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Transport and inhibition mechanisms of human VMAT2

Nature volume 626, pages 427–434 (2024)Cite this article

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Abstract

Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission1,2,3. Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson’s disease, hyperkinetic movement disorders and depression4,5,6. Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington’s disease7,8, respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5–2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents.

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Fig. 1: Functional characterization and structure of human VMAT2.
Fig. 2: Structural basis of 5-HT recognition by VMAT2.
Fig. 3: Structural basis of VMAT2 inhibition by reserpine.
Fig. 4: Structural basis of VMAT2 inhibition by TBZ.
Fig. 5: Structural basis of VMAT2 inhibition by KET.
Fig. 6: Structural basis of VMAT2 state transitions.

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Data availability

The three-dimensional cryo-EM density maps of human VMAT25-HT, VMAT2RES, VMAT2TBZ and VMAT2KET have been deposited into the Electron Microscopy Data Bank under accession numbers EMD-36628, EMD-36640, EMD-36638 and EMD-36637, respectively. The coordinates of human VMAT25-HT, VMAT2RES, VMAT2TBZ and VMAT2KET have been deposited into the Protein Data Bank under accession codes 8JSW, 8JTC, 8JTA and 8JT9, respectively. Source data are provided with this paper.

References

  1. Schuldiner, S., Shirvan, A. & Linial, M. Vesicular neurotransmitter transporters: from bacteria to humans. Physiol. Rev. 75, 369–392 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Blakely, R. D. & Edwards, R. H. Vesicular and plasma membrane transporters for neurotransmitters. Cold Spring Harb. Perspect. Biol. 4, a005595 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Henry, J. P., Sagne, C., Bedet, C. & Gasnier, B. The vesicular monoamine transporter: from chromaffin granule to brain. Neurochem. Int. 32, 227–246 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Charney, D. S. Monoamine dysfunction and the pathophysiology and treatment of depression. J. Clin. Psychiatry 59, 11–14 (1998).

    CAS  PubMed  Google Scholar 

  5. Bernstein, A. I., Stout, K. A. & Miller, G. W. The vesicular monoamine transporter 2: an underexplored pharmacological target. Neurochem. Int. 73, 89–97 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Carlsson, M. & Carlsson, A. Interactions between glutamatergic and monoaminergic systems within the basal ganglia–implications for schizophrenia and Parkinson’s disease. Trends Neurosci. 13, 272–276 (1990).

    Article  CAS  PubMed  Google Scholar 

  7. Stitzel, R. E. The biological fate of reserpine. Pharmacol. Rev. 28, 179–208 (1976).

    CAS  PubMed  Google Scholar 

  8. Chen, J. J., Ondo, W. G., Dashtipour, K. & Swope, D. M. Tetrabenazine for the treatment of hyperkinetic movement disorders: a review of the literature. Clin Ther 34, 1487–1504 (2012).

    Article  PubMed  Google Scholar 

  9. Liu, Y. et al. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell 70, 539–551 (1992).

    Article  CAS  PubMed  Google Scholar 

  10. Erickson, J. D. & Eiden, L. E. Functional identification and molecular cloning of a human brain vesicle monoamine transporter. J. Neurochem. 61, 2314–2317 (1993).

    Article  CAS  PubMed  Google Scholar 

  11. Erickson, J. D., Eiden, L. E. & Hoffman, B. J. Expression cloning of a reserpine-sensitive vesicular monoamine transporter. Proc. Natl Acad. Sci. USA 89, 10993–10997 (1992).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Peter, D. et al. Differential expression of two vesicular monoamine transporters. J. Neurosci. 15, 6179–6188 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Weihe, E., Schafer, M. K., Erickson, J. D. & Eiden, L. E. Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat. J. Mol. Neurosci. 5, 149–164 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Erickson, J. D., Schafer, M. K., Bonner, T. I., Eiden, L. E. & Weihe, E. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter. Proc. Natl Acad. Sci. USA 93, 5166–5171 (1996).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Knoth, J., Zallakian, M. & Njus, D. Stoichiometry of H+-linked dopamine transport in chromaffin granule ghosts. Biochemistry 20, 6625–6629 (1981).

