Abstract
Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na(+)/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na(+) binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na(+)/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.
Highlights
The uphill transport of negatively charged amino acid substrates catalyzed by high affinity plasma membrane glutamate transporters is driven by the coupled downhill movement of three sodium ions and one potassium ion across the membrane [1,2,3]
Glutamate-induced steady-state currents are abolished by the mutation, a rapid inwardly directed transient EAAC1E373Q current is observed at the time of the glutamate application (Fig. 2A) that is absent in non-transfected control cells (Fig. 2C)
We addressed this question by using site-directed mutagenesis, focusing on the EAAC1 glutamic acid residue 373, which is highly conserved in the EAAT family, but not in the related Hϩ-independent ASC transporters that are selective for neutral amino acids
Summary
Mined whether Glu-373 contributes to the charge neutralization provided by the empty EAAC1 cation binding sites, and whether its charge moves across the electric field during the charge translocating conformational change of the transporter. Based on the facts that glutamate binding to EAAC1E373Q is completely pH-independent and homoexchange is unimpaired, we propose that Glu-373 is part of the proton-translocating machinery of EAAC1
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