Functional Characterization of the Betaine/γ-Aminobutyric Acid Transporter BGT-1 Expressed in Xenopus Oocytes*

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter γ-aminobutyric acid (BGT-1) was expressed inXenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or γ-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 = β-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. TheK 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 of22Na+-, 36Cl−-, and14C-labeled GABA and 14C-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.

Cl ؊ -, 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.
Because osmotic equilibrium across most cell membranes occurs rapidly via water flux down its gradient, cells regulate their volume by adjusting their solute content. To this end they employ a variety of mechanisms including ion transport, formation or degradation of glycogen and proteins, as well as accumulation of organic osmolytes (1,2). The osmolytes most frequently utilized by mammalian cells include the polyols sorbitol and myo-inositol, amino acids and amino acid derivatives such as taurine, as well as methylamines such as glycerophosphorylcholine and betaine (3,4). In contrast to inorganic ions, the organic osmolytes do not destabilize proteins and are thus compatible with cellular protein function even at very high concentrations (5,6). Whereas sorbitol and glycerophosphorylcholine are accumulated by cellular formation, myo-inositol, taurine, and betaine are largely accumulated by Na ϩcoupled transport processes (4 -6).
In recent years considerable progress has been made toward understanding the molecular mechanisms underlying osmotic regulation. Hypertonicity-regulated signal transduction pathways have been identified (for review, see Ref. 1), and several osmolyte transporters have been cloned, such as BGT-1 1 for betaine/GABA (7)(8)(9)(10), SCT/TAUT for taurine (11,12), and SMIT for myo-inositol (13). BGT-1 was originally cloned from Madin-Darby canine kidney cells (7) but has subsequently been found to be expressed ubiquitously in most mammalian tissues including the central nervous system (8 -10, 14). BGT-1 and SCT/TAUT belong to the family of Na ϩ -and Cl Ϫ -coupled transporters for neurotransmitters, amino acids, and osmolytes. Four different GABA transporters have been cloned (7,10,(15)(16)(17)(18) and are named GAT-1, BGT-1, GAT-2, and GAT-3 in rat and man or GAT-1, GAT-2, GAT-3, and GAT-4 in mice, respectively. Most of these transporters accomplish either neuronal or glial transport of GABA in the central nervous system, whereas BGT/GAT-2 is also widely expressed outside the central nervous system (14). The mechanism of GABA uptake via GAT-1 has been studied in detail in cRNA-injected Xenopus laevis oocytes and transfected mammalian cells (19 -24). Considerable differences have been found with respect to ion-cotransport stoichiometry, binding order, and leak-and transport-associated currents in this family (25,26), but not much is known about the functional properties of BGT-1. Additionally, pharmacological properties of BGT-1 are less well studied than those other three known GAT transporter isoforms (14,15), and only little is known about the functional properties of BGT-1 in general. In this study the canine BGT-1 was expressed in Xenopus oocytes and examined using the two-electrode voltage clamp technique and radioactive tracer studies. * The study was supported in part by Deutsche Forschungsgemeinschaft Grants La 315/4-3, Bu 704/7-2, and Br 1318/2-2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Contributed equally to this paper and therefore share first authorship. ʈ

