The serotonin transporter (SERT) terminates serotonergic signaling through the sodium and chloride dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signaling. Here we report x-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8, and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and TMs 1, 6, 10, and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT and provide blueprints for future drug design.

SUMMARY The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium-and chloride-dependent recycling of serotonin into presynaptic neurons1–3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors (SSRIs), small molecules that competitively block substrate binding, prolonging neurotransmitter action2,4. The dopamine and norepinephrine transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. Cocaine and amphetamines inhibit or modulate the transport activities of NSSs2,3 and genetic variants are associated with multiple neuropsychiatric disorders including attention deficit hyperactivity disorder, autism, and bipolar disorder2,5. Studies of bacterial NSS homologs, including LeuT, have shown how transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane1,6–12. However, the conformational changes associated with transport in eukaryotic NSSs remain obscure. To elucidate structure-based mechanisms for transport in SERT, we turned to complexes with ibogaine, a centuries old hallucinogenic natural product with psychoactive and anti-addictive properties13,14 (Fig. 1a). Interestingly, ibogaine displays non-competitive inhibition of transport, yet it exhibits competitive binding toward SSRIs15,16. Here we report cryo-EM structures of SERT-ibogaine complexes captured in outward-open, occluded, and inward-open conformations. Ibogaine binds to the central binding site and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and partial unwinding of TM5, which together create a permeation pathway enabling substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from outward-open to the inward-open conformations, providing insight into the mechanism of neurotransmitter transport and ibogaine inhibition.

P 4 -ATPases comprise a relatively new subfamily of P-type ATPases implicated in the energy-dependent translocation of aminophospholipids across cell membranes. In this study, we report on the localization and functional properties of Atp8a2, a member of the P 4 -ATPase subfamily that has not been studied previously. Reverse transcription-PCR revealed high expression of atp8a2 mRNA in the retina and testis. Within the retina, immunofluorescence microscopy and subcellular fractionation studies localized Atp8a2 to outer segment disc membranes of rod and cone photoreceptor cells. Atp8a2 purified from photoreceptor outer segments by immunoaffinity chromatography exhibited ATPase activity that was stimulated by phosphatidylserine and to a lesser degree phosphatidylethanolamine but not by phosphatidylcholine or other membrane lipids. Purified Atp8a2 was reconstituted into liposomes containing fluorescent-labeled phosphatidylserine to measure the ability of Atp8a2 to flip phosphatidylserine across the lipid bilayer. Fluorescence measurements showed that Atp8a2 flipped fluorescent-labeled phosphatidylserine from the inner leaflet of liposomes (equivalent to the exocytoplasmic leaflet of cell membranes) to the outer leaflet (equivalent to cytoplasmic leaflet) in an ATP-dependent manner. Our studies provide the first direct biochemical evidence that purified P 4 -ATPases can translocate aminophospholipids across membranes and further implicates Atp8a2 in the generation and maintenance of phosphatidylserine asymmetry in photoreceptor disc membranes.

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