Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter ␥-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or ␥-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine ‫؍‬ GABA > diaminobutyric acid ‫؍‬ ␤-alanine > proline ‫؍‬ quinidine > dimethylglycine > glycine > sarcosine. Both the V max and K m of GABA-and betaine-induced currents were voltage-dependent, and GABA-and betaine-induced currents and radioactive tracer uptake were strictly Na ؉ -dependent but only partially dependent on the presence of Cl ؊ . The apparent affinity of GABA decreased with decreasing Na ؉ concentrations. The K m of Na ؉ also depended on the GABA and Cl ؊ concentration. A decrease of the Cl ؊ concentration reduced the apparent affinity for Na ؉ and GABA, and a decrease of the Na ؉ concentration reduced the apparent affinity for Cl ؊ and GABA. A comparison of 22 Na ؉ -, 36Cl ؊ -, and 14 C-labeled GABA and 14 C-labeled betaine fluxes and GABA-and betaine-induced currents yielded a coupling ratio of Na ؉ /Cl ؊ /organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na ؉ second, and Cl ؊ third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.

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Glutamine release from astrocytes is an essential part of the glutamate-glutamine cycle in the brain. Uptake of glutamine into cultured rat astrocytes occurs by at least four different routes. In agreement with earlier studies, a significant contribution of amino acid transport systems ASC, A, L, and N was detected. It has not been determined whether these systems are also involved in glutamine efflux or whether specific efflux transporters exist. We show here that ASCT2, a variant of transport system ASC, is strongly expressed in rat astroglia-rich primary cultures but not in neuron-rich primary cultures. The amino acid sequence of rat astroglial ASCT2 is 83% identical to that of mouse ASCT2. In Xenopus laevis oocytes expressing rat ASCT2, we observed high-affinity uptake of [U-14 C]glutamine (K m ϭ 70 M) that was Na ϩ -dependent, concentrative, and unaffected by membrane depolarization. When oocytes were preloaded with [U-14 C]glutamine, no glutamine efflux was detected in the absence of extracellular amino acids. Neither lowering intracellular pH nor raising the temperature elicited efflux. However, addition of 0.1 mM unlabeled alanine, serine, cysteine, threonine, glutamine, or leucine to the extracellular solution resulted in a rapid release of glutamine from the ASCT2-expressing oocytes. Amino acids that are not recognized as substrates by ASCT2 were ineffective in this role. Extracellular glutamate stimulated glutamine release weakly at pH 7.5 but was more effective on lowering pH to 5.5, consistent with the pH dependence of ASCT2 affinity for glutamate. Our findings suggest a significant role of ASCT2 in glutamine efflux from astrocytes by obligatory exchange with extracellular amino acids. However, the relative contribution of this pathway to glutamine release from cells in vivo or in vitro remains to be determined.