title: Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation.
abstract: The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT.
authors.affiliation: From the Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen and.
authors.forename: Hafsteinn
authors.initials: H
authors.lastname: Rannversson
authors.affiliation: From the Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen and.
authors.forename: Pamela
authors.initials: P
authors.lastname: Wilson
authors.affiliation: the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, DK-8240 Risskov, Denmark.
authors.forename: Kristina Birch
authors.initials: KB
authors.lastname: Kristensen
authors.affiliation: the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, DK-8240 Risskov, Denmark.
authors.forename: Steffen
authors.initials: S
authors.lastname: Sinning
authors.affiliation: From the Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen and.
authors.forename: Anders Skov
authors.initials: AS
authors.lastname: Kristensen
authors.affiliation: From the Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen and.
authors.forename: Kristian
authors.initials: K
authors.lastname: Strømgaard
authors.affiliation: From the Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen and jaa@sund.ku.dk.
authors.forename: Jacob
authors.initials: J
authors.lastname: Andersen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology
chemical_list: D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; C493230:SLC6A4 protein, human
keywords: conformational change; molecular pharmacology; neurotransmitter transport; selective serotonin reuptake inhibitor (SSRI); serotonin transporter