title: Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1.
abstract: Glutamate transporters actively take up glutamate into the cell, driven by the co-transport of sodium ions down their transmembrane concentration gradient. It was proposed that glutamate binds to its binding site and is subsequently transported across the membrane in the negatively charged form. With the glutamate binding site being located partially within the membrane domain, the possibility has to be considered that glutamate binding is dependent on the transmembrane potential and, thus, is electrogenic. Experiments presented in this report test this possibility. Rapid application of glutamate to the wild-type glutamate transporter subtype EAAC1 (excitatory amino acid carrier 1) through photo-release from caged glutamate generated a transient inward current, as expected for the electrogenic inward movement of co-transported Na(+) In contrast, glutamate application to a transporter with the mutation A334E induced transient outward current, consistent with movement of negatively charged glutamate into its binding site within the dielectric of the membrane. These results are in agreement with electrostatic calculations, predicting a valence for glutamate binding of -0.27. Control experiments further validate and rule out other possible explanations for the transient outward current. Electrogenic glutamate binding can be isolated in the mutant glutamate transporter because reactions, such as glutamate translocation and/or Na(+) binding to the glutamate-bound state, are inhibited by the A334E substitution. Electrogenic glutamate binding has to be considered together with other voltage-dependent partial reactions to cooperatively determine the voltage dependence of steady-state glutamate uptake and glutamate buffering at the synapse.
authors.affiliation: From the Department of Chemistry Binghamton University, Binghamton, New York 13902 and.
authors.forename: Rose
authors.initials: R
authors.lastname: Tanui
authors.affiliation: From the Department of Chemistry Binghamton University, Binghamton, New York 13902 and.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Tao
authors.affiliation: the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada Faculty of Medicine, Hebrew University, Jerusalem 91120, Israel.
authors.forename: Nechama
authors.initials: N
authors.lastname: Silverstein
authors.affiliation: the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada Faculty of Medicine, Hebrew University, Jerusalem 91120, Israel.
authors.forename: Baruch
authors.initials: B
authors.lastname: Kanner
authors.affiliation: From the Department of Chemistry Binghamton University, Binghamton, New York 13902 and cgrewer@binghamton.edu.
authors.forename: Christof
authors.initials: C
authors.lastname: Grewer
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D004594:Electrophysiology; D050580:Excitatory Amino Acid Transporter 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D050580:Excitatory Amino Acid Transporter 3; C493400:SLC1A1 protein, human; D018698:Glutamic Acid; D012964:Sodium
keywords: electrophysiology; molecular dynamics; mutagenesis; neurotransmitter transport; pre-steady-state kinetics