Is the glutamate residue Glu-373 the proton acceptor of the excitatory amino acid carrier 1?
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.
Grewer
Christof
C
Max-Planck-Institut für Biophysik, Kennedyallee 70, Frankfurt D-60596, Germany. grewer@mpibp-frankfurt.mpg.de
Watzke
Natalie
N
Rauen
Thomas
T
Bicho
Ana
A
D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D016014:Linear Models; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011188:Potassium / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D027322:Amino Acid Transport System X-AG; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D007477:Ions; C493400:SLC1A1 protein, human; C493398:Slc1a1 protein, rat; D027981:Symporters; D005973:Glutamine; D018698:Glutamic Acid; D011188:Potassium