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 competitive12,13 and noncompetitive14,15 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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We thank D. C. LaPorte, R. D. Blakely, and G. Rudnick for insightful comments; M. Post for information on TCA crystallographic numbering; C. Piscitelli for assistance with LeuT purification and electrostatic calculations; L. Vaskalis for Fig. 4a, d; and the staff at beamlines X26C and X29A of the National Synchrotron Light Source and beamline 8.2.2 of the Advanced Light Source for assistance with X-ray data collection. S.K.S. was supported by an individual NIH National Research Service Award. This work was supported by the NIH. E.G. is an investigator with the Howard Hughes Medical Institute.
Coordinates and structure factors for the alanine–sodium–clomipramine and the leucine–sodium–clomipramine, –imipramine and –desipramine complexes have been deposited in the Protein Data Bank under accession codes 2QEI, 2Q6H, 2Q72 and 2QB4, respectively.
This file contains Supplementary Figures S1-S4 with Legends, Supplementary Tables 1 and 2, Supplementary Methods and Supplementary Notes with additional references.
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Scientific Reports (2016)