Abstract
The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT and act as non-competitive inhibitors of transport. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Antidepressive Agents, Second-Generation / metabolism
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Antidepressive Agents, Second-Generation / pharmacology*
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Antidepressive Agents, Tricyclic / metabolism
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Antidepressive Agents, Tricyclic / pharmacology*
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Bacterial Proteins / antagonists & inhibitors
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Binding, Competitive / drug effects
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Biogenic Amines / metabolism*
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Chlorides / metabolism
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Crystallography, X-Ray
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Humans
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Mazindol / metabolism
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Mazindol / pharmacology
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Models, Molecular
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Mutation
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Norepinephrine / metabolism
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Plasma Membrane Neurotransmitter Transport Proteins* / antagonists & inhibitors
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Plasma Membrane Neurotransmitter Transport Proteins* / chemistry
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Plasma Membrane Neurotransmitter Transport Proteins* / genetics
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Plasma Membrane Neurotransmitter Transport Proteins* / metabolism
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Protein Conformation / drug effects
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Recombinant Fusion Proteins / chemistry*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Reproducibility of Results
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Selective Serotonin Reuptake Inhibitors / metabolism
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Selective Serotonin Reuptake Inhibitors / pharmacology*
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Serotonin Plasma Membrane Transport Proteins / chemistry*
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Serotonin Plasma Membrane Transport Proteins / genetics
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Serotonin Plasma Membrane Transport Proteins / metabolism*
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Sertraline / metabolism
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Sertraline / pharmacology
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Sodium / metabolism
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Structure-Activity Relationship
Substances
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Antidepressive Agents, Second-Generation
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Antidepressive Agents, Tricyclic
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Bacterial Proteins
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Biogenic Amines
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Chlorides
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Plasma Membrane Neurotransmitter Transport Proteins
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Recombinant Fusion Proteins
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Serotonin Plasma Membrane Transport Proteins
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Serotonin Uptake Inhibitors
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Sodium
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Mazindol
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Sertraline
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Norepinephrine
Associated data
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PDB/4MM4
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PDB/4MM5
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PDB/4MM6
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PDB/4MM7
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PDB/4MM8
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PDB/4MM9
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PDB/4MMA
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PDB/4MMB
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PDB/4MMC
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PDB/4MMD
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PDB/4MME
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PDB/4MMF