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Zinc ions (Zn2+) are stored in synaptic vesicles with glutamate in a number of regions of the brain. When released into the synapse, Zn2+ modulates the activity of various receptors and ion channels. Excitatory amino acid transporters (EAATs) maintain extracellular glutamate concentrations below toxic levels and regulate the kinetics of glutamate receptor activation. We have investigated the actions of Zn2+ on two of the most abundant human excitatory amino acid transporters, EAAT1 and EAAT2. Zn2+ is a noncompetitive, partial inhibitor of glutamate transport by EAAT1 with an IC50 value of 9.9 +/- 2.3 microM and has no effect on glutamate transport by EAAT2 at concentrations up to 300 microM. Glutamate and aspartate transport by EAAT1 are associated with an uncoupled chloride conductance, but Zn2+ selectively inhibits transport and increases the relative chloride flux through the transporter. We have investigated the molecular basis for differential inhibition of EAAT1 and EAAT2 by Zn2+ using site-directed mutagenesis and demonstrate that histidine residues of EAAT1 at positions 146 and 156 form part of the Zn2+ binding site. EAAT2 contains a histidine residue at the position corresponding to histidine 146 of EAAT1, but at the position corresponding to histidine 156 of EAAT1, EAAT2 has a glycine residue. Mutation of this glycine residue in EAAT2 to histidine generates a Zn2+ sensitive transporter, further confirming the role of this residue in conferring differential Zn2+ sensitivity.

1. Glutamate is the predominant excitatory neurotransmitter in the brain, but it is also a potent neurotoxin. Following release of glutamate from presynaptic vesicles into the synapse and activation of a variety of ionotropic and metabotropic glutamate receptors, glutamate is removed from the synapse. This is achieved through active uptake of glutamate by transporters located pre- and also post-synaptically or, alternatively, glutamate can diffuse out of the synapse and be taken up by transporters located on the cell surface of glial cells. 2. Complementary DNA encoding a number of glutamate transporters have recently been cloned and form a family of structurally related membrane proteins with a high degree of amino acid sequence conservation. Expression of the cloned glutamate transporters in various cell types has aided in the characterization of the functional properties of the different transporter subtypes. 3. Glutamate transport is coupled to sodium, potassium and pH gradients across the cell membrane creating an electrogenic process. This allows transport to be measured using electrophysiological techniques, which has greatly aided in understanding some of the basic mechanisms of the transport process and has also allowed a detailed understanding of the molecular pharmacology of the different transporter subtypes. 4. In the present review I shall discuss some of the recent advances in understanding the molecular basis for glutamate transporter function and then highlight some of the unanswered questions concerning the physiological roles of these proteins and suggest possible strategies for pharmacological manipulation of transporters for the treatment of neurological disorders.

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.