    Article  CAS  PubMed  Google Scholar 

  16. Johnson, R. G., Carty, S. E. & Scarpa, A. Proton: substrate stoichiometries during active transport of biogenic amines in chromaffin ghosts. J. Biol. Chem. 256, 5773–5780 (1981).

    Article  CAS  PubMed  Google Scholar 

  17. Eiden, L. E., Schafer, M. K., Weihe, E. & Schutz, B. The vesicular amine transporter family (SLC18): amine/proton antiporters required for vesicular accumulation and regulated exocytotic secretion of monoamines and acetylcholine. Pflugers Arch. 447, 636–640 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Arvidsson, U., Riedl, M., Elde, R. & Meister, B. Vesicular acetylcholine transporter (VAChT) protein: a novel and unique marker for cholinergic neurons in the central and peripheral nervous systems. J. Comp. Neurol. 378, 454–467 (1997).

    Article  CAS  PubMed  Google Scholar 

  19. Hiasa, M. et al. Identification of a mammalian vesicular polyamine transporter. Sci. Rep. 4, 6836 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Pao, S. S., Paulsen, I. T. & Saier, M. H. Jr. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 62, 1–34 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Drew, D., North, R. A., Nagarathinam, K. & Tanabe, M. Structures and general transport mechanisms by the major facilitator superfamily (MFS). Chem. Rev. 121, 5289–5335 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yaffe, D., Forrest, L. R. & Schuldiner, S. The ins and outs of vesicular monoamine transporters. J. Gen. Physiol. 150, 671–682 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yaffe, D., Radestock, S., Shuster, Y., Forrest, L. R. & Schuldiner, S. Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2. Proc. Natl Acad. Sci. USA 110, E1332–E1341 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yaffe, D., Vergara-Jaque, A., Forrest, L. R. & Schuldiner, S. Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis. Proc. Natl Acad. Sci. USA 113, E7390–E7398 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wimalasena, K. Vesicular monoamine transporters: structure-function, pharmacology, and medicinal chemistry. Med. Res. Rev. 31, 483–519 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Henry, J. P. et al. Biochemistry and molecular biology of the vesicular monoamine transporter from chromaffin granules. J. Exp. Biol. 196, 251–262 (1994).

    Article  CAS  PubMed  Google Scholar 

  27. Zheng, G., Dwoskin, L. P. & Crooks, P. A. Vesicular monoamine transporter 2: role as a novel target for drug development. AAPS J. 8, E682–692 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Scherman, D. & Henry, J. P. Reserpine binding to bovine chromaffin granule membranes. Characterization and comparison with dihydrotetrabenazine binding. Mol. Pharmacol. 25, 113–122 (1984).

    CAS  PubMed  Google Scholar 

  29. Howell, M. et al. Cloning and functional expression of a tetrabenazine sensitive vesicular monoamine transporter from bovine chromaffin granules. FEBS Lett. 338, 16–22 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Kanner, B. I., Fishkes, H., Maron, R., Sharon, I. & Schuldiner, S. Reserpine as a competitive and reversible inhibitor of the catecholamine transporter of bovine chromaffin granules. FEBS Lett. 100, 175–178 (1979).

    Article  CAS  PubMed  Google Scholar 

  31. Rudnick, G., Steiner-Mordoch, S. S., Fishkes, H., Stern-Bach, Y. & Schuldiner, S. Energetics of reserpine binding and occlusion by the chromaffin granule biogenic amine transporter. Biochemistry 29, 603–608 (1990).

    Article  CAS  PubMed  Google Scholar 

  32. Leysen, J. E., Niemegeers, C. J., Van Nueten, J. M. & Laduron, P. M. [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin 2 receptor binding sites. Binding properties, brain distribution, and functional role. Mol. Pharmacol. 21, 301–314 (1982).

    CAS  PubMed  Google Scholar 

  33. Darchen, F., Scherman, D., Laduron, P. M. & Henry, J. P. Ketanserin binds to the monoamine transporter of chromaffin granules and of synaptic vesicles. Mol. Pharmacol. 33, 672–677 (1988).

    CAS  PubMed  Google Scholar 

  34. Henry, J. P. & Scherman, D. Radioligands of the vesicular monoamine transporter and their use as markers of monoamine storage vesicles. Biochem. Pharmacol. 38, 2395–2404 (1989).

