Mutation of Tyrosine 470 of Human Dopamine Transporter is Critical for HIV-1 Tat-Induced Inhibition of Dopamine Transport and Transporter Conformational Transitions
Mutation of Tyrosine 470 of Human Dopamine Transporter is Critical for HIV-1 Tat-Induced Inhibition of Dopamine Transport and Transporter Conformational Transitions
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DOI：
10.1007/s11481-013-9464-6
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发表时间：
2013-09-01
影响因子：
6.2
通讯作者：
Zhu, Jun
中科院分区：
文献类型：
--
作者：
Midde, Narasimha M.;Huang, Xiaoqin;Zhu, Jun
HIV-1达特蛋白在多巴胺（DA）系统的扰动中起着至关重要的作用。我们以前的研究表明，达特降低DA摄取，并变构调节DA转运蛋白（DAT）的功能。在本研究中，我们发现达特直接与DAT相互作用，导致DAT功能的抑制。通过计算机模拟，预测了达特与人DAT（hDAT）的潜在识别结合位点。酪氨酸470（Y 470 H）的突变减弱了Tat诱导的DA转运抑制，暗示了该残基与达特结合hDAT的功能相关性。Y 470 H降低了[H-3]DA摄取的最大速度，而DA抑制DA摄取的Km和IC 50值没有变化，但增加了可卡因和GBR 12909的DA摄取效力，表明该残基与hDAT中底物的结合位点不重叠，但对这些抑制剂至关重要。此外，Y 470 H还通过影响锌对DA摄取和WIN 35，428结合的调节以及增强基础DA流出而导致转运蛋白构象转变。总的来说，这些研究结果表明Tyr 470作为一个功能性识别残基在hDAT的Tat诱导的抑制DA转运和转运蛋白构象转换。该残基突变的结果是阻断达特与hDAT的功能性结合而不影响生理性DA转运。
HIV-1 Tat protein plays a crucial role in perturbations of the dopamine (DA) system. Our previous studies have demonstrated that Tat decreases DA uptake, and allosterically modulates DA transporter (DAT) function. In the present study, we have found that Tat interacts directly with DAT, leading to inhibition of DAT function. Through computational modeling and simulations, a potential recognition binding site of human DAT (hDAT) for Tat was predicted. Mutation of tyrosine470 (Y470H) attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of this residue for Tat binding to hDAT. Y470H reduced the maximal velocity of [H-3]DA uptake without changes in the K-m and IC50 values for DA inhibition of DA uptake but increased DA uptake potency for cocaine and GBR12909, suggesting that this residue does not overlap with the binding sites in hDAT for substrate but is critical for these inhibitors. Furthermore, Y470H also led to transporter conformational transitions by affecting zinc modulation of DA uptake and WIN35,428 binding as well as enhancing basal DA efflux. Collectively, these findings demonstrate Tyr470 as a functional recognition residue in hDAT for Tat-induced inhibition of DA transport and transporter conformational transitions. The consequence of mutation at this residue is to block the functional binding of Tat to hDAT without affecting physiological DA transport.