Antidepressant binding site in a bacterial homologue of neurotransmitter transporters

Nature. 2007 Aug 23;448(7156):952-6. doi: 10.1038/nature06038. Epub 2007 Aug 8.

Abstract

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 A above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antidepressive Agents, Tricyclic / metabolism*
  • Antidepressive Agents, Tricyclic / pharmacology
  • Bacteria / chemistry*
  • Binding Sites
  • Clomipramine / metabolism*
  • Clomipramine / pharmacology
  • Crystallography, X-Ray
  • Drug Design
  • Kinetics
  • Models, Molecular
  • Plasma Membrane Neurotransmitter Transport Proteins / antagonists & inhibitors
  • Plasma Membrane Neurotransmitter Transport Proteins / chemistry*
  • Plasma Membrane Neurotransmitter Transport Proteins / metabolism*
  • Protein Binding
  • Protein Conformation
  • Sequence Homology
  • Sodium / metabolism
  • Sodium / pharmacology

Substances

  • Antidepressive Agents, Tricyclic
  • Plasma Membrane Neurotransmitter Transport Proteins
  • Sodium
  • Clomipramine

Associated data

  • PDB/2Q6H
  • PDB/2Q72
  • PDB/2QB4
  • PDB/2QEI