LeuT-Desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake

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 Å 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. Na + /Cl − -dependent neurotransmitter transporters for serotonin (SERT), norepinephrine (NET) and dopamine (DAT) in the presynaptic plasma membrane terminate neuronal signal transmission in the central nervous system through a reuptake mechanism (1-6). These systems have been shown to modulate mood, emotion, sleep and appetite (7). Depression, arguably the most prevalent psychiatric disorder, is directly associated with perturbation of serotonergic neurotransmission (8, 9), and drugs blocking serotonin reuptake have been used successfully for its treatment. One class of these drugs, tricyclic antidepressants (TCAs) such as desipramine and imipramine, binds to serotonin and norepinephrine transporters with affinities of nanomolar to tens of nanomolar concentrations and blocks transport activity (10). The response rate of patients to TCAs is typically 60-70% (11). More recently, highly selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac) have also been developed and are increasingly prescribed to treat depression (12). The molecular The human SERT, DAT and NET proteins all belong to a family of transporters for amino acids and their derivatives, the Neurotransmitter:Sodium Symporter (NSS) family (2)(3)(4)(5)14). Whilst the dopamine transporters from human, bovine or rat are inhibited by TCAs at a K i of micromolar concentrations, the DAT proteins from C. elegans (15) and D. melanogaster (16) are inhibited by TCAs at a K i of nanomolar and sub-micromolar concentrations, respectively (17). As bacterial NSS proteins share up to 30 % sequence identity with human SERT and NET as well as worm and fly DATs, we hypothesized that bacterial NSS proteins also possess high binding affinity to TCAs and could provide opportunities for studying proteindrug interactions. We therefore chose a bacterial NSS protein, the leucine transporter (LeuT) from Aquifex aeolicus, to study the molecular mechanism of neurotransmitter transporter binding to TCAs (18). LeuT shares 2...

Mechanism for alternating access in neurotransmitter transporters

“…Transporters for the amine neurotransmitters dopamine, norepinephrine, and serotonin are targets for important therapeutic agents and drugs of abuse, including antidepressants, cocaine, and amphetamines. The structural basis for the function of these proteins and their interactions with drugs has been a topic of intense recent interest (16,17).…”

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

“…Solute translocation is driven by a Na ϩ -and Cl Ϫ -dependent alternating access mechanism in which the proteins cycle through outwardly and inwardly facing conformations that bind and release substrates on opposite sides of the membrane (14). Major insights into structural mechanisms of NSS proteins have come from the homologous prokaryotic leucine transporter (LeuT) from Aquifex aeolicus, which has been crystallized in conformations corresponding to outwardly facing, occluded, inwardly facing, and inhibitor-bound forms (15)(16)(17)(18)(19)(20). These structures reveal that outwardly facing transporters possess an aqueously accessible external vestibule that opens to a compact central/primary substrate-binding site (S1) composed of residues from TM domains 1, 3, 6, and 8 (15).…”

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

“…Earlier computational studies suggested that APC members possess the so called "5 ϩ 5"-fold (24), commonly found in the crystal struc-tures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities (25)(26)(27)(28). By using a sensitive homology threading approach, a three-dimensional model of PrnB was previously generated, using as template the crystal structure of the LeuT transporter of Aquifex aeolicus (23).…”