title: Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry.
abstract: The serotonin transporter (SERT) regulates neurotransmission by the biogenic monoamine neurotransmitter serotonin (5-HT, 5-hydroxytryptamine) in the central nervous system, and drugs inhibiting SERT are widely used for the treatment of a variety of central nervous system diseases. The conformational dynamics of SERT transport function and inhibition is currently poorly understood. We used voltage-clamp fluorometry to study conformational changes in human SERT (hSERT) during 5-HT transport and inhibitor binding. Cys residues were introduced at 12 positions in hSERT to enable covalent attachment of a rhodamine-based fluorophore. Transport-associated changes in fluorescence from fluorophore-labeled hSERT expressed in Xenopus oocytes could be robustly detected at four positions in hSERT: endogenous Cys109 in the top of transmembrane domain (TM) 1b, Cys substituted for Thr323 in the top of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram. At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations.
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Pella C
authors.initials: PC
authors.lastname: Söderhielm
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jacob
authors.initials: J
authors.lastname: Andersen
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Lachlan
authors.initials: L
authors.lastname: Munro
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Anne T
authors.initials: AT
authors.lastname: Nielsen
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark ask@sund.ku.dk.
authors.forename: Anders S
authors.identifier: http://orcid.org/0000-0001-8955-6790
authors.initials: AS
authors.lastname: Kristensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D005260:Female; D005470:Fluorometry / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques / Q000379:methods; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017367:Selective Serotonin Reuptake Inhibitors / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology; D014982:Xenopus laevis
chemical_list: D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; C493230:SLC6A4 protein, human