Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non-nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each EAAT subtype were determined in transfected mammalian cells by radio-label uptake and in microinjected oocytes by voltage-clamp measurements. The affinities of the EAAT subtypes for L-glutamate were similar, with Km determinations varying from 48 to 97 microM in the mammalian cell assay and from 18 to 28 microM in oocytes. Glutamate uptake inhibitors were used to compare the pharmacologies of the EAAT subtypes. The EAAT2 subtype was distinguishable from the EAAT1/EAAT3 subtypes by the potency of several inhibitors, but most notably by sensitivity to kainic acid (KA) and dihydrokainic acid (DHK). KA and DHK potently inhibited EAAT2 transport, but did not significantly affect transport by EAAT1/EAAT3. Using voltage-clamp measurements, most inhibitors were found to be substrates that elicited transport currents. In contrast, KA and DHK did not evoke currents and they were found to block EAAT2-mediated transport competitively. This selective interaction with the EAAT2 subtype could be a significant factor in KA neurotoxicity. These studies provide a foundation for understanding the role of glutamate transporters in human excitatory neurotransmission and in neuropathology.

Synaptic transmission is commonly terminated by diffusion and reuptake of neurotransmitter from the synaptic cleft. Glutamate reuptake prevents neurotoxicity and sets the lower limit for the concentration of extracellular glutamate, so it is important to understand the thermodynamics of this process. Here we use voltage clamping with a pH-sensitive fluorescent dye to monitor electrical currents and pH changes associated with flux of glutamate mediated by the human neuronal glutamate transporter EAAT3. In contrast to a previous model, we find that three sodium ions and one proton are cotransported with each glutamate ion into the cell, while one potassium ion is transported out of the cell. This coupling can support a transmembrane glutamate concentration gradient ([Glu]in/[Glu]out) exceeding 10(6) under equilibrium conditions, and would allow the transporter to continue removing glutamate over a wide range of ionic conditions.

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-D-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate f lux and twoelectrode voltage clamp recording. EAAT5-mediated Lglutamate uptake is sodium-and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium-and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.The uptake of glutamate and other excitatory amino acids is mediated by a gene family of high affinity sodium-dependent transporters that includes four known mammalian subtypes; in humans, we have termed these glutamate transporters excitatory amino acid transporter (EAAT) 1 through 4 (1, 2). The transport of glutamate is driven by the cotransport of sodium ions and the countertransport of potassium ions down their electrochemical gradients, and recent studies suggest that this complex process involves the cotransport of protons as well (3-6). Because there is net inward movement of positive charge with the transport of each molecule of glutamate, the transport process is readily studied in the Xenopus oocyte expression system by observing the associated current. In addition to these transport currents, however, we have found that application of substrate to the transporter also gates an uncoupled, passive flux of chloride ions (2, 7). The relative magnitude of this associated chloride conductance varies with each cloned EAAT subtype; for EAAT1-EAAT3, the magnitude of the chloride current at physiological membrane potentials is similar to that of the electrogenic cotransport current, but the currents generated by EAAT4 are almost entirely due to the flux of chloride ions. In vivo, a glutamatedependent current that has a transporter-like pharmacology is carried largely by chloride ions in retinal cone (8) and rod (9) photoreceptors and bipolar cells (10). In bipolar cells, this chloride current has been proposed to mediate the cone component of the ON bipolar cell light response (10). Although the properties of EAAT4 are similar to the glu...

Flux of substrate and charge mediated by three cloned excitatory amino acid transporters widely expressed in human brain were studied in voltage-clamped Xenopus oocytes. Superfusion of L-glutamate or D-aspartate resulted in currents due in part to electrogenic Na+ cotransport, which contributed 1 net positive charge per transport cycle. A significant additional component of the currents was due to activation of a reversible anion flux that was not thermodynamically coupled to amino acid transport. The selectivity sequence of this ligand-activated conductance was NO3- > 1- > Br- > Cl- > F-. The results suggest that these proteins mediate both transporter- and channel-like modes of permeation, providing a potential mechanism for dampening cell excitability, in addition to removal of transmitter.

Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and marine amphotropic retrovirus (Ram-i) Ram-i (10) and human Glvr-1 (7) cDNAs were cloned into pGEM-7Z (Promega) with their 5' ends adjacent to the SP6 promoter. For mRNA synthesis, the plasmids were linearized and transcribed with SP6 polymerase in the presence of m7G(5')ppp(5')G caps according to the manufacturer's directions (Pharmacia). Xenopus laevis oocytes were injected with 50 nl of mRNA (1 ng/nl) or with an equal volume of H20 and were incubated for 4-6 days at 17'C; then two-microelectrode voltage-clamp recordings or radiolabel uptake assays were performed at room temperature as described (23).Abbreviations: GALV, gibbon ape leukemia virus; Glvr-1, cell surface receptor for GALV; Ram-i, cell surface receptor for amphotropic murine retrovirus; HIV, human immunodeficiency virus; MLV, murine leukemia virus; Mo-MLV, Moloney MLV.

Electrophysiological and pharmacological studies of a cloned human dopamine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Using two-electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate "transport-associated" current. (2) The velocity of DA uptake measured in oocytes clamped at various membrane potentials was voltage-dependent, increasing with hyperpolarization. Concurrent measurement of transport-associated current and substrate flux in individual oocytes revealed that charge movement during substrate translocation was greater than would be expected for a transport mechanism with fixed stoichiometry of 2 Na+ and 1 Cl- per DA+ molecule. In addition to the transport-associated current, hDAT also mediates a constitutive leak current, the voltage and ionic dependencies of which differ markedly from those of the transport-associated current. Ion substitution experiments suggest that alkali cations and protons are carried by the hDAT leak conductance. In contrast to the transport-associated functions, the leak does not require Na+ or Cl-, and DAT ligands readily interact with the transporter even in the absence of these ions. The currents that hDAT mediates provide a functional assay that readily distinguishes the modes of action of amphetamine-like "DA-releasing" drugs from cocaine-like translocation blockers. In addition, the voltage dependence of DA uptake suggests a mechanism through which presynaptic DA autoreceptor activation may accelerate the termination of dopaminergic neurotransmission in vivo.

Currents mediated by a glutamate transporter cloned from human motor cortex were measured in Xenopus oocytes. In the absence of glutamate, voltage jumps induced Na(+)-dependent capacitive currents that were blocked by kainate, a competitive transport antagonist. The pre-steady-state currents can be described by an ordered binding model in which a voltage-dependent Na+ binding is followed by a voltage-independent kainate binding. At -80 mV, two charges are translocated per molecule of glutamate, with a cycling time of approximately 70 ms, which is significantly slower than the predicted time course of synaptically released glutamate. The results suggest that glutamate diffusion and binding to transporters, rather than uptake, are likely to dominate the synaptic concentration decay kinetics.

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