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LeuT-desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake

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LeuT-desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake

Zheng Zhou et al. Science.

Abstract

Tricyclic antidepressants exert their pharmacological effect-inhibiting the reuptake of serotonin, norepinephrine, and dopamine-by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 angstroms of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters.

Figures

Fig. 1
Fig. 1
Binding of various antidepressants and other compounds to LeuT. (A). Affinity of desipramine, imipramine, fluoxetine and nortriptyline. Data are shown as mean ± SEM (vertical bars, N = 3). The IC50 values for inhibition [3H]leucine binding to LeuT were 80±5, 244±12, 858±64 and 75±14μM, respectively. (B). Mechanism of inhibition of [3H]leucine binding to LeuT by desipramine and nortriptyline. The plot shows that desipramine and nortriptyline are not competitive inhibitors of leucine binding to LeuT. A representative experiment is shown (N = 3).
Fig. 2
Fig. 2
Structure of the LeuT-desipramine complex and molecular mechanism of LeuT inhibition by desipramine. (A). Structure shown as ribbon diagram viewed from within the membrane plane. An Fo-Fc map contoured at 3 σ is superimposed on the structural model. The EL4 hairpin is colored green, and the rest of the protein pink. The helices TM6 and TM11 are removed for clarity. (B). 2Fo-Fc map contoured at 1 σ showing the desipramine binding site in LeuT, viewed from within the membrane plane. Residues R30, Y108 and F253 form the extracellular gate that separates the leucine substrate from the bound desipramine. (C). Local structural changes of LeuT induced by desipramine binding. The structure with desipramine bound is shown in pink and green, without desipramine bound in cyan and blue. Upon desipramine binding, the side chain of R30 rotates toward D404 and forms a salt bridge with the latter, and the EL4 hairpin, along with A319 and F320, is pushed toward to the extracellular space. (D). Molecular contacts between LeuT and the bound desipramine molecule. The chemical structure of desipramine is shown together with LeuT residues that are in direct contact with the drug. Residues from the EL4 hairpin are shown in the green box, while residues from the rest of the protein are shown in pink boxes.
Fig. 3
Fig. 3
Homology models and electrostatic surface potential of desipramine-binding sites in human SERT, NET and DAT. (A). Desipramine-binding site in the LeuT-desipamine crystal structure. Homology model and electrostatic surface potential of desipramine-binding site in (B). hSERT, (C). hNET and (D). hDAT, viewed from within the membrane plane. The equivalent residues of those in LeuT that are in direct contact with desipramine are indicated.
Fig. 4
Fig. 4
Measurements of inhibition of [3H]dopamine and [3H]serotonin uptake by desipramine for human DAT and SERT mutants in HEK-293 cells. Wild-type (WT) hDAT, hSERT and various mutant constructs are denoted on the left. Results are expressed as percent of uptake measured with vehicle (% control). Data are shown as mean ± SEM (horizontal bars, N = 3–5). Asterisk indicates P < 0.05 (compared with corresponding WT at the same concentration of desipramine, one-way analysis of variance followed by Dunnett multiple comparison test for hDAT, and Student’s t-test for hSERT).

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