title: Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter.
abstract: Vesicular glutamate transporters (VGLUTs) accumulate the neurotransmitter glutamate in synaptic vesicles. Transport depends on a V-ATPase-dependent electrochemical proton gradient (ΔμH+) and requires chloride ions, but how chloride acts and how ionic and charge balance is maintained during transport is controversial. Using a reconstitution approach, we used an exogenous proton pump to drive VGLUT-mediated transport either in liposomes containing purified VGLUT1 or in synaptic vesicles fused with proton-pump-containing liposomes. Our data show that chloride stimulation can be induced at both sides of the membrane. Moreover, chloride competes with glutamate at high concentrations. In addition, VGLUT1 possesses a cation binding site capable of binding H+ or K+ ions, allowing for proton antiport or K+ / H+ exchange. We conclude that VGLUTs contain two anion binding sites and one cation binding site, allowing the transporter to adjust to the changing ionic conditions during vesicle filling without being dependent on other transporters or channels.
authors.affiliation: Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Preobraschenski
authors.affiliation: AG Functional Cell Biology, Institute for Integrative Neuroanatomy, Charité University Medicine Berlin, Germany.
authors.forename: Johannes-Friedrich
authors.initials: JF
authors.lastname: Zander
authors.affiliation: Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan; Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: AG Functional Cell Biology, Institute for Integrative Neuroanatomy, Charité University Medicine Berlin, Germany.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Ahnert-Hilger
authors.affiliation: Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany. Electronic address: rjahn@gwdg.de.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Jahn
mesh_terms: D000818:Animals; D000838:Anions; D001665:Binding Sites; D002414:Cations, Monovalent / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D018377:Neurotransmitter Agents / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*; D050598:Vesicular Glutamate Transport Protein 1 / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000838:Anions; D002414:Cations, Monovalent; D002712:Chlorides; D008081:Liposomes; D018377:Neurotransmitter Agents; D050598:Vesicular Glutamate Transport Protein 1; D018698:Glutamic Acid; D011188:Potassium