VMAT2的神经递质转运和药物抑制机制-动脉网

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2

Nature 等信源发布 2023-10-31 23:57

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AbstractMonoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3,4,5.

摘要多巴胺和5-羟色胺等单胺神经递质控制着重要的脑通路，包括运动，睡眠，奖励和情绪1。单胺能回路的功能障碍与各种神经退行性疾病和神经精神疾病有关2。囊泡单胺转运蛋白（VMATs）将单胺包装成囊泡用于突触释放，并且对神经传递至关重要3,4,5。

VMATs are also therapeutic drug targets for a number of different conditions6,7,8,9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin.

VMAT也是许多不同病症的治疗药物靶标6,7,8,9。尽管这些转运蛋白很重要，但VMAT的底物转运和药物抑制机制仍然难以捉摸。在这里，我们报告了人类囊泡单胺转运蛋白VMAT2与抗肿瘤药物丁苯那嗪，抗高血压药物利血平或底物5-羟色胺复合物的低温电子显微镜结构。

Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access.

值得注意的是，这两种药物使用完全不同的抑制机制。丁苯那嗪以面向管腔的构型结合VMAT2，将管腔门控盖锁定在闭塞状态以阻止运输周期。相比之下，利血平以面向细胞质的构象结合，扩大前庭并阻止底物进入。

Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters..

VMAT2的结构分析还揭示了转运蛋白异构化后驱动底物转运到囊泡中的构象变化。这些发现为理解突触小泡转运蛋白包装神经递质的生理学和药理学提供了结构框架。。

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Fig. 1: Cryo-EM structures of VMAT2 in complex with therapeutic drugs.Fig. 2: Inhibition mechanism of TBZ.Fig. 3: Inhibition mechanism of reserpine.Fig. 4: Serotonin binding and recognition.Fig. 5: Mechanism of substrate translocation.

图1:VMAT2与治疗药物复合的Cryo-EM结构。图2:TBZ的抑制机制图3：利血平的抑制机制。图4：血清素结合和识别。图5：底物易位的机制。

Data availability

数据可用性

Cryo-EM maps and coordinates have been deposited in the EMDB and wwPDB, respectively, with accession numbers EMD-41066 and PDB 8T69 (VMAT2 with TBZ bound); EMD-41067 and PDB 8T6A (VMAT2 with reserpine bound); and EMD-41068 and PDB 8T6B (VMAT2 with 5-HT bound).

Cryo-EM图和坐标分别存储在EMDB和wwPDB中，登录号为EMD-41066和PDB 8T69（结合TBZ的VMAT2）；EMD-41067和PDB 8T6A（结合利血平的VMAT2）；和EMD-41068和PDB 8T6B（具有5-HT结合的VMAT2）。

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Download referencesAcknowledgementsWe thank scientists in the Cryo-EM Center of St. Jude Children’s Research Hospital for their support in data collection. We thank scientists in the Cell and Tissue Imaging Center of St. Jude Children’s Research Hospital and P. Zheng, P. Du and S. Jian for their support in cellular imaging.

下载参考文献致谢我们感谢圣裘德儿童研究医院Cryo-EM中心的科学家对数据收集的支持。我们感谢圣裘德儿童研究医院细胞和组织成像中心的科学家以及P.Zheng，P。Du和s.Jian对细胞成像的支持。

We thank Y. Wang for cell culture. We thank the members of the Lee and Zhang laboratories and F. Liu for helpful discussions; Z. Luo for assistance in preparing cartoon diagrams; and I. Chen for editing the manuscript. We thank the Roussel laboratory and the Schuetz laboratory at St. Jude for sharing equipment for radioisotope experiments.

我们感谢Y.Wang的细胞培养。我们感谢Lee和Zhang实验室的成员以及F.Liu的有益讨论；Z、罗协助准备漫画；和I.Chen编辑手稿。我们感谢Roussel实验室和St.Jude的Schuetz实验室共享放射性同位素实验设备。

We thank the National Center for Protein Science at Peking University for other technical support. This work was supported by the National Key Research and Development Program of China (2021YFA1302300 to Z.Z.), the National Natural Science Foundation of China (32171201 to Z.Z.), the Center for Life Science, School of Life Science (SLS) of Peking University (to Z.Z.), the SLS-Qidong innovation fund (to Z.Z.), the Li Ge-Zhao Ning Life Science Youth Research Foundation (to Z.Z.) and the State Key Laboratory of Membrane Biology of China (to Z.Z.) and by National Institutes of Health (R01GM143282 to C.-H.L.) and ALSAC (to C.-H.L.).Author informationAuthor notesThese authors contributed equally: Shabareesh Pidathala, Shuyun Liao, Yaxin DaiAuthors and AffiliationsDepartment of Structural Biology, St.

我们感谢北京大学国家蛋白质科学中心的其他技术支持。这项工作得到了国家重点研究发展计划（2021YFA1302300至Z.Z.），国家自然科学基金（32171201至Z.Z.），北京大学生命科学学院生命科学中心（至Z.Z.），SLS Qidong创新基金（至Z.Z.）的支持，赵立革生命科学青年研究基金会（至Z.Z.）和中国膜生物学国家重点实验室（至Z.Z.）以及国立卫生研究院（R01GM143282至C.-H.L.）和ALSAC（至C.-H.L.）。作者信息作者注：这些作者的贡献相同：Shabareesh Pidathala，廖淑云，Daixin作者和附属机构圣路易斯结构生物学系。

Jude Children’s Research Hospital, Memphis, TN, USAShabareesh Pidathala, Yaxin Dai & Chia-Hsueh LeeState Key Laboratory of Membrane Biology, Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, School of Life Sciences, Peking University, Beijing, ChinaShuyun Liao, Changkun Long & Zhe Zhang.