MATERIALS AND METHODS
cRNA encoding the canine BGT-1 was synthesized in vitro as described previously (7). Dissection of X. laevis ovaries and collection and handling of the oocytes have been described in detail elsewhere (27). Oocytes were injected with 15 ng of cRNA and 50 nl of water/oocyte; noninjected oocytes served as controls. All experiments were performed at room temperature 3-8 days after injection.
Flux studies have been performed utilizing the radiochemicals [ 14 C]GABA, 22 NaCl, and H 36 Cl (NEN Life Science Products, Brussels, Belgium) and [ 14 C]betaine (Biotrend, Cologne, Germany). BGT-1-expressing oocytes and noninjected control oocytes were washed twice with ice-cold OR 2ϩ buffer (calcium-containing oocyte Ringer OR 2ϩ : 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 1 mM Na 2 HPO 4 , 1 mM CaCl 2 , 5 mM HEPES, pH 7.8 (28)). Uptake was initiated by addition of 100 l of OR 2ϩ buffer supplemented with 1 mM GABA and one of the radioactive substrates [ 14 C]GABA, [ 14 C]betaine (final specific activity 63.0 Bq/ nmol), or 22 NaCl (final specific activity 21.9 Bq/nmol). Chloride uptake was studied in transport buffer in which unlabeled NaCl was replaced by H 36 Cl (final specific activity 6.7 Bq/nmol) titrated with NaOH to a pH of about 8; in addition, the HEPES concentration was elevated to 20 mM to increase buffering capacity. Groups of seven oocytes were incubated for each experimental condition in 5-ml polypropylene tubes containing 100 l of supplemented OR 2ϩ buffer. Uptake was stopped after 30 min or 1 h by washing the oocytes with ice-cold OR 2ϩ buffer. Single oocytes were lysed by adding 200 l of 10% SDS; 3 ml of scintillation fluid was added, and radioactivity was determined by liquid scintillation counting. Each measurement was performed at least twice. Current induced by GABA was measured by two-electrode voltage clamp on the same batch of oocytes and on the same day the transport measurements were performed.
Two-electrode voltage clamp recordings were performed at a holding potential of Ϫ50 mV if not otherwise specified. The data were filtered at 10 Hz and recorded with MacLab digital-to-analog converter and software for data acquisition and analysis (AD Instruments, Castle Hill, Australia). The external control solution (superfusate/ND96) contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES, pH 7.4. For experiments studying the dependence on external sodium or chloride, sodium was replaced by choline or N-methyl-D-glucamine and chloride by gluconate, NO 3 Ϫ , or SO 4 2Ϫ . When necessary, the osmolarity was adjusted by adding glucose. Betaine or GABA was added to the solutions at the indicated concentrations. The final solutions were titrated to the pH indicated using HCl or KOH. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s. The currents given are the maximal values measured during a 30-s substrate superfusion. Usually the maximal current was reached after 25 substrate superfusion. To rule out effects of the different solution used (OR 2ϩ versus ND96) control exper-  Mean values Ϯ S.E. of substrate-induced currents in BGT-1-expressing oocytes are shown. Substrates were added at a concentration of 1 mM and at a holding potential of Ϫ50 mV for a period of 30 s. Not shown are serotonin, histamine, dopamine, L-DOPA, and noradrenaline, which did not induce any currents. In noninjected oocytes no currents were found for all substrates tested. TEA, tetraethylammonium.
iments were performed, and currents and uptake were measured under both conditions. No significant difference was found.
Data are provided as means Ϯ S.E., and n represents the number of oocytes investigated. The magnitude of the induced currents varied 2-5-fold, depending on the time period after cRNA injection and on the batch of oocytes (from different animals). Therefore, throughout the paper we show experimental data obtained on the same day for each specific set of experiments. All experiments were repeated with at least two or three batches of oocytes; in all repetitions qualitatively similar data were obtained. All data were tested for significance using the paired Student's t test, and only results with p Ͻ 0.05 were considered statistically significant.

RESULTS
In current clamp studies superfusion of BGT-1 expressing Xenopus oocytes with 1 mM GABA or betaine caused a depolarization of 27.6 Ϯ 2.0 mV (n ϭ 5) and 24.2 Ϯ 1.7 mV (n ϭ 5), respectively. In the voltage clamp modus 1 mM GABA or betaine induced a mean inward current of Ϫ73.9 Ϯ 3.8 nA and Ϫ75.3 Ϯ 4.1 nA, respectively, as illustrated in Fig. 1A. In noninjected control oocytes GABA did not induce significant currents (0.9 Ϯ 0.3 nA, n ϭ 5), whereas betaine led to an inward current of Ϫ7.2 Ϯ 1.5 nA (n ϭ 5). The betaine-induced current in noninjected oocytes was independent of the presence of