Identification of functional domains of the human glutamate transporters EAAT1 and EAAT2.
Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S, 4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.
Mitrovic
A D
AD
Department of Pharmacology, University of Sydney, New South Wales 2006, Australia.
Amara
S G
SG
Johnston
G A
GA
Vandenberg
R J
RJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D004594:Electrophysiology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D027342:Excitatory Amino Acid Transporter 2; D015870:Gene Expression / Q000235:genetics; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid / Q000493:pharmacokinetics*; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017981:Receptors, Neurotransmitter / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D014982:Xenopus laevis
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D018690:Excitatory Amino Acid Agonists; D027342:Excitatory Amino Acid Transporter 2; D005971:Glutamates; D017981:Receptors, Neurotransmitter; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; C036040:serine O-sulfate; D007608:Kainic Acid