美国田纳西州孟菲斯裘德儿童研究医院，美国北京大学生命科学学院生命科学研究院生命科学中心膜生物学国家重点实验室，北京，ChinaShuyun Liao，Changkun Long＆Zhe Zhang。

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PubMed Google ScholarContributionsS.P. performed and analysed radioligand binding/transport assays. S.P. expressed and purified protein samples. S.P. and Y.D. performed cryo-EM structural experiments. S.P., S.L. and Y.D. analysed structural data. S.L. and Y.D. performed fluorescent substrate transport assays.

PubMed Google学术贡献SS.P.执行并分析了放射性配体结合/转运分析。S、 P.表达和纯化的蛋白质样品。S、 P.和Y.D.进行了冷冻EM结构实验。S、 P.，S.L。和Y.D.分析了结构数据。S、 L.和Y.D.进行了荧光底物转运测定。

X.L., S.P., Y.D., S.L. and C.-L.C. performed colocalization experiments. C.L. performed initial construct characterization and biochemical experiments. Z.Z. and C.-H.L. conceived the research and supervised the project. Z.Z. and C.-H.L. wrote the manuscript with input from all authors.Corresponding authorsCorrespondence to.

十、 L.，S.P.，Y.D.，S.L。和C.-L.C.进行了共定位实验。C、 L.进行了初始构建体表征和生化实验。Z、 Z.和C.-H.L.构思了这项研究并监督了该项目。Z、 Z.和C.-H.L.在所有作者的帮助下撰写了手稿。通讯作者对应。

Zhe Zhang or Chia-Hsueh Lee.Ethics declarations

Zhe Zhang或Chia-Hsueh Lee。道德声明

Competing interests

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The authors declare no competing interests.

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Nature thanks Shimon Schuldiner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

自然感谢Shimon Schuldiner和其他匿名审稿人对本文同行评审的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended data figures and tablesExtended Data Fig. 1 Construct design and sequence alignment of VMAT2.a, Time course of 3H-dopamine uptake in permeabilized VMAT2-transfected or untransfected cells.

其他信息发布者的说明Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据图和表扩展数据图1 VMAT2的构建体设计和序列比对a，透化的VMAT2转染或未转染的细胞中3H-多巴胺摄取的时间过程。

The inset shows the uptake of the first 10 min. Data are shown as mean ± s.d.; n = 3 biological replicates. b, Schematic of the VMAT2EM construct. c, Representative size exclusion chromatography profile and SDS-PAGE analysis of purified VMAT2EM in saposin nanodisc. For gel source data, see Supplementary Fig.

插图显示了前10个 min。数据显示为平均值±s.d。；北 = 3个生物学重复。b、 VMAT2EM结构示意图。c、 saposin nanodisc中纯化的VMAT2EM的代表性尺寸排阻色谱图和SDS-PAGE分析。对于凝胶源数据，见Supplementary Fig。

1. The trace and gel image are representative of 3 experimental replicates. d, Sequence alignment of the SLC18 family. Residues with functional roles are highlighted with different colors: green, residues that contribute to TBZ selectivity toward VMAT2; blue, residues forming the cytoplasmic gates; yellow, residues forming the luminal gates and the lid; red, acidic residues potentially involved in proton coupling.Extended Data Fig.

痕量和凝胶图像代表3个实验重复。d、 SLC18家族的序列比对。具有功能作用的残基以不同的颜色突出显示：绿色，有助于TBZ对VMAT2选择性的残基；蓝色，残基形成细胞质门；黄色，残留物形成管腔门和盖子；红色酸性残基可能与质子偶联有关。扩展数据图。

2 Cryo-EM analyses of VMAT2 in complex with TBZ.a, Summary of image processing procedures of VMAT2EM in complex with TBZ. All procedures were done with cryoSPARC, except for particle polishing which was done with RELION. b, Representative micrograph (left) (out of 26,389 similar micrographs) and 2D class averages (right).

2与TBZ复合的VMAT2的Cryo-EM分析.a，与TBZ复合的VMAT2EM的图像处理程序总结。除使用RELION进行颗粒抛光外，所有步骤均使用cryoSPARC完成。b、代表性显微照片（左）（26389张类似显微照片中）和2D类平均值（右）。

c, Fourier shell correlation (FSC) curves between two half maps. d, Angular distribution of particles for the final 3D reconstructions. e, Local resolution of the cryo-EM map. f, Cryo-EM densities of the transmembrane helices. Map contour level = 0.15–0.24 in ChimeraX.Extended Data Fig. 3 Cryo-EM analyses of VMAT2 in complex with reserpine.a, Summary of image processing pr.

c、两个半图之间的傅里叶壳相关（FSC）曲线。d、用于最终3D重建的粒子的角度分布。e、低温电磁图的局部分辨率。f、跨膜螺旋的低温电磁密度。ChimeraX中的地图轮廓水平=0.15-0.24。扩展数据图3与利血平复合的VMAT2的Cryo-EM分析.a，图像处理摘要pr。

Nature (2023). https://doi.org/10.1038/s41586-023-06727-9Download citationReceived: 16 June 2023Accepted: 09 October 2023Published: 01 November 2023DOI: https://doi.org/10.1038/s41586-023-06727-9Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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Cryoelectron microscopyTransporters in the nervous system

神经系统中的低温电子显微镜转运蛋白

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