Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2

Nature volume 623, pages 1086–1092 (2023)Cite this article

Subjects

Abstract

Monoamine 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. 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. 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. 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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Data availability

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).

References

  1. Kandel, E. R., Koester, J. D., Mack, S. H. & Siegelbaum, S. A. Principles of Neural Science 6th edn (McGraw Hill Professional, 2021).

  2. Ng, J., Papandreou, A., Heales, S. J. & Kurian, M. A. Monoamine neurotransmitter disorders—clinical advances and future perspectives. Nat. Rev. Neurol. 11, 567–584 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Eiden, L. E. & Weihe, E. VMAT2: a dynamic regulator of brain monoaminergic neuronal function interacting with drugs of abuse. Ann. N.Y. Acad. Sci. 1216, 86–98 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. 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 

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

    Article  CAS  PubMed  Google Scholar 

  7. Davis, M. C., Miller, B. J., Kalsi, J. K., Birkner, T. & Mathis, M. V. Efficient trial design—FDA approval of valbenazine for tardive dyskinesia. N. Engl. J. Med. 376, 2503–2506 (2017).

    Article  PubMed  Google Scholar 

  8. Huntington Study Group. Effect of deutetrabenazine on chorea among patients with Huntington disease: a randomized clinical trial. J. Am. Med. Assoc. 316, 40–50 (2016).

    Article  Google Scholar 

  9. Siddiqui, M., Bhatt, H., Judd, E. K., Oparil, S. & Calhoun, D. A. Reserpine substantially lowers blood pressure in patients with refractory hypertension: a proof-of-concept study. Am. J. Hypertens. 33, 741–747 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Takahashi, N. et al. VMAT2 knockout mice: heterozygotes display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity. Proc. Natl Acad. Sci. USA 94, 9938–9943 (1997).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang, Y.-M. et al. Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine. Neuron 19, 1285–1296 (1997).

    Article  CAS  PubMed  Google Scholar 

  12. Fon, E. A. et al. Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19, 1271–1283 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Saida, K. et al. Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: a study in 42 affected individuals. Genet. Med. 25, 90–102 (2023).

    Article  CAS  PubMed  Google Scholar 

  14. Drew, D. & Boudker, O. Shared molecular mechanisms of membrane transporters. Annu. Rev. Biochem. 85, 543–572 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. 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 

  16. Yan, N. Structural biology of the major facilitator superfamily transporters. Annu. Rev. Biophys. 44, 257–283 (2015).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. 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 

  19. 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 

  20. Weihe, E., Schäfer, 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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 

  23. Nikkhah, A. A brief review on the role of vesicular monoamine transporter2 inhibitors in hyperkinetic movement disorders. Iran. J. Child Neurol. 15, 29–33 (2021).

    PubMed  PubMed Central  Google Scholar 

  24. 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 

  25. Harriott, N. D., Williams, J. P., Smith, E. B., Bozigian, H. P. & Grigoriadis, D. E. VMAT2 inhibitors and the path to Ingrezza (valbenazine). Prog. Med. Chem. 57, 87–111 (2018).

    Article  PubMed  Google Scholar 

  26. Weir, M. R. Reserpine: a new consideration of an old drug for refractory hypertension. Am. J. Hypertens. 33, 708–710 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Fraser, H. S. Reserpine: a tragic victim of myths, marketing, and fashionable prescribing. Clin. Pharmacol. Ther. 60, 368–373 (1996).

    Article  CAS  PubMed  Google Scholar 

  28. Lobay, D. Rauwolfia in the treatment of hypertension. Integr. Med. 14, 40–46 (2015).

    Google Scholar 

  29. 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 

  30. Darchen, F., Scherman, D. & Henry, J. P. Reserpine binding to chromaffin granules suggests the existence of two conformations of the monoamine transporter. Biochemistry 28, 1692–1697 (1989).

    Article  CAS  PubMed  Google Scholar 

  31. Schuldiner, S., Liu, Y. & Edwards, R. H. Reserpine binding to a vesicular amine transporter expressed in Chinese hamster ovary fibroblasts. J. Biol. Chem. 268, 29–34 (1993).