    Article  CAS  PubMed  Google Scholar 

  35. Li, F. et al. Ion transport and regulation in a synaptic vesicle glutamate transporter. Science 368, 893–897 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Deng, D. et al. Crystal structure of the human glucose transporter GLUT1. Nature 510, 121–125 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Merickel, A., Kaback, H. R. & Edwards, R. H. Charged residues in transmembrane domains II and XI of a vesicular monoamine transporter form a charge pair that promotes high affinity substrate recognition. J. Biol. Chem. 272, 5403–5408 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Merickel, A., Rosandich, P., Peter, D. & Edwards, R. H. Identification of residues involved in substrate recognition by a vesicular monoamine transporter. J. Biol. Chem. 270, 25798–25804 (1995).

    Article  CAS  PubMed  Google Scholar 

  39. Yaffe, D. et al. Functionally important carboxyls in a bacterial homologue of the vesicular monoamine transporter (VMAT). J. Biol. Chem. 289, 34229–34240 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Steiner-Mordoch, S., Shirvan, A. & Schuldiner, S. Modification of the pH profile and tetrabenazine sensitivity of rat VMAT1 by replacement of aspartate 404 with glutamate. J. Biol. Chem. 271, 13048–13054 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Varoqui, H. & Erickson, J. D. Active transport of acetylcholine by the human vesicular acetylcholine transporter. J. Biol. Chem. 271, 27229–27232 (1996).

    Article  CAS  PubMed  Google Scholar 

  42. Deupree, J. D. & Weaver, J. A. Identification and characterization of the catecholamine transporter in bovine chromaffin granules using [3H]reserpine. J. Biol. Chem. 259, 10907–10912 (1984).

    Article  CAS  PubMed  Google Scholar 

  43. Finn, J. P. 3rd & Edwards, R. H. Individual residues contribute to multiple differences in ligand recognition between vesicular monoamine transporters 1 and 2. J. Biol. Chem. 272, 16301–16307 (1997).

    Article  CAS  PubMed  Google Scholar 

  44. Erickson, J. D. et al. Functional identification of a vesicular acetylcholine transporter and its expression from a “cholinergic” gene locus. J. Biol. Chem. 269, 21929–21932 (1994).

    Article  CAS  PubMed  Google Scholar 

  45. Paulsen, I. T., Brown, M. H. & Skurray, R. A. Proton-dependent multidrug efflux systems. Microbiol. Rev. 60, 575–608 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Jiang, D. et al. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. Proc. Natl Acad. Sci. USA 110, 14664–14669 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  47. Jessen-Marshall, A. E., Paul, N. J. & Brooker, R. J. The conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease. J. Biol. Chem. 270, 16251–16257 (1995).

    Article  CAS  PubMed  Google Scholar 

  48. Parsons, S. M. Transport mechanisms in acetylcholine and monoamine storage. FASEB J. 14, 2423–2434 (2000).

    Article  CAS  PubMed  Google Scholar 

  49. Muth, T. R. & Schuldiner, S. A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE. EMBO J. 19, 234–240 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Heng, J. et al. Substrate-bound structure of the E. coli multidrug resistance transporter MdfA. Cell Res. 25, 1060–1073 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhong, P. et al. Structural insights into two distinct nanobodies recognizing the same epitope of green fluorescent protein. Biochem. Biophys. Res. Commun. 565, 57–63 (2021).

    Article  CAS  PubMed  Google Scholar 

  52. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, https://doi.org/10.1016/j.jsb.2015.11.003 (2016).

  55. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  57. Wang, N. et al. Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates. Cell 184, 370–383.e313 (2021).

    Article  CAS  PubMed  Google Scholar 

  58. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  59. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  ADS  PubMed  Google Scholar 

  60. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  61. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  62. Olsson, M. H., Sondergaard, C. R., Rostkowski, M. & Jensen, J. H. PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J. Chem. Theory Comput. 7, 525–537 (2011).

    Article  CAS  PubMed  Google Scholar 

  63. Ma, L., Ouyang, Q., Werthmann, G. C., Thompson, H. M. & Morrow, E. M. Live-cell microscopy and fluorescence-based measurement of luminal pH in intracellular organelles. Front. Cell Dev. Biol. 5, 71 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Li, Y. et al. Structure of human NaV1.6 channel reveals Na+ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin. Nat. Commun. 14, 1030 (2023).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. Van Der Spoel, D. et al. GROMACS: fast, flexible, and free. J. Comput. Chem. 26, 1701–1718 (2005).

