将多巴胺转运蛋白靶向丝状伪足需要转运蛋白的朝外构象。,Scientific Reports

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将多巴胺转运蛋白靶向丝状伪足需要转运蛋白的朝外构象。
Scientific Reports ( IF 3.8 ) Pub Date : 2017-07-14 , DOI: 10.1038/s41598-017-05637-x
Shiqi Ma ₁ , Mary H Cheng ₂ , Daryl A Guthrie ₃ , Amy H Newman ₃ , Ivet Bahar ₂ , Alexander Sorkin ₁

Affiliation

多巴胺转运蛋白（DAT）已显示在神经元和非神经元细胞的丝状伪足中积聚。为了检查DAT尾巴靶向的机制，我们使用了定量活细胞荧光显微镜，并比较了DAT抑制剂可卡因及其荧光类似物JHC1-64对野生型DAT和两种非功能性DAT的质膜分布的影响丝状伪足中不积累的突变体R60A和W63A。W63A不结合JHC1-64，而R60A结合，尽管与野生型DAT相比效率较低。分子动力学模拟预测，R60A优先通过补偿性细胞内盐桥形成而呈现向外（OF）构象，这反过来又有利于可卡因的结合。影像学分析表明，与JHC1-64结合的R60A突变体主要位于丝状伪足中，而游离的R60A分子则均匀地分布在质膜中。可卡因结合在丝状伪足中显着增加了R60A的密度，但没有增加W63A的密度。此外，锌结合（已知可稳定OF状态）也增加了丝状伪足中R60A的浓度。最后，安非他明被认为会破坏DAT OF构象，从而降低了丝状伪足中野生型DAT的浓度。总之，这些数据表明OF构象是DAT有效靶向丝状伪足并在丝状伪足中积累所必需的。认为这会破坏DAT OF构象，降低丝状伪足中野生型DAT的浓度。总之，这些数据表明OF构象是DAT有效靶向丝状伪足并在丝状伪足中积累所必需的。认为这会破坏DAT OF构象，降低丝状伪足中野生型DAT的浓度。总而言之，这些数据表明OF构象是DAT有效靶向丝状伪足并在丝状伪足中积累所必需的。

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Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter.

Dopamine transporter (DAT) has been shown to accumulate in filopodia in neurons and non-neuronal cells. To examine the mechanisms of DAT filopodial targeting, we used quantitative live-cell fluorescence microscopy, and compared the effects of the DAT inhibitor cocaine and its fluorescent analog JHC1-64 on the plasma membrane distribution of wild-type DAT and two non-functional DAT mutants, R60A and W63A, that do not accumulate in filopodia. W63A did not bind JHC1-64, whereas R60A did, although less efficiently compared to the wild-type DAT. Molecular dynamics simulations predicted that R60A preferentially assumes an outward-facing (OF) conformation through compensatory intracellular salt bridge formation, which in turn favors binding of cocaine. Imaging analysis showed that JHC1-64-bound R60A mutant predominantly localized in filopodia, whereas free R60A molecules were evenly distributed within the plasma membrane. Cocaine binding significantly increased the density of R60A, but not that of W63A, in filopodia. Further, zinc binding, known to stabilize the OF state, also increased R60A concentration in filopodia. Finally, amphetamine, that is thought to disrupt DAT OF conformation, reduced the concentration of wild-type DAT in filopodia. Altogether, these data indicate that OF conformation is required for the efficient targeting of DAT to, and accumulation in, filopodia.

更新日期：2017-07-14

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