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 transpo rters (EAATs). However, even in the absence of L-glutamate, the membra ne conductance of oocytes expressing EAAT1 was significantly increased relative to oocytes expressing EAAT2 or control oocytes. Whereas tran sport mediated by EAAT2 is blocked by the non-transported competitive glutamate analog kainate (K-i = 14 mu M), EAAT1 is relatively insensit ive K-i > 3 mM). Substitution of a block of 76 residues from EAAT2 int o EAAT1, in which 18 residues varied from EAAT1, conferred high affini ty kainate binding to EAAT1, and application of kainate to oocytes exp ressing the chimeric transporter blocked a pre-existing monovalent cat ion conductance that displayed a permeability sequence K+ > Na+ > Li>> choline(+). The results identify a structural domain of glutamate t ransporters that influences kainate binding and demonstrate the presen ce of a constitutive ion-selective pore in the transporter.