    Article  CAS  PubMed  Google Scholar 

  32. Liu, Y. & Edwards, R. H. The role of vesicular transport proteins in synaptic transmission and neural degeneration. Annu. Rev. Neurosci. 20, 125–156 (1997).

    Article  CAS  PubMed  Google Scholar 

  33. Peter, D., Jimenez, J., Liu, Y., Kim, J. & Edwards, R. H. The chromaffin granule and synaptic vesicle amine transporters differ in substrate recognition and sensitivity to inhibitors. J. Biol. Chem. 269, 7231–7237 (1994).

    Article  CAS  PubMed  Google Scholar 

  34. Støve, S. I., Skjevik, Å. A., Teigen, K. & Martinez, A. Inhibition of VMAT2 by β2-adrenergic agonists, antagonists, and the atypical antipsychotic ziprasidone. Commun. Biol. 5, 1283 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Ugolev, Y., Segal, T., Yaffe, D., Gros, Y. & Schuldiner, S. Identification of conformationally sensitive residues essential for inhibition of vesicular monoamine transport by the noncompetitive inhibitor tetrabenazine. J. Biol. Chem. 288, 32160–32171 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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 

  37. 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 

  38. 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 

  39. 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 

  40. 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 

  41. 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 

  42. 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 

  43. 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 

  44. Chen, H. et al. Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate. Cell 186, 2644–2655 (2023).

    Article  CAS  PubMed  Google Scholar 

  45. Binz, H. K. et al. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22, 575–582 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Hu, G. et al. New fluorescent substrate enables quantitative and high-throughput examination of vesicular monoamine transporter 2 (VMAT2). ACS Chem. Biol. 8, 1947–1954 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Weaver, J. A. & Deupree, J. D. Conditions required for reserpine binding to the catecholamine transporter on chromaffin granule ghosts. Eur. J. Pharmacol. 80, 437–438 (1982).

    Article  CAS  PubMed  Google Scholar 

  48. Stern-Bach, Y., Greenberg-Ofrath, N., Flechner, I. & Schuldiner, S. Identification and purification of a functional amine transporter from bovine chromaffin granules. J. Biol. Chem. 265, 3961–3966 (1990).

    Article  CAS  PubMed  Google Scholar 

  49. 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 

  50. 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 

  51. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    Article  MathSciNet  CAS  PubMed  Google Scholar 

  53. Rana, M. S., Wang, X. & Banerjee, A. An improved strategy for fluorescent tagging of membrane proteins for overexpression and purification in mammalian cells. Biochemistry 57, 6741–6751 (2018).

    Article  CAS  PubMed  Google Scholar 

  54. 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 

  55. 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 

  56. Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  58. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  60. 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 

  61. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  62. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D 74, 519–530 (2018).

    Article  ADS  CAS  Google Scholar 

  63. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  ADS  CAS  Google Scholar 

  64. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    Article  ADS  CAS  PubMed  Google Scholar 

  65. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  66. Black, C. A. et al. Assessing vesicular monoamine transport and toxicity using fluorescent false neurotransmitters. Chem. Res. Toxicol. 34, 1256–1264 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We 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. 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. 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 information

Author notes

  1. These authors contributed equally: Shabareesh Pidathala, Shuyun Liao, Yaxin Dai

Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Shabareesh Pidathala, Yaxin Dai & Chia-Hsueh Lee

  2. State Key Laboratory of Membrane Biology, Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, School of Life Sciences, Peking University, Beijing, China

    Shuyun Liao, Changkun Long & Zhe Zhang

  3. Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Xiao Li & Chi-Lun Chang

Authors
  1. Shabareesh Pidathala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuyun Liao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yaxin Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Changkun Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chi-Lun Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhe Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chia-Hsueh Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.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. 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 authors

Correspondence to Zhe Zhang or Chia-Hsueh Lee.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

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

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Construct design and sequence alignment of VMAT2.

a, Time course of 3H-dopamine uptake in permeabilized VMAT2-transfected or untransfected cells. 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. 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. 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). 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 procedures of VMAT2EM Y418S in complex with reserpine. All procedures were done with cryoSPARC, except for particle polishing which was done with RELION. b, Representative micrograph (left) (out of 25,947 similar micrographs) and 2D class averages (right). 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 = 0.15–0.23 in ChimeraX.

