Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

Journal Article · Mon Jan 01 00:00:00 EST 2007 · Nature

DOI:https://doi.org/10.1038/nature06038· OSTI ID:930530

Singh, S; Yamashita, A; Gouaux, E

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 {angstrom} 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.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 930530
• Report Number(s): BNL-80557-2008-JA; TRN: US0901409
• Journal Information: Nature, Vol. 448; ISSN 0028-0836
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
74 ATOMIC AND MOLECULAR PHYSICS
ANTICONVULSANTS
ANTIDEPRESSANTS
CATALYSIS
DISSOCIATION
DIURETICS
EPILEPSY
HYPOTHYROIDISM
KINETICS
LEUCINE
LIPIDS
MEMBRANE PROTEINS
NUTRIENTS
SEROTONIN
SODIUM
SODIUM IONS
SUBSTRATES
national synchrotron light source