title: Intersubunit interactions in EAAT4 glutamate transporters.
abstract: Excitatory amino acid transporters (EAATs) play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian CNS. A functional transporter is assembled as oligomer consisting of three subunits, each of which appears to transport glutamate independently from the neighboring subunits. EAATs do not only sustain a secondary-active glutamate transport but also function as anion channel. We here address the question whether intersubunit interactions play a role in pore-mediated anion conduction. We expressed a neuronal isoform, EAAT4, heterologously in Xenopus oocytes and mammalian cells and measured glutamate flux and anion currents under various concentrations of Na+ and glutamate. EAAT4 anion channels are active in the absence of both substrates, and increasing concentrations activate EAAT4 anion currents with a sigmoidal concentration dependence. Because only one glutamate molecule is cotransported per uptake cycle, the cooperativity between glutamate binding sites most likely arises from an interaction between different carrier domains. This interaction is modified by two point mutations close to the putative glutamate binding site, G464S and Q467S. Both mutations alter the dissociation constants and Hill coefficient of the substrate dependence of anion currents, leaving the concentration dependence of glutamate uptake unaffected. Our results demonstrate that glutamate carriers cooperatively interact during anion channel activation.
authors.affiliation: Abteilung Neurophysiologie, Medizinische Hochschule, 30625 Hannover, Germany.
authors.forename: Delany
authors.initials: D
authors.lastname: Torres-Salazar
authors.forename: Christoph
authors.initials: C
authors.lastname: Fahlke
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D050581:Excitatory Amino Acid Transporter 4 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D014981:Xenopus
chemical_list: D050581:Excitatory Amino Acid Transporter 4; D007473:Ion Channels; D021122:Protein Subunits; C493389:Slc1a6 protein, rat; D018698:Glutamic Acid; D012964:Sodium