Extended Data Fig. 4 Topology and architecture of VMAT2.

a, Schematic of VMAT2 topology. VMAT2 adopts a canonical MFS fold with 12 TMs. The N-domain (TM1–6) and the C-domain (TMs 7–12) are connected by a cytosolic loop and a short amphipathic helix. Each domain is made up from 3-TM structurally inverted repeats, represented by triangles. b, Architecture of VMAT2. The TBZ-bound structure is shown. For clarity, TBZ is not shown. Left, viewed parallel to the membrane plane. Right, viewed from the luminal side of the vesicle.

Extended Data Fig. 5 Ligands-VMAT2 interaction diagrams.

a, Schematic of TBZ binding interactions. b, Schematic of reserpine binding interactions. c, Schematic of 5-HT binding interactions. Ligand interactions are analyzed using Schrödinger Maestro.

Extended Data Fig. 6 Mutations on the TBZ binding site affect TBZ sensitivity.

a, TBZ sensitivity of VMAT2 variants with alanine mutations in the binding pocket. For each construct, 100% uptake is defined as the average FFN206 signal in the absence of TBZ. Data are shown as mean ± s.d.; n = 3 biological replicates. The IC50 of VMAT2WT is 19 nM, in line with reported values for human and rat VMAT2 (18–37 nM)34,66. The IC50 values of F135A and Y433A are 514.5 and 230 nM, respectively. b, TBZ sensitivity of VMAT2 variants with VMAT1 substitutions in the binding pocket. For each construct, 100% uptake is defined as the average FFN206 signal in the absence of TBZ. Data are shown as mean ± s.d.; n = 3 biological replicates. The IC50 of L37F, I308V, Y433F, and L37F/I308V/Y433F is 82.4, 42.9, 26.1, and 1034 nM, respectively. c, FFN206 uptake of VMAT2 variants. 100% uptake is defined as the average FFN206 signal of VMAT2WT. Data are shown as mean ± s.d.; n = 3 biological replicates. d, Representative images of the FFN206 uptake assay. The FFN206 images have been brightened for enhanced visibility. The raw fluorescent intensities without adjustments are displayed in the lower right. e, TBZ sensitivity of VMAT2WT-transfected or untransfected cells in the FFN206 uptake assay. Data are shown as mean ± s.d.; n = 3 biological replicates.

Extended Data Fig. 7 Cryo-EM analyses of VMAT2 in complex with 5-HT.

a, Summary of image processing procedures of VMAT2EM Y418S in complex with 5-HT. All procedures were done with cryoSPARC, except for particle polishing and the final 3D classification which were done with RELION. b, Representative micrograph (left) (out of 19,023 similar micrographs) and 2D class averages (right). 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 and 5-HT. Map contour level = 0.15–0.19 or 0.13 (TM12) in ChimeraX.

Extended Data Fig. 8 Conformational changes of the luminal gate from the cytoplasm-facing to lumen-facing occluded state.

Structural transition in the luminal gates of VMAT, viewed from the luminal side. The TM7 segment that undergoes secondary structural changes is highlighted in red.

Extended Data Fig. 9 Acidic residues in VMAT2 as potential proton sites.

a, Local interaction clusters of the potential proton sites in TBZ-bound, reserpine-bound, or 5-HT-bound state. b, 3H-dopamine uptake of VMAT2 variants with mutations in local interaction clusters of the potential proton sites. Uptake data of E312 mutants are plotted in Fig. 4e. 100% uptake is defined as the average 3H-dopamine signal of VMAT2WT. Data are shown as mean ± s.d.; n = 3 biological replicates. Average measurements in 10 µM cold reserpine were used for background correction.

Extended Data Table 1 Cryo-EM data collection, processing, and refinement statistics
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Figs. 1–5.

Reporting Summary

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pidathala, S., Liao, S., Dai, Y. et al. Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2. Nature 623, 1086–1092 (2023). https://doi.org/10.1038/s41586-023-06727-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-023-06727-9

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing