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.

Low-stringency hybridization with human glucocorticoid receptor (hGR) complementary DNA was used to isolate a new gene encoding a predicted 107-kilodalton polypeptide. Expression studies demonstrate its ability to bind aldosterone with high affinity and to activate gene transcription in response to aldosterone, thus establishing its identity as the human mineralocorticoid receptor (hMR). This molecule also shows high affinity for glucocorticoids and stimulates a glucocorticoid-responsive promoter. Together the hMR and hGR provide unexpected functional diversity in which hormone-binding properties, target gene interactions, and patterns of tissue-specific expression may be used in a combinatorial fashion to achieve complex physiologic control.

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...