title: A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters.
abstract: In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.
authors: Department of Biochemistry and Molecular Biology, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.
authors: Noa
authors: N
authors: Rosental
authors: Department of Chemistry, Binghamton University, Binghamton, New York 13902.
authors: Armanda
authors: A
authors: Gameiro
authors: Department of Chemistry, Binghamton University, Binghamton, New York 13902.
authors: Christof
authors: C
authors: Grewer
authors: Department of Biochemistry and Molecular Biology, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel. Electronic address: kannerb@cc.huji.ac.il.
authors: Baruch I
authors: BI
authors: Kanner
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D050580:Excitatory Amino Acid Transporter 3 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D040681:Structural Homology, Protein; D014982:Xenopus laevis
chemical_list: D050580:Excitatory Amino Acid Transporter 3; D009419:Nerve Tissue Proteins; C493400:SLC1A1 protein, human; C493398:Slc1a1 protein, rat; D001224:Aspartic Acid