1. The Vollum Institute and,
2. Howard Hughes Medical Institute, Oregon Health and Science University, 3181 S.W. Sam Jackson Road, Portland, Oregon 97239, USA
3. Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USA
4. Present address: RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo, Hyogo 679-5148, Japan.

Correspondence to: Eric Gouaux^1,2 Correspondence and requests for materials should be addressed to E.G. (Email: gouauxe@ohsu.edu).

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 bilayer¹. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption², congenital hypothyroidism³, Bartter's syndrome⁴, epilepsy⁵, depression⁶, autism⁷ and obsessive-compulsive disorder⁸. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics⁹, anticonvulsants¹⁰ and antidepressants¹¹, 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^{12, 13} and noncompetitive^{14, 15} 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 site¹⁶. 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 Å 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.

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