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Binding mechanism and antagonism of the vesicular acetylcholine transporter VAChT

Nature Structural & Molecular Biology volume 32, pages 818–827 (2025)Cite this article

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Abstract

The vesicular acetylcholine transporter (VAChT) has a pivotal role in packaging and transporting acetylcholine for exocytotic release, serving as a vital component of cholinergic neurotransmission. Dysregulation of its function can result in neurological disorders. It also serves as a target for developing radiotracers to quantify cholinergic neuron deficits in neurodegenerative conditions. Here we unveil the cryo-electron microscopy structures of human VAChT in its apo state, the substrate acetylcholine-bound state and the inhibitor vesamicol-bound state. These structures assume a lumen-facing conformation, offering a clear depiction of architecture of VAChT. The acetylcholine-bound structure provides a detailed understanding of how VAChT recognizes its substrate, shedding light on the coupling mechanism of protonation and substrate binding. Meanwhile, the vesamicol-bound structure reveals the binding mode of vesamicol to VAChT, laying the structural foundation for the design of the next generation of radioligands targeting VAChT.

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Fig. 1: Functional characterization and architecture of human VAChTapo.
Fig. 2: Cryo-EM structure of VAChTACh complex.
Fig. 3: Cryo-EM structure of the VAChTVES complex.
Fig. 4: Conformational changes between lumen-facing and cytoplasm-facing states of VAChT.

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Data availability

The three-dimensional cryo-EM density maps of human VAChTapo, VAChTACh and VAChTVES were deposited to the EM Data Bank under accession codes EMD-38652, EMD-38651 and EMD-38653. The coordinates for human VAChTapo, VAChTACh and VAChTVES were deposited to the PDB under accession codes 8XTX, 8XTW and 8XTY, respectively. Source data are provided with this paper.

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Acknowledgements

We thank B. Xu at the Cryo-EM Center of the School of Advanced Agricultural Sciences of Peking University and X. Huang, B. Zhu, X. Li, L. Chen and other staff members at the Center for Biological Imaging, Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science for support in cryo-EM data collection. We thank H. Zhang at the Core Facility of Protein Research, Institute of Biophysics, Chinese Academy of Sciences for support with functional experiment. We thank Y. Wu for his research assistance services and Y.Z. laboratory members for helpful discussions. This work was funded by the Chinese National Programs for Brain Science and Brain-like Intelligence Technology (grant no. 2022ZD0205800 to Y.Z.), the National Key Research and Development Program of China (grant no. 2021YFA1301501 to Y.Z.), the Chinese Academy of Sciences Strategic Priority Research Program (grant no. XDB37030304 to Y.Z.), the National Natural Science Foundation of China (grant nos. 92157102 to Y.Z., 32301026 to Y.D. and 32100773 to K.M.) and the China Postdoctoral Science Foundation.

Author information

Author notes

  1. These authors contributed equally: Qiao Ma, Kunpeng Ma, Yanli Dong, Yufei Meng, Jun Zhao.

Authors and Affiliations

  1. Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Qiao Ma, Yanli Dong, Yufei Meng, Renjie Li, Qinru Bai & Yan Zhao

  2. University of Chinese Academy of Sciences, Beijing, China

    Qiao Ma, Yufei Meng, Renjie Li, Qinru Bai, Di Wu, Daohua Jiang & Jianyuan Sun

  3. Interdisciplinary Center for Brain Information, The Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Kunpeng Ma & Jianyuan Sun

  4. Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences at Weifang, Weifang, China

    Jun Zhao

  5. Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

    Di Wu & Daohua Jiang

  6. CAS Key Laboratory of Brain Connectome and Manipulation, Shenzhen-Hong Kong Institute of Brain Science, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Jianyuan Sun

  7. Beijing Key Laboratory of Mental Disorders, National Clinical Research Center for Mental Disorders and National Center for Mental Disorders, Beijing Anding Hospital, Capital Medical University, Beijing, China

    Yan Zhao

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Contributions

Y.Z. conceptualized and supervised the project. Q.M. and Y.D. prepared the samples for the cryo-EM study and made all the constructs. R.L., Y.D., J.Z., D.W. and D.J. collected the cryo-EM data. Y.D., Q.M. and Q.B. processed the data and built and refined the models. Y.D., Q.M., Y.M. and Y.Z. analyzed the structures and prepared the figures. K.M., Y.M., J.S. and Y.Z designed, performed and analyzed the radioligand-binding assays and slice imaging experiments. Y.Z. wrote the paper with input and support from all coauthors. All authors reviewed and revised the paper.

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Correspondence to Yan Zhao.

