A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters

Noa Rosental, Armanda Gameiro, Christof Grewer, Baruch I. Kanner*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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.

Original languageEnglish
Pages (from-to)41381-41390
Number of pages10
JournalJournal of Biological Chemistry
Volume286
Issue number48
DOIs
StatePublished - 2 Dec 2011

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