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.
Highlights
The cation binding sites of glutamate transporters are not solved
Transport Activity of Asp-440 Mutants—To test the role of the conserved aspartate residue located at position 440 in EAAC1, we first measured D-[3H]aspartate uptake in HeLa cells expressing mutants where this residue was replaced by glutamate, asparagine, glutamine, serine, or cysteine
Under physiological conditions in intact cells, D440E behaves as if it is locked in the exchange mode, effectively carrying out the sodium-coupled glutamic acid half-cycle but not the potassium-dependent relocation step (Fig. 1A)
Summary
The cation binding sites of glutamate transporters are not solved. Results: Mutation of a conserved aspartate residue alters apparent cation affinity. We have obtained functional evidence that the conserved tyrosine residue, corresponding to Tyr-317 of GltPh, represents a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1 [10]. In the GltPh structure, a conserved aspartate residue (Asp-390) is located adjacent to the conserved tyrosine residue [16] (Fig. 1B), suggesting the possibility that these two residues could participate together in the formation of a new and as yet unidentified cation binding site. The functional characteristics of this mutant are consistent with the possibility that the conserved aspartate and tyrosine residues could be involved in the formation of a novel cation binding site
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