Midde Narasimha M, Yuan Yaxia, Quizon Pamela M, Sun Wei-Lun, Huang Xiaoqin, Zhan Chang-Guo, Zhu Jun
Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, 715 Sumter Street, Columbia, SC, 29208, USA.
J Neuroimmune Pharmacol. 2015 Mar;10(1):122-35. doi: 10.1007/s11481-015-9583-3. Epub 2015 Jan 22.
HIV-1 transactivator of transcription (Tat) protein disrupts the dopamine (DA) neurotransmission by inhibiting DA transporter (DAT) function, leading to increased neurocognitive impairment in HIV-1 infected individuals. Through integrated computational modeling and pharmacological studies, we have demonstrated that mutation of tyrosine470 (Y470H) of human DAT (hDAT) attenuates Tat-induced inhibition of DA uptake by changing the transporter conformational transitions. The present study examined the functional influences of other substitutions at tyrosine470 (Y470F and Y470A) and tyrosine88 (Y88F) and lysine92 (K92M), two other relevant residues for Tat binding to hDAT, in Tat-induced inhibitory effects on DA transport. Y88F, K92M and Y470A attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of these residues for Tat binding to hDAT. Compared to wild type hDAT, Y470A and K92M but not Y88F reduced the maximal velocity of [(3)H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [(3)H]DA uptake and [(3)H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [(3)H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [(3)H]WIN35,428 binding. Moreover, only Y470A and K92M enhanced DA efflux relative to wild type hDAT, suggesting mutations of these residues differentially modulate transporter conformational transitions. These results demonstrate Tyr88 and Lys92 along with Tyr470 as functional recognition residues in hDAT for Tat-induced inhibition of DA transport and provide mechanistic insights into identifying target residues on the DAT for Tat binding.
人类免疫缺陷病毒1型转录反式激活因子（Tat）蛋白通过抑制多巴胺（DA）转运体（DAT）功能来破坏DA神经传递，导致HIV-1感染个体的神经认知障碍增加。通过整合计算模型和药理学研究，我们已经证明人类DAT（hDAT）的酪氨酸470（Y470H）突变通过改变转运体构象转变减弱了Tat诱导的DA摄取抑制。本研究检测了酪氨酸470处其他替代位点（Y470F和Y470A）以及酪氨酸88（Y88F）和赖氨酸92（K92M）（Tat与hDAT结合的另外两个相关残基）对Tat诱导的DA转运抑制作用的功能影响。Y88F、K92M和Y470A减弱了Tat诱导的DA转运抑制，这表明这些残基与Tat结合hDAT的功能相关性。与野生型hDAT相比，Y470A和K92M但不是Y88F降低了[³H]DA摄取的最大速度，而Km没有变化。Y88F和K92M提高了DA对[³H]DA摄取和[³H]WIN35,428结合的抑制IC50值，但降低了可卡因和GBR12909对[³H]DA摄取抑制的IC50值，表明这些残基对底物和这些抑制剂至关重要。Y470F、Y470A、Y88F和K92M减弱了锌诱导的[³H]WIN35,428结合增加。此外，相对于野生型hDAT，只有Y470A和K92M增强了DA外流，表明这些残基的突变差异调节转运体构象转变。这些结果证明了酪氨酸88和赖氨酸92以及酪氨酸470是hDAT中Tat诱导的DA转运抑制的功能识别残基，并为确定DAT上Tat结合的靶残基提供了机制性见解。
