Abstract
The dopamine transporter has a crucial role in regulation of dopaminergic neurotransmission by uptake of dopamine into neurons and contributes to the abuse potential of psychomotor stimulants1,2,3. Despite decades of study, the structure, substrate binding, conformational transitions and drug-binding poses of human dopamine transporter remain unknown. Here we report structures of the human dopamine transporter in its apo state, and in complex with the substrate dopamine, the attention deficit hyperactivity disorder drug methylphenidate, and the dopamine-uptake inhibitors GBR12909 and benztropine. The dopamine-bound structure in the occluded state precisely illustrates the binding position of dopamine and associated ions. The structures bound to drugs are captured in outward-facing or inward-facing states, illuminating distinct binding modes and conformational transitions during substrate transport. Unlike the outward-facing state, which is stabilized by cocaine, GBR12909 and benztropine stabilize the dopamine transporter in the inward-facing state, revealing previously unseen drug-binding poses and providing insights into how they counteract the effects of cocaine. This study establishes a framework for understanding the functioning of the human dopamine transporter and developing therapeutic interventions for dopamine transporter-related disorders and cocaine addiction.
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Data availability
Three-dimensional cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-38850 (hDATapo), EMD-38851 (hDATDA), EMD-38852 (hDATBZT), EMD-38853 (hDATGBR) and EMD-38854 (hDATMPH). Coordinates have been deposited in Protein Data Bank under accession codes 8Y2C (hDATapo), 8Y2D (hDATDA), 8Y2E (hDATBZT), 8Y2F (hDATGBR) and 8Y2G (hDATMPH). Source data are provided with this paper.
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Acknowledgements
The authors thank B. Xu for support with cryo-EM data collection; H. Zhang for support with radioactivity experiments; and Y. Wu for research assistance. This work is funded by 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.) and the National Natural Science Foundation of China (grant no. 92157102 to Y.Z.).
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Y.Z. conceived and supervised the project. X.W. carried out molecular cloning and Y.L., P.Y., J.H., K.H. and Y.Q. made the mutant constructs. Y.L., X.W. and N.L. expressed and purified proteins and prepared samples for cryo-EM study. Y.M. and T.H. performed functional assays. J.Z. and R.L. carried out cryo-EM data collection. Y.L., Y.Z. and R.L. processed the cryo-EM data. Y.L., Q.B., T.H. and Y.W. built and refined the atomic model. Y.L., P.Y. and Y.Z. analysed the structures and prepared the figures. Y.Z. and Y.L. wrote and revised the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Functional characterization and purification of hDAT.
a, Representative HPLC profiles of full-length hDAT (DATWT) and hDATEM in the LMNG detergent buffer. b, Kinetic DA uptake activities of DATEM as compared to DATWT. Data represented as mean ± S.E.M. (n = 3 biological independent experiments) and curve has been normalized to the surface expression level (c). The Km and Vmax value are 0.66 ± 0.16 μM, 2243 ± 108.8 CPM and 0.83 ± 0.43 μM, 1957 ± 214.9 CPM for DATWT and DATEM, respectively. There is no significant difference in the comparison of Km (P = 0.7328) and Vmax (P = 0.3003) using two-sided unpaired t-test. c, Cell surface biotinylation of DATWT and DATEM in HEK293F cells. The samples of whole cell lysate (lane 1) and surface protein (lane 2–4) are labeled with biotin and recovered using streptavidin beads. No protein was detected in unbiotinylated cell lysate sample (lane 5). The experiments were repeated three times, yielding similar results. d-f, Concentration-inhibition curves of [3H]DA uptake by MPH (d), GBR12909 (e) and BZT (f) for DATWT and DATEM. Data represent the mean ± S.E.M. (n = 3 biological independent experiments). The [3H]DA uptake is normalized to the value measured in the absence of investigated drugs. There is no significant difference between the IC50 values of MPH, GBR12909 and BZT for DATWT and DATEM, supported by P-values of 0.4723, 0.6728 and 0.9161, respectively, calculated by two-sided unpaired t-test. g, The melting curve of DATEM in 0.1% LMNG in comparison to DATEM in 0.1% DDM. h, Size exclusion chromatography (SEC) profiles of purified DATEM in the LMNG buffer and in MSP1D1E3 nanodiscs. i, The Coomassie-stained SDS-PAGE gel of the DATEM–MSP1D1E3 cryo-EM samples. The experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 Representative structural determination of hDATDA.
a, Cryo-EM data processing flowchart of hDATDA. Representative micrograph of hDATDA was shown with the white scale bar equaling 100 nm. Briefly, 3,862,637 particles were picked from 6,138 collected movies. After several rounds of 2D classification, an initial map was reconstructed by ab-initio reconstruction. The initial map then severed as a reference for heterogeneous refinement of all particles. Following two more rounds of ab-initio reconstruction and non-uniform refinement, the map resolution was improved to 3.06 Å. The final map (2.80 Å) was obtained through 3D classification without alignment and local refinement. Particles and resolutions at each stage of data processing are labeled. For details, refer to ‘Cryo-EM data analysis’ section in the Methods. b, Local resolution distribution of the final map of the hDATDA. c, Euler angle distribution of the 3D reconstruction for hDATDA. d, Gold-standard Fourier Shell Correlations (FSC) curves of model versus map (black), unmasked map (blue) and masked map (red), marked with resolutions corresponding to FSC = 0.5 and 0.143. e, Cryo-EM densities superimposed on atomic model of the 12 transmembrane helices of hDATDA.
