title: Neutralizing aspartate 83 modifies substrate translocation of excitatory amino acid transporter 3 (EAAT3) glutamate transporters.
abstract: Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission by removing glutamate from the synaptic cleft into neuronal and glial cells. EAATs are not only secondary active glutamate transporters but also function as anion channels. Gating of EAAT anion channels is tightly coupled to transitions within the glutamate uptake cycle, resulting in Na(+)- and glutamate-dependent anion currents. A point mutation neutralizing a conserved aspartic acid within the intracellular loop close to the end of transmembrane domain 2 was recently shown to modify the substrate dependence of EAAT anion currents. To distinguish whether this mutation affects transitions within the uptake cycle or directly modifies the opening/closing of the anion channel, we used voltage clamp fluorometry. Using three different sites for fluorophore attachment, V120C, M205C, and A430C, we observed time-, voltage-, and substrate-dependent alterations of EAAT3 fluorescence intensities. The voltage and substrate dependence of fluorescence intensities can be described by a 15-state model of the transport cycle in which several states are connected to branching anion channel states. D83A-mediated changes of fluorescence intensities, anion currents, and secondary active transport can be explained by exclusive modifications of substrate translocation rates. In contrast, sole modification of anion channel opening and closing is insufficient to account for all experimental data. We conclude that D83A has direct effects on the glutamate transport cycle and that these effects result in changed anion channel function.
authors.affiliation: Institut für Neurophysiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Hotzy
authors.forename: Jan-Philipp
authors.initials: JP
authors.lastname: Machtens
authors.forename: Christoph
authors.initials: C
authors.lastname: Fahlke
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D050580:Excitatory Amino Acid Transporter 3 / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D008954:Models, Biological; D020125:Mutation, Missense; D014982:Xenopus laevis
chemical_list: D050580:Excitatory Amino Acid Transporter 3; C493400:SLC1A1 protein, human; D001224:Aspartic Acid; D018698:Glutamic Acid