and the transport pathway (divalent metal ions) is proposed. The possible physiological advantages of proton slippage through DCT1 and of Na(+) slippage through Smf1p are discussed.
过渡金属对许多代谢过程至关重要，其体内平衡对生命至关重要。金属离子转运蛋白在维持活细胞中各种金属离子的正确浓度方面发挥着主要作用。关于真核细胞转运金属离子的机制知之甚少。通过追踪各种金属离子的摄取以及使用注射了这些转运蛋白基因的RNA拷贝（c-RNA）的非洲爪蟾卵母细胞进行电生理实验，对哺乳动物转运蛋白DCT1和酵母转运蛋白Smf1p的研究获得了一些见解。这两种转运蛋白都催化质子依赖性二价阳离子的摄取，并伴有每种转运蛋白特有的不同单价阳离子的“滑移”现象。在这里，我们进一步表征了DCT1和Smf1p的转运活性、它们的底物特异性和转运特性。我们观察到，即使锌离子抑制其他金属离子的转运并使质子能够“滑移”通过DCT1转运蛋白，但两种转运蛋白都不能将锌离子转运穿过非洲爪蟾卵母细胞的膜。制备了一种特殊构建体（Smf1p-s）以增强卵母细胞中Smf1p的活性，从而能够对表达Smf1p-s的细胞进行电生理研究。在各种保持电位下测量了Smf1p-s对54Mn(2+)的摄取。在没有钠离子且pH为5.5的情况下，金属离子摄取不受负保持电位变化的影响。将培养基的pH提高到6.5会导致金属离子摄取受到保持电位的影响：当电位降低时离子摄取增加。钠离子根据保持电位的升高抑制金属离子摄取。提出了一种新的离子滑移离合器机制，该机制通过驱动力途径（氢离子）和转运途径（二价金属离子）之间连续可变的化学计量比起作用。讨论了质子通过DCT1滑移和钠离子通过Smf1p滑移可能的生理优势。
