Structures of cocaine and human dopamine transporters revealed, unlocking insights into addiction

A recent study published in the journal NatureResearchers have published the molecular structure of the dopamine transporter (DAT) complexed with cocaine.

Cocaine is highly addictive and, if abused, can exacerbate psychiatric disorders. Cocaine exerts its effects by binding to monoamine transporters, but inhibition of the DAT underlies its addictive and rewarding properties. Currently, there are no human DAT (hDAT) structures. However, there are known mechanisms underlying the inhibition of solute carrier 6 (SLC6) family members and Drosophila melange DAT (dDAT) provides structural information.

study: Structure of the human dopamine transporter complexed with cocaineImage credit: Naeblys / Shutterstock

These reveal a consensus structure of 12 transmembrane helices (TM1–TM12) within a pseudosymmetric fold (the LeuT fold), with the substrate-binding site located in the core. In all SLC6 family members, the central substrate-binding site is accessible from either the intracellular or extracellular membrane face, and the energy for substrate transport is provided by sodium ions (Na⁺) cotransport. Most of them transport chloride ions (Cl^–), Cl^– Whether it will be combined/transported is unknown.

chlorine^– Binding to the DAT may allosterically reduce the affinity of the second sodium site (Na2), which may be required for isomerization of the transporter to an inward-facing state before substrate release and subsequent transition to an outward-facing open conformation. In the dDAT-cocaine complex, cocaine binds to the central substrate-binding site. However, dDAT has greater sequence homology to the human noradrenaline transporter (hNET) than does hDAT.

Research and Results

In this study, the researchers performed cryo-electron microscopy (cryo-EM) reconstructions of cocaine-bound hDAT. First, they used Expi293F cells to express Twin-Strep-tagged hDAT (hDAT^twin) was purified in glycodiosgenin (GDN) micelles. The tag reduced overall dopamine uptake and hDAT^twin When transiently expressed in COS7 cells, expression was lower than that of wild-type hDAT.

Next, the structure of hDAT^twin The structure of the cocaine-bound protein was solved at 2.66 Å resolution by cryo-electron microscopy. The protein had an open outward conformation with 12 TMs forming a LeuT fold. TMs 11 and 12 were located outside the core domain, and TM12 was bent almost halfway. Extracellular loop 4 (EL4) formed a hairpin-like structure that partially covered the exit from the central binding site (S1).

The overall structure was similar to that of the human serotonin transporter (hSERT) and dDAT facing outward. The researchers investigated whether the purification procedure affected the overall structure. They measured binding to cocaine analogues and found no difference in affinity for hDAT when expressed in COS7 cells and purified in detergent micelles.

Similarly, there was no significant difference in affinity for cocaine.^twin Reconstituted into proteoliposomes containing Na⁺ The gradient and time-dependent dopamine uptake were measured. Specific uptake was measured and hDAT^twin Even after purification, it is capable of adopting all the conformational states required for substrate transport.

Cocaine inhibited transport activity with an inhibition constant similar to that in COS7 cells. The cocaine molecule was located in the hDAT.^twin The core is within interaction distance of the S1 residue. The S1 site has three subsites (A–C). The tertiary amine of the tropane ring of cocaine formed an ionic interaction with Asp79 in subsite A. In addition, cocaine bound to subsite B through the benzene ring and formed an edge-to-face π–π interaction with Tyr156.

Three water molecules formed hydrogen bonds around the benzene ring, two of which are absent in the dDAT structure. Furthermore, only the chloride and Na2 sites were occupied, while the first sodium site (Na1) either collapsed upon cocaine binding or was not established at all. The difference with hDAT is that^twin -Cocaine and porcine SERT (pSERT)- and dDAT-cocaine complexes were in the EL4 position.

In the pSERT and dDAT complexes, the loops occupied similar positions, whereas in the hDAT^twin In the complex, TM1b was displaced by 3.7 A and Trp84 Cα was displaced by 2.2 A. Furthermore, a mantle of non-protein density was observed at the interface between GDN micelles and hDAT.^twin-cocaine complexes have not been reported in cryo-EM reconstructions of monoamine transporters. These densities were heterogeneous and primarily present in the periphery of the hDAT.^twin.

Four of these densities were comparable in size and shape. Three were located at previously reported cholesterol sites (CHOL1, CHOL2, and CHOL3), and one (CHOL4) was beneath CHOL3. The researchers fitted cholesteryl hemisuccinate molecules to CHOL2 and cholesterol molecules to the CHOL3 density. They also fitted a cholesterol model to the CHOL4 density. Molecular dynamics simulations indicated the presence of a CHOL4 analogue in hNETs.

Conclusion

Together, the researchers solved a cryo-electron microscopy structure of the hDAT-cocaine complex at 2.66 Å. The structure contains one Na⁺ It exists at the Na2 site and has a density equivalent to that of Cl.^– “The stimulant and addictive properties of cocaine have been detected at the chloride site. Remarkably, this structure was solved without the use of molecular criteria that are often used in cryo-electron microscopy of small membrane proteins. These findings may improve our understanding of the stimulant and addictive properties of cocaine and aid in the development of medicines that target addiction.”