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Nature Structural & Molecular Biology thanks Shimon Schuldiner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Biochemical characterization and construction strategy of the hVAChT.

a. Schematic construct and topology diagram of VAChT from N-terminal E27 to the C-terminal T476. Unresolved regions are depicted with dotted lines. TM numbers are labeled, and the plasma membrane is indicated with dashed lines. Inverted repeat motifs TM1-3 (deep blue), TM4-6 (light blue), TM7-9 (red), and TM10-12 (pink) are shown as gray triangles. b. Size-exclusion chromatogram (Superose 6 Increase) profile of the purified VAChT sample. Peak fractions between dashes were used for cryo-EM sample preparation. c. Coomassie-blue-stained SDS-PAGE gel of purified VAChT sample for cryo-EM data collection. VAChT appeared as two bands due to glycosylation. The experiments were independently repeated more than three times, yielding consistent results.

Source data

Extended Data Fig. 2 Cryo-EM data processing of VAChTapo.

a. Flow chart for cryo-EM data processing of VAChTapo. A total of 3,030 micrographs were motion-corrected and dose-weighted using MotionCor2 with 7 × 10 patching in RELION-3.1. A representative motion-corrected micrograph of this dataset is shown here (scale bar = 30 nm). The particles were picked using template picker, and 2D classification was performed to remove junk particles in cryoSPARC. After three rounds of heterogeneous refinements, 218,702 particles displaying transmembrane helices were classified and subjected to Bayesian polishing in RELION-3.1 to further improve the map quality. The final map was reported at 3.4-Å resolution according to the golden standard Fourier shell correlation (GSFSC) criterion. Details of data processing can be found in Method. b. The angular distribution of the final reconstruction. c. Local resolution distribution of VAChTapo. d. Fourier Shell Correlations (FSC) of the final map of the VAChTapo, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM map and the structural model shown in black. e. Representative overlay of cryo-EM density and transmembrane helices of VAChTapo. The cryo-EM maps are shown as gray surface.

Extended Data Fig. 3 Cryo-EM data processing of VAChTACh.

a. Flow chart for cryo-EM data processing of VAChTACh. A total of 6,600 micrographs were motion-corrected using patch motion correction, and CTF parameters were determined using patch CTF estimation in CryoSPARC. A representative motion-corrected micrograph of this dataset is shown here (scale bar = 30 nm). The particles were picked using template picker, and 2D classification was performed to remove junk particles. After three rounds of heterogeneous refinements, followed by ab-initio reconstruction and non-uniform refinement, a 3.8-Å resolution map was obtained. The 201,774 good particles were used as the “seed” and subjected to seed-facilitated 3D classification to further improve the resolution. Details of data processing can be found in Method. b. The angular distribution of the final reconstruction. c. Local resolution distribution of VAChTACh. d. Fourier Shell Correlations (FSC) of the final map of the VAChTACh, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM map and the structural model shown in black. e. Representative overlay of cryo-EM density and transmembrane helices of VAChTACh. The cryo-EM maps are shown as grey surface.

Extended Data Fig. 4 Cryo-EM data processing of VAChTVES.

a. Flow chart for cryo-EM data processing. A total of 1,762 movie stacks were collected and motion-corrected. A representative motion-corrected micrograph of this dataset is shown here (scale bar = 30 nm). A total of 2,257,399 particles were picked using template picker in CryoSPARC, and were used for 2D classification. 2D class averages of distinct secondary structure features from different views of VAChTVES are shown. Only classes featuring transmembrane helices were selected and subjected to heterogeneous refinements to remove junk particles, followed by local refinement to improve map quality. The final map was reported at 2.7 Å according to the GSFSC criterion. Details of data processing can be found in Method. b. The angular distribution of the final reconstruction. c. Local resolution distribution of VAChTVES. d. Fourier Shell Correlations (FSC) of the final map of the VAChTVES, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM density and the structural model shown in black. e. Representative overlay of cryo-EM density and transmembrane helices of VAChTVES. The cryo-EM maps are shown as grey surface.

Extended Data Fig. 5 Comparison of VAChTapo with VMAT25HT and VGLUT2apo.

a-b. Superposition of the overall structures of VAChTapo (purple and pink) and VMAT25HT (gray, PDB ID: 8JSW) with substrates represented as sticks viewed parallel to the membrane plane (a) and from the luminal side (b), respectively. c. Superposition of the N-domain (TM1-6, purple) structure between VAChTapo and VMAT25HT viewed parallel to the membrane plane. d. Superposition of the C-domain (TM7-12, pink) structure between VAChTapo and VMAT25HT viewed parallel to the membrane plane. e-f. Superposition of the overall structures of VAChTapo (purple and pink) and VGLUT2apo (green, PDB ID: 8SBE) viewed parallel to the membrane plane (e) and from the luminal side (f), respectively. g. Superposition of the N-domain (TM1-6, purple) structure between VAChTapo and VGLUT2apo viewed parallel to the membrane plane. h. Superposition of the C-domain (TM7-12, pink) structure between VAChTapo and VGLUT2apo viewed parallel to the membrane plane. i-k. The estimated electrostatic potential of the substrate binding pocket of VAChTapo, VMAT25HT, and VGLUT2apo viewed from the luminal side, respectively. The central cavities are outlined by the dark dashed circle. The positively and negatively charged surfaces are colored blue and red, respectively, and the electro-neutral (or nonpolar/hydrophobic) surface is colored white.

