title: Mechanisms of anion conduction by coupled glutamate transporters.
abstract: Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.
authors: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52428 Jülich, Germany; Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany; Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. Electronic address: jan-philipp.machtens@gmx.de.
authors: Jan-Philipp
authors: JP
authors: Machtens
authors: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52428 Jülich, Germany.
authors: Daniel
authors: D
authors: Kortzak
authors: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52428 Jülich, Germany.
authors: Christine
authors: C
authors: Lansche
authors: Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors: Ariane
authors: A
authors: Leinenweber
authors: Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors: Petra
authors: P
authors: Kilian
authors: Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors: Birgit
authors: B
authors: Begemann
authors: School of Engineering, Physics, and Mathematics and Division of Computational Biology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors: Ulrich
authors: U
authors: Zachariae
authors: Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors: David
authors: D
authors: Ewers
authors: Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors: Bert L
authors: BL
authors: de Groot
authors: Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors: Rodolfo
authors: R
authors: Briones
authors: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52428 Jülich, Germany. Electronic address: c.fahlke@fz-juelich.de.
authors: Christoph
authors: C
authors: Fahlke
mesh_terms: D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000378:metabolism*; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D050491:Glutamate Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D043902:Pyrococcus horikoshii / Q000737:chemistry*; D051381:Rats; D016415:Sequence Alignment
chemical_list: D027322:Amino Acid Transport System X-AG; D000838:Anions; D019843:Archaeal Proteins; D050491:Glutamate Plasma Membrane Transport Proteins