Extended Data Fig. 3 Structural analysis of C-terminal tails among hDAT, dDAT, GAT1 and GlyT1.
a, Interactions between the C-terminal tail and TMs of hDATDA. Crucial residues are shown as sticks and labeled. b-d, Structural comparison of the C-terminal tails between hDAT and dDAT (b), hDAT and GAT1 (c), hDAT and GlyT1 (d). hDATapo, dDAT (PDB: 4XP1), GAT1 (PDB: 7Y7V), and GlyT1 (PDB: 8WFJ) are represented by yellow, gray, cyan, and deep blue cylinders and cartoons, respectively.
Extended Data Fig. 4 Transport activity and surface expression of DATWT and its mutants.
a, Quantitation of the transport activity in DATWT and its mutants using the [3H]DA uptake assay. The transport activity of DATWT and its mutants is normalized to their respective expression level and the transport activity of DATWT was set to 100%. The uptake duration is set to 2 min to ensure that the uptake occurs within the linear range. Data represent the mean ± S.E.M. n = 3 biologically independent experiments. b, Representative western blots of the surface DATWT and its mutants. Biotinylated Na+/K+ ATPase on the cell surface was used as the loading control. Experiment was repeated three times independently with similar results. For gel source data, see Supplementary Fig. 1. c, Densitometric quantification of surface expression for DATWT and its mutants. The gels derived from the same experiment were processed in parallel. The surface expression of DATWT and its mutants was first normalized to Na+/K+ ATPase and then to the expression of DATWT. Each circular symbol represents an individual data point. Data are represented as mean ± S.E.M. n = 3 biologically independent experiments.
Extended Data Fig. 5 Structural comparison of hDATDA and dDATDA.
a, Structural alignment of hDATDA and dDATDA (PDB: 4XP1) based on the scaffold domain. The transmembrane helixes (TMs) of hDATDA and dDATDA are depicted as violet and gray cylinders, respectively. Conformational changes from dDATDA to hDATDA are highlighted with red arrows. b-c, Superposition of the substrate binding sites of hDATDA and dDATDA. Key residues involved in substrate binding are visualized as sticks. DAs are shown as dark blue and gray sticks in hDATDA and dDATDA, respectively. Water molecules are represented as red spheres.
Extended Data Fig. 6 Sequence alignment of hDAT, hSERT, hNET and dDAT.
Sequence alignment of hDAT (UniProt Q01959), hSERT (UniProt P31645), hNET (UniProt P23975) and dDAT (UniProt Q7K4Y6). Transmembrane (TM) helices, intracellular loops (IL) and extracellular loops (EL) of hDAT are indicated above the alignment. Highly conserved amino acids are highlighted with grey shadows, while positively and negatively charged amino acids are shaded in blue and red, respectively. Residues involved in the interactions with Na+ and Cl− are labeled above the hDAT sequence.
Extended Data Fig. 7 Structural comparison of DAT at outward-facing and occluded conformational states.
a, Structural comparisons between hDATDA (violet) and hDATMPH (green). TM1a, TM1b, and TM10 of hDATDA and hDATMPH are depicted as cartoons, while other transmembrane helices are shown as cylinders. Residues involve in the interaction between TM1b and TM10 are illustrated as sticks, and the interaction region is outlined in a dashed box. b-c, Enlarged view of the interactions of TM1b and TM10 of hDATDA in the occluded (b) and outward-facing (c) conformations. Key residues and interactions are represented by sticks and dashed lines, respectively. The distances of Cα between the D476 and R85 are indicated by dashed lines and labeled. d, Cryo-EM density and model of the sodium and chloride binding pocket. e-f, Analysis of Na1 (e), Na2 (f), and Cl (e) coordination in hDATMPH. The key residues for interaction are presented as sticks and labeled. g, Analysis of Na1, Na2, and Cl coordination in hDAT and dDAT. The key residues for interaction are presented as sticks and labeled. h, Comparison of the binding sites of Na1, Na2, and Cl between hDATDA and hDATMPH. TM1 and TM6 are shown as cartoons. The changes are labeled by red arrows. i-k, Expanded comparison view of the binding sites of Na1 (i), Na2 (j), and Cl (k) between hDATDA and hDATMPH.
Extended Data Fig. 8 Structural analysis of the benztropine binding site.
a-b, Structural superposition of hDATBZT and cocaine bound dDAT (dDATCOC, PDB: 4XP4). The structures of hDATBZT and dDATCOC are colored in blue and green, respectively. Benztropine and cocaine are depicted as pink and green sticks, respectively. The surface of hDATBZT is colored in gray. c-d, Comparison of the hDATBZT (blue) and hDATDA (violet). TM1 and TM6 are represented by colored helixs, and others are depicted as cylinders. Benztropine and residues are shown as sticks and labeled. e-f, Alignment of inward-facing structures of hDATBZT (blue) and hSERT5HT (gray, PDB: 7LI9). Benztropine and key residues are depicted as sticks and labeled.
Extended Data Fig. 9 Schematic diagrams for structures of dDAT and hDAT.
a, The structure of dDAT is stabilized in outward-facing conformation. TM1 and TM6 are highlighted in yellow, while TM5, TM10, TM12, and the C-terminal tail are colored in gray. b, The structures of hDAT are determined in outward-facing, occluded, and inward-facing conformations. TM1 and TM6 are depicted in brown and purple, respectively, while TM5, TM10, TM12, and the C-terminal tail are colored in cyan. Substrates and inhibitors are depicted as sticks. Sodium and chloride ions are represented as spheres.
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Li, Y., Wang, X., Meng, Y. et al. Dopamine reuptake and inhibitory mechanisms in human dopamine transporter. Nature 632, 686–694 (2024). https://doi.org/10.1038/s41586-024-07796-0
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DOI: https://doi.org/10.1038/s41586-024-07796-0
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