Extended Data Fig. 6 Mapping the pathogenic mutations and critical residues on the VAChT structure.

a. The side-view of the structure of hVAChT with pathogenic mutations. The Cα atoms of the pathogenic mutations are shown as spheres. b. Currently reported pathogenic mutations and associated disease. CMS: Congenital myasthenic syndromes. The mutations labeled with ‘*’ represent nonsense variants. c. The representative residues surrounding the positions of various pathogenic mutations. N-domain and C-domain are colored in purple and pink, respectively. d. Mapping of four residues A228, G233, S252, and C391 on the hVAChT structure derived from Caenorhabditis elegans mutants. The Cα atoms of the residues are shown as spheres. Vesamicol and acetylcholine are represented as gold and cyan sticks, respectively. e. The positions of residues A228, G233, S252, and C391 relative to the ligand-binding pocket of VAChT. N-domain and C-domain are colored in purple and pink, respectively.

Extended Data Fig. 7 Sequence alignment of Vesicular Acetylcholine Transporter homologues.

Sequence alignment among human VAChT (hVAChT), human VMAT1 (hVMAT1), human VMAT2 (hVMAT2), Mus musculus VAChT (mVAChT), Rattus norvegicus VAChT (rVAChT), Drosophila melanogaster VAChT (dVAChT), Tetronarce californica VAChT (tcVAChT), and Torpedo torpedo VAChT (ttVAChT). The secondary structural elements of hVAChT are depicted above the sequence alignment, with unmodeled regions represented as dashed lines and transmembrane domains represented as grey rectangle boxes. Conserved acidic, basic, and other types of amino acid residues in the protein are highlighted using red, blue, and grey, respectively.

Extended Data Fig. 8 Structural comparison of VAChTACh, VMAT25HT and VMAT2MPP+.

a. Superposition of the overall structures of VAChTACh (purple and pink) and VMAT25HT (grey). Acetylcholine and 5-HT are represented as cyan and gray spheres respectively. b. Comparison of acetylcholine and 5-HT binding sites between VAChTACh and VMAT25HT. The substrates and residues involved in the interactions are shown as sticks. c. Superposition of the overall structures of VAChTACh (purple and pink) and VMAT2MPP+ (olive, PDB ID: 8XOA), with acetylcholine and MPP+ represented as cyan and olive spheres, respectively. d. Comparison of acetylcholine and 5-HT binding sites between VAChTACh and VMAT2MPP+. The substrates and residues involved in the interactions are shown as sticks. e. Superposition of the overall structures between VAChTACh and VMAT25HT with the region of potential protonated sites outlined by a dark dashed circle. f. Comparison of the potential protonated sites between VAChTACh and VMAT25HT. The putative protonated residues are represented as sticks.

Extended Data Fig. 9 Structural comparison between VAChT and VMAT2 in different conformations.

a-c. Superposition of the overall structures of VAChTVES (purple and pink) and VMAT2RES (light blue) viewed parallel to the membrane plane (a), from the luminal side (b), and cytoplasmic side (c). The N-domain shown in cartoon is used as a reference for structural superposition. C-domains are shown as cylinders and an approximate 32-degree rotation between the two structures is indicated by black arrows. d-f. Superposition of the overall structures of VAChTVES (purple and pink) and VMAT2KET (pale cyan, PDB ID: 8JT9) shown in cylinders, viewed parallel to the membrane plane (d). The cytoplasmic gates are outlined by a black dashed circle. The critical residues and salt bridges involved in forming the gate of VAChTVES (e) and VMAT2KET (f) are depicted in sticks and black dashed lines, respectively. g-i. Superposition of the overall structures of VAChTCyto shown in cylinders (purple and pink, cytoplasm-facing model of VAChT) and VMAT2RES (pale cyan). The luminal gates are outlined by a black dashed circle. The critical residues and salt bridges involved in forming the gate of VAChTCyto (e) and VMAT2RES (f) are depicted in sticks and black dashed lines, respectively.

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Ma, Q., Ma, K., Dong, Y. et al. Binding mechanism and antagonism of the vesicular acetylcholine transporter VAChT. Nat Struct Mol Biol 32, 818–827 (2025). https://doi.org/10.1038/s41594-024-01462-9

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