    Article  PubMed  Google Scholar 

  66. Harris, C. R. et al. Array programming with NumPy. Nature 585, 357–362 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank D. Sun at the SM10 Cryo-EM Facility at the Institute of Physics, Chinese Academy of Sciences (IOP, CAS), B. Xu at the Cryo-EM Center of 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 (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science (IBP, CAS) for support in cryo-EM data collection; H. Zhang and T. Sun; X. C. Zhang for helpful discussions; and Y. Wu and W. Fan for assistance. This work is funded by the National Natural Science Foundation of China (T2221001 and 32271272 to D.J., 92157102 to Y.Z.), 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 XDB37030304 to Y.Z.), the Institute of Physics, Chinese Academy of Sciences (E0VK101 and E2V4101 to D.J.).

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Author notes

  1. These authors contributed equally: Di Wu, Qihao Chen, Zhuoya Yu, Bo Huang, Jun Zhao

Authors and Affiliations

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

    Di Wu, Rui Yan & Daohua Jiang

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

    Di Wu, Qihao Chen, Zhuoya Yu, Yuhang Wang, Jiawei Su, Yan Zhao & Daohua Jiang

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

    Qihao Chen, Zhuoya Yu, Yuhang Wang, Jiawei Su & Yan Zhao

  4. Beijing StoneWise Technology, Beijing, China

    Bo Huang & Feng Zhou

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

    Jun Zhao

  6. Heart Center and Beijing Key Laboratory of Hypertension, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China

    Na Li

  7. Songshan Lake Materials Laboratory, Dongguan, China

    Daohua Jiang

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Contributions

D.J. and Y.Z. conceived and designed the experiments. D.W., Q.C., N.L. and R.Y. prepared samples for the cryo-EM study and made all the constructs. D.W. and J.Z. collected cryo-EM data. D.W., Z.Y., B.H. and D.J. processed the data and built and refined the models. Z.Y., D.W., Y.W. and Q.C. prepared figures. Q.C. and J.S. collected the transport activity data. B.H. and F.Z. performed the molecular dynamics simulation experiments. All authors analysed and interpreted the results. Y.Z. and D.J. wrote the paper, and all authors reviewed and revised the paper.

Corresponding authors

Correspondence to Yan Zhao or Daohua Jiang.

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Extended data figures and tables

Extended Data Fig. 1 Purification of human VMAT2.

a, Representative size-exclusion chromatography (SEC) profile of the detergent-purified VMAT2WT samples. Peak fractions shown in the left panel were visualized by SDS-PAGE with Coomassie blue staining (right). b, Representative reference-free 2D class averages of VMAT2WT. Scale bar, 5 nm. c, Topology diagram of VMAT2 from the N-terminal S18 to the C-terminal P474. Unsolved regions are shown with dashed lines. Gray triangles represent the inverted repeats formed by TM1-3 (deep blue), TM4-6 (sky blue), TM7-9 (yellow), and TM10-12 (red). Green and yellow dashed boxes represent GFP and GFP-nanobody, respectively. d and e, Representative SEC profiles of the purified VMAT2EM protein samples in detergents (d) and in nanodiscs (e). Peak fractions shown in the left panel were visualized by SDS-PAGE with Coomassie blue staining (right). Peak fractions between red dashed lines were collected and concentrated for cryo-EM sample preparation. Red labels show the samples corresponding to the peak fractions between the red dashed lines. The SEC profile and gel image are representative of 3 experimental replicates in a,d,e.

Source Data

Extended Data Fig. 2 Cryo-EM analysis of human VMAT25HT.

a, A representative motion-corrected EM micrograph (out of 4,745 micrographs) of VMAT25HT. Scale bar, 50 nm. b, Flowchart for cryo-EM data processing of VMAT25HT. The scale bar for the representative reference-free 2D class averages is 8 nm. c, Particle angular distribution calculated in cryoSPARC for the final reconstruction of VMAT25HT. d, Fourier-shell correlation (FSC) of the final map, calculated between two independently refined half-maps before (blue) and after (red) post-processing (FSC cut-off = 0.143). The FSC curve calculated between the EM map and the model is shown in black (FSC cut-off = 0.5). e, Representative EM densities of VMAT25HT. Structural elements are labelled, and side-chains are shown as sticks. f. Local resolution distribution for the final VMAT25HT map. The EM density for serotonin is indicated by a red arrow.

