Constitutive ion fluxes and substrate binding domains of human glutamate transporters.
Application of L-glutamate activates ionic currents in voltage-clamped Xenopus oocytes expressing cloned human excitatory amino acid transporters (EAATs). However, even in the absence of L-glutamate, the membrane conductance of oocytes expressing EAAT1 was significantly increased relative to oocytes expressing EAAT2 or control oocytes. Whereas transport mediated by EAAT2 is blocked by the non-transported competitive glutamate analog kainate (Ki = 14 microM), EAAT1 is relatively insensitive (Ki > 3 mM). Substitution of a block of 76 residues from EAAT2 into EAAT1, in which 18 residues varied from EAAT1, conferred high affinity kainate binding to EAAT1, and application of kainate to oocytes expressing the chimeric transporter blocked a pre-existing monovalent cation conductance that displayed a permeability sequence K+ > Na+ > Li+ >> choline+. The results identify a structural domain of glutamate transporters that influences kainate binding and demonstrate the presence of a constitutive ion-selective pore in the transporter.
Vandenberg
R J
RJ
Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
Arriza
J L
JL
Amara
S G
SG
Kavanaugh
M P
MP
D018528:ATP-Binding Cassette Transporters / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D006801:Humans; D017136:Ion Transport; D007608:Kainic Acid / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D007608:Kainic Acid