Excitatory amino acid transporters (EAATs) regulate glutamate concentrations in the brain to maintain normal excitatory synaptic transmission. A widely accepted view of transporters is that they consist of a pore with alternating access to the intracellular and extracellular solutions, which serves to couple ion movement to the movement of substrate. However, recent observations that EAATs, and also a number of other neurotransmitter transporters, can also function as ligand-gated chloride channels have blurred the distinctions between transporters and ion channels. Here we show that mutations in the second transmembrane domain (TM2) of EAAT1 alter anion permeation properties without affecting glutamate transport and that a number of TM2 residues are accessible to the external aqueous solution. Furthermore, we demonstrate that the extracellular edge of TM2 is in close proximity to a membrane-associated domain that influences glutamate transport. This study will provide the foundation for beginning to understand how transporters can function as both transporters and ion channels.In the mammalian central nervous system, glutamate is the predominant excitatory neurotransmitter, and EAATs 1 regulate synaptic glutamate concentrations to maintain a dynamic signaling process between neurons. EAATs are secondary active transporters in which glutamate is co-transported with 3 Na ϩ and 1 H ϩ followed by the counter-transport of 1 K ϩ (1), which enables EAATs to maintain a 10 6 -fold glutamate concentration gradient across the cell membrane. In addition to this coupled transport conductance, glutamate also activates a thermodynamically uncoupled chloride conductance through the transporter (2-4), which may serve a number of functions such as modulation of cell excitability, regulation of the rate of voltage-dependent glutamate uptake, and influencing ion homeostasis (4 -7). A number of other neurotransmitter transporters, including transporters for dopamine, serotonin, noradrenaline, and ␥-aminobutyric acid, also allow uncoupled fluxes of ions (8 -11), and in the case of the dopamine transporter the substrate-activated chloride conductance regulates presynaptic excitability (8). At present there is very little understanding of the molecular basis for how transporters can support these separate functions.Five EAAT subtypes have been characterized (3,(12)(13)(14)(15)(16), and the highly conserved carboxyl-terminal half of EAATs has been identified, by the use of chimeric transporters and site-directed mutagenesis, as forming the glutamate binding and/or translocation domain (17-23) (see Fig. 1A). However, a number of mutations that disrupt transport do not affect chloride permeation (for example see Refs. 18 and 24), and several recent studies have reported selective modulation of the transport component without affecting the chloride component of transport (20 -22), implying there are separate molecular determinants for glutamate transport and chloride permeation. In order to investigate this idea further, it is necessary t...

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SUMMARYWe have investigated the mechanism of action of a series of glutamate derivatives on the cloned excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), expressed in Xenopus laevis oocytes. The compounds were tested as substrates and competitive blockers of the glutamate transporters. A number of compounds showed contrasting mechanisms of action on EAAT1 compared with EAAT2. In particular, (2S,4R)-4-methylglutamate and 4-methylene-glutamate were transported by EAAT1, with K m values of 54 M and 391 M, respectively, but potently blocked glutamate transport by EAAT2, with K b values of 3.4 M and 39 M, respectively. Indeed, (2S,4R)-4-methylglutamate is the most potent blocker of EAAT2 yet described. (Ϯ)-Threo-3-methylglutamate also potently blocked glutamate transport by EAAT2 (K b ϭ 18 M), but was inactive on EAAT1 as either a substrate or a blocker at concentrations up to 300 M. In contrast to (2S,4R)-4-methylglutamate, L-threo-4-hydroxyglutamate was a substrate for both EAAT1 and EAAT2, with K m values of 61 M and 48 M, respectively. It seems that the chemical nature and also the orientation of the group at the 4-position of the carbon backbone of glutamate is crucial in determining the pharmacological activity. The conformations of these molecules have been modeled to understand the structural differences between, firstly, compounds that are blockers versus substrates of EAAT2 and, secondly, the pharmacological differences between EAAT1 and EAAT2.

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.

We report the synthesis of a series of 3-carboxy-, 3-(carboxymethyl)-, 3-(omega-phosphonoalkyl)-1-aminocyclobutane-1-carboxylic acids for evaluation as agonists or antagonists of neurotransmission at excitatory amino acid receptors, particularly N-methyl-D-aspartic acid (NMDA) receptors. The compounds were evaluated as agonists on their ability to depolarize the rat brain cortical wedge preparation or as antagonist of the actions of the selective agonists NMDA, quisqualic acid, and kainic acid. The chain-elongated glutamate derivatives with potential antagonist activity proved to be weak and frequently nonselective antagonists in this assay. The most noteworthy result was that trans isomer 7b was a very potent agonist, approximately 20 times more active than NMDA at NMDA receptors, while the cis isomer was 1/3 as potent as NMDA.