title: Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.
abstract: Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.
authors.affiliation: From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Christian B
authors.initials: CB
authors.lastname: Billesbølle
authors.affiliation: From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Mie B
authors.initials: MB
authors.lastname: Krüger
authors.affiliation: Department of Physiology and Biophysics and The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, New York, New York 10065.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: Center for Molecular Recognition and Departments of Psychiatry and Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032.
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.affiliation: Department of Physiology and Biophysics and.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Physiology and Biophysics and.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Stolzenberg
authors.affiliation: From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Kniazeff
authors.affiliation: From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Kamil
authors.initials: K
authors.lastname: Gotfryd
authors.affiliation: From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Jonas S
authors.initials: JS
authors.lastname: Mortensen
authors.affiliation: Center for Molecular Recognition and Departments of Psychiatry and Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032 Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, and.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.affiliation: Department of Physiology and Biophysics and The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, Cornell University, New York, New York 10065.
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.affiliation: From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Claus J
authors.initials: CJ
authors.lastname: Loland
authors.affiliation: From the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark, gether@sund.ku.dk.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000737:chemistry* / Q000378:metabolism; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D011994:Recombinant Proteins; D012235:Rhodamines; C005358:tetramethylrhodamine; D012964:Sodium; D009638:Norepinephrine
keywords: amino acid transport; conformational change; dopamine transporter; fluorescence quenching; fluorescence spectroscopy; gating; mechanisms of membrane transport; membrane protein; monoamine transporter; neurotransmitter transport