title: A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin.
abstract: Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.
authors.affiliation: From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark.
authors.forename: Henriette
authors.initials: H
authors.lastname: Bjerregaard
authors.affiliation: From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Severinsen
authors.affiliation: From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark.
authors.forename: Saida
authors.initials: S
authors.lastname: Said
authors.affiliation: From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark.
authors.forename: Ove
authors.initials: O
authors.lastname: Wiborg
authors.affiliation: From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark steffen.sinning@clin.au.dk.
authors.forename: Steffen
authors.initials: S
authors.lastname: Sinning
mesh_terms: D002784:Cholesterol / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008563:Membrane Lipids; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D002784:Cholesterol
keywords: Cholesterol; LeuT; Membrane Protein; Monoamine Transporter; Neurotransmitter; Neurotransmitter Transport; Protein Conformation; Serotonin; Serotonin Transporter; Transport