title: An intracellular interaction network regulates conformational transitions in the dopamine transporter.
abstract: Neurotransmitter:sodium symporters (NSS)(1) mediate sodium-dependent reuptake of neurotransmitters from the synaptic cleft and are targets for many psychoactive drugs. The crystal structure of the prokaryotic NSS protein, LeuT, was recently solved at high resolution; however, the mechanistic details of regulation of the permeation pathway in this class of proteins remain unknown. Here we combine computational modeling and experimental probing in the dopamine transporter (DAT) to demonstrate the functional importance of a conserved intracellular interaction network. Our data suggest that a salt bridge between Arg-60 in the N terminus close to the cytoplasmic end of transmembrane segment (TM) 1 and Asp-436 at the cytoplasmic end of TM8 is stabilized by a cation-pi interaction between Arg-60 and Tyr-335 at the cytoplasmic end of TM6. Computational probing illustrates how the interactions may determine the flexibility of the permeation pathway, and mutagenesis within the network and results from assays of transport, as well as the state-dependent accessibility of a substituted cysteine in TM3, support the role of this network in regulating access between the substrate binding site and the intracellular milieu. The mechanism that emerges from these findings may be unique to the NSS family, where the local disruption of ionic interactions modulates the transition of the transporter between the outward- and inward-facing conformations.
authors.affiliation: Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Kniazeff
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Claus J
authors.initials: CJ
authors.lastname: Loland
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018377:Neurotransmitter Agents / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D018377:Neurotransmitter Agents; D012964:Sodium