Functional analysis of the high affinity, Na(+)-dependent glutamate transporter GLAST-1 by site-directed mutagenesis.
The reuptake of excitatory amino acids, such as glutamate, terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. The L-glutamate/L-aspartate transporter (GLAST-1) is the first member of the recently discovered glutamate transporter family, which includes GLT-1 and EAAC1. The neutral amino acid carrier ASCT1 is structurally closely related to this new family of membrane proteins. Transmembrane transport of neutral amino acids is expected to differ in its binding site from that of the acidic excitatory amino acids glutamate and aspartate. Three positively charged amino acid residues, Arg-122, Arg-280, Arg-479, and one polar Tyr-405 are conserved in all glutamate transporters. They are replaced by apolar amino acid residues in the ASCT1 sequence. We exchanged these residues in the GLAST-1-specific cDNA by site-directed mutagenesis. cRNAs of these mutants were expressed in the Xenopus oocyte system. The functional characterization of the mutants R122I and R280V and the double mutant R122I, R280V revealed that the mutations have no influence on the intrinsic properties and kinetics of glutamate transport but alter the Km-values for L-aspartate and the competitive inhibitor D,L-threo-3-hydroxy aspartate. Substitutions of Tyr-405 by Phe (Y405F) and Arg-479 (R479T) by Thr completely inactivate the glutamate transporter. Immunoprecipitations of [35S]methionine-labeled transporter molecules indicate similar expression levels of wild-type and mutant transporters. Immunostaining of oocyte sections clearly proves the correct targeting to and integration of the mutant GLAST-1 proteins in the plasma membrane. Our results suggest the pivotal function of the hydroxy group of the highly conserved Tyr-405 and the positively charged Arg-479 in the binding of the negatively charged acidic neurotransmitter glutamate.
Conradt
M
M
Institute of Biochemistry, Medical Faculty, University of Cologne, Koeln, Germany.
Stoffel
W
W
D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012964:Sodium / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014981:Xenopus
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D006023:Glycoproteins; D018698:Glutamic Acid; D012964:Sodium