|
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.
|
