Abstract
Transporters of the major excitatory neurotransmitter glutamate play a crucial role in glutamatergic neurotransmission by removing their substrate from the synaptic cleft. The transport mechanism involves co-transport of glutamic acid with three Na(+) ions followed by countertransport of one K(+) ion. Structural work on the archeal homologue Glt(Ph) indicates a role of a conserved asparagine in substrate binding. According to a recent proposal, this residue may also participate in a novel Na(+) binding site. In this study, we characterize mutants of this residue from the neuronal transporter EAAC1, Asn-451. None of the mutants, except for N451S, were able to exhibit transport. However, the K(m) of this mutant for l-aspartate was increased ∼30-fold. Remarkably, the increase for d-aspartate and l-glutamate was 250- and 400-fold, respectively. Moreover, the cation specificity of N451S was altered because sodium but not lithium could support transport. A similar change in cation specificity was observed with a mutant of a conserved threonine residue, T370S, also implicated to participate in the novel Na(+) site together with the bound substrate. In further contrast to the wild type transporter, only l-aspartate was able to activate the uncoupled anion conductance by N451S, but with an almost 1000-fold reduction in apparent affinity. Our results not only provide experimental support for the Na(+) site but also suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance.
Highlights
The cation binding sites of brain glutamate transporters are not yet identified
We reported that the T370S mutant has an altered cation selectivity of radioactive transport and of the transport currents induced by L-aspartate
The sodium concentration dependence of the transport currents by N451S appeared dependent on the nature of the substrate and half of the current measured at 96 mM was observed at ϳ40, 55, and 65 mM for L-aspartate, D-aspartate, and L-glutamate, respectively (Fig. 5C)
Summary
The cation binding sites of brain glutamate transporters are not yet identified. Results: Mutation of a conserved asparagine residue changed cation selectivity and apparent substrate affinity. A similar change in cation specificity was observed with a mutant of a conserved threonine residue, T370S, implicated to participate in the novel Na؉ site together with the bound substrate. Our results provide experimental support for the Na؉ site and suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance. Because of the limited resolution of the GltPh structure, Tlϩ ions, which exhibit a robust anomalous scattering signal, have been used in an attempt to visualize the sodium sites in this homologue [23], which uses three Naϩ ions per transported substrate molecule [32]. The Na3 site was proposed to be formed by the side-chains of conserved threonine and asparagine residues, from TMs 7 and 8, respectively as well as by carboxyl oxygens of the acidic amino acid substrate [35]. The new data suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance
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