Extended Data Fig. 3 Cryo-EM data processing of VMAT2RES.

a, A representative motion-corrected EM micrograph (out of 2,850 micrographs) of VMAT2RES. Scale bar, 30 nm. b, Representative reference-free 2D class averages. Scale bar, 10 nm. c, Flowchart for cryo-EM data processing of VMAT2RES. d, Particle angular distribution calculated in cryoSPARC for the final reconstruction of VMAT2RES. e, FSC of the final map, calculated between two independently refined half-maps before (blue) and after (red) post-processing (FSC cut-off = 0.143). The FSC curve calculated between the EM map and the model is shown in black (FSC cut-off = 0.5). f, Representative EM densities of VMAT2RES. Side chains are shown as sticks and labelled. g, Local resolution distribution of VMAT2RES. The EM density for RES is indicated by a red arrow. h, Chemical structure and EM density of RES. The density is shown in blue meshed and contoured at 10 σ.

Extended Data Fig. 4 Cryo-EM data processing of VMAT2TBZ and VMAT2KET.

a and g, Representative motion-corrected EM micrographs of VMAT2TBZ (a) (out of 2,438 micrographs) and VMAT2KET (g) (out of 5,196 micrographs). Scale bar, 50 nm. b and h, Flowchart for cryo-EM data processing of VMAT2TBZ (b) and VMAT2KET (h). c and i, Selected reference-free 2D class averages of VMAT2TBZ (c) and VMAT2KET (i). Scale bar, 5 nm. d and j, FSC of the final map, calculated between two independently refined half-maps before (blue) and after (red) post-processing (FSC cut-off = 0.143) for VMAT2TBZ (d) and VMAT2KET (j). The FSC curve calculated between the EM map and the model is shown in black (FSC cut-off = 0.5). e and k, Local resolution distribution of VMAT2TBZ (e) and VMAT2KET (k). f and l, Chemical structure and EM density of TBZ (f) and KET (l). The density is shown in blue meshed and contoured at 10 σ. m, Representative EM densities of VMAT2TBZ. Side chains are shown as sticks and labelled.

Extended Data Fig. 5 Structures of VMAT2 in three distinct states.

a–c, Cryo-EM density maps of VMAT2 complexed with reserpine (a), tetrabenazine (b), and ketanserin (c). The N- and C-domains are colored in red and green, respectively. d–f, Cartoon representation of VMAT2 complexed with reserpine (d), tetrabenazine (e), and ketanserin (f). Reserpine, tetrabenazine, and ketanserin are shown in yellow, orange, and cyan spheres, respectively. The width of the open cavity is defined by the distance between CαG218 and CαP404 in VMAT2RES and CαP42 and CαP316 in VMAT2KET. g–i, Cut-open surface representation of VMAT2RES (g), VMAT2TBZ (h), and VMAT2KET (i) viewed from the cytoplasm side. k–m, Cut-open surface representation of VMAT2RES (k), VMAT2TBZ (l), and VMAT2KET (m) viewed from the lumen side. The opening of the pocket is depicted by black dashed lines in d,f,g,m.

Extended Data Fig. 6 Comparison of the hydrogen bond network of VMAT2 in different states.

a, Relative position of the 5-HT binding pocket and the hydrogen bond network region in VMAT25HT, shown in two views: parallel to the membrane (upper) and perpendicular to the membrane from the lumen side (bottom). The residues involved in the hydrogen bond network are represented as sticks and the 5-HT molecule is shown as spheres. The N- and C-domains are colored in red and green, respectively. b–d, Comparison of the hydrogen bond network of VMAT2 in three different states: 5-HT bound lumen-facing state (b), TBZ bound occluded state (c), and reserpine bound cytosol-facing state (d). The potential hydrogen bonds involved are indicated with dotted lines, and the distances are labelled. e–f, The distance between R189TM4 from the N-domain and E312TM8 from the C-domain in the lumen-facing VMAT25HT (e) and the cytosol-facing VMAT2RES (f). R189TM4 and E312TM8 are shown as sticks. The distances between R189TM4 and E312TM7 are indicated. The N-domain and C-domain of the two structures are colored in red and green, respectively. Serotonin and reserpine molecules are shown as ball sticks.

