Generation of an activating Zn2+ switch in the dopamine transporter:: Mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle
Generation of an activating Zn2+ switch in the dopamine transporter:: Mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle
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DOI：
10.1073/pnas.032386299
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发表时间：
2002-02-05
影响因子：
11.1
通讯作者：
Gether, U
中科院分区：
文献类型：
--
作者：
Loland, CJ;Norregaard, L;Gether, U
Zn 2+与人多巴胺转运蛋白中的内源性Zn 2+结合位点的结合导致[H-3]多巴胺摄取的有效抑制。在这里，我们表明，突变的细胞内酪氨酸丙氨酸（Y335 A）转换成一个激活的Zn 2+开关，允许Zn 2+依赖激活的转运蛋白的抑制性Zn 2+开关。酪氨酸是保守的YXXphi运输基序的一部分（X是任何残基，phi是具有庞大疏水基团的残基），但Y335 A在表面靶向或蛋白激酶C介导的内化中没有显示出改变。尽管具有野生型表面表达水平，但Y335 A的[（3）H]多巴胺摄取速度（V-max）显着降低至野生型的1%以下。此外，Y335 A表现出高达150倍的可卡因，马吲哚和相关抑制剂的表观亲和力下降，而几个底物的表观亲和力增加。然而，在微摩尔浓度的Zn 2+的存在下，V-最大值增加了24倍，并部分恢复了表观亲和力。Zn 2+恢复转运的能力与Tyr-335突变后转运循环中构象状态分布的可逆组成性转变一致。我们提出，这种转变是由破坏分子内相互作用的重要稳定的转运蛋白的构象中，细胞外底物可以结合和启动运输，因此，酪氨酸-335是至关重要的调节异构化之间的离散状态的运输周期。
Binding of Zn2+ to the endogenous Zn2+ binding site in the human dopamine transporter leads to potent inhibition of [H-3]dopamine uptake. Here we show that mutation of an intracellular tyrosine to alanine (Y335A) converts this inhibitory Zn2+ switch into an activating Zn2+ switch, allowing Zn2+-dependent activation of the transporter. The tyrosine is part of a conserved YXXphi trafficking motif (X is any residue and phi is a residue with a bulky hydrophobic group), but Y335A did not show alterations in surface targeting or protein kinase C-mediated internalization. Despite wild-type levels of surface expression, Y335A displayed a dramatic decrease in [(3) H]dopamine uptake velocity (V-max ) to less than 1% of the wild type. In addition, Y335A showed up to 150-fold decreases in the apparent affinity for cocaine, mazindol, and related inhibitors whereas the apparent affinity for several substrates was increased. However, the presence of Zn2+ in micromolar concentrations increased the V-max up to 24-fold and partially restored the apparent affinities. The capability of Zn2+ to restore transport is consistent with a reversible, constitutive shift in the distribution of conformational states in the transport cycle upon mutation of Tyr-335. We propose that this shift is caused by disruption of intramolecular interactions important for stabilizing the transporter in a conformation in which extracellular substrate can bind and initiate transport, and accordingly that Tyr-335 is critical for regulating isomerization between discrete states in the transport cycle.