Extended Data Fig. 7 MD simulation of 5HT binding to the lumen-facing VMAT2.

a, Estimated pKa values for the acidic residues in the VMAT25HT. The blue and orange bars represent the pKa values of the residues on the lumen side and the cytosol side, respectively. The cytoplasmic and luminal pH values are indicated with orange and blue dashed lines, respectively. b, The solvent accessibility of D460 and D262 in the MD system. The green box indicates the water channels through which solvents from the lumen side can access D460 and D262. The enlarged view of these water channels is highlighted on the right. c, 5-HT binding free energy contribution by key residues in the substrate binding pocket. None of E312, D33, D426, and D399 were designated as protonated in the system. Data are the mean ± S.D.; n = 3 experimental replicates. d, The binding free energy contribution by the key residues in the pocket, red indicating a high contribution and white indicating a low contribution. e, Binding free energy of 5-HT to VMAT2 with varying protonation states of E312, D33, D426, and D399. Data are the mean ± S.D.; n = 3 experimental replicates. f, The protein backbone RMSD plots for each replicate of the simulations for the 16 systems in panel e. Prior to calculating the RMSD, all protein structures within each trajectory were aligned with the initial structure of that trajectory using the Least Squares algorithm. Protonated residues are listed for each system, and the RMSD plots are color-coded as blue, gray, and orange for the three replicates. The protein exhibits a stable backbone RMSD plot after 50 ns, characterized by a flat stage with fluctuations of less than 1.0 Å.

Source Data

Extended Data Fig. 8 Sequence alignment of SLC18 transporters.

a, Sequence alignment of human VMATs (SLC18A1-2), rat VMAT2, and human VAChT (SLC18A3). The key residues involved in the binding of 5-HT and the three inhibitors are labelled by icons (triangle for 5-HT, star for KET, circle for reserpine, and hexagon for TBZ). b, Sequence alignment of SLC18 transporters from humans, rats, mice and bovines. The sequences involved in stabilizing the conformations were used for alignment. The dashed lines represent the salt bridges. Acidic, basic, and hydrophobic residues are highlighted in red, blue, and gray, respectively. A motif-A-like sequence of GS(X3)E(X2)GK is labelled above the alignment.

Extended Data Fig. 9 Function and transport model of VMAT2.

a,b, The binding of [3H]reserpine (a) and [3H]tetrabenazine (b) to VMAT2WT and mutants. GFP-expressing cells served as controls. Data are normalized to VMAT2WT and presented as mean values ± s.e.m.; n = 3 biological replicates. One-way ANOVA; ****p < 0.0001. c, Concentration-dependent inhibition of [3H]5-HT uptake by KET in VMAT2WT (blue), VMAT2V232L (orange), and VMAT2L37Y (green). Data are mean values ± s.e.m.; n = 3 biological replicates. Curves were fitted using nonlinear regression. d, The lumen-facing VMAT25HT structure revealed the binding site of 5-HT and the hydrogen-bound network (top). In the lumen-facing conformation, one proton competes with the substrate for binding to D399TM10, thereby facilitating the release of the substrate into the lumen (right). The other proton interacts with the hydrogen bond network, triggering a conformational transition. During the transition to the cytosol-facing conformation, R189TM4 potentially forms an interaction with E312TM7, which is supposed to stabilize the cytosol-facing conformation (bottom). In the cytosol-facing conformation, protons are released to the cytosol, and the substrate binds to the high-affinity site and induces local conformational changes including side chain displacement of R189TM4 and E312TM7, leading to the disruption of this interaction and causing a conformational change in the transporter (left). The N- and C-domains are represented by red and green ellipses, respectively. The critical acidic and basic residues are depicted as sticks. Residues involved in the hydrogen-bond network within the N-domain are marked as red spheres. Hydrogen bonds are illustrated using dashed lines. 5-HT is depicted as black sticks.

Source Data

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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This file contains the uncropped blots relating to Extended Data Figure 1.

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Wu, D., Chen, Q., Yu, Z. et al. Transport and inhibition mechanisms of human VMAT2. Nature 626, 427–434 (2024). https://doi.org/10.1038/s41586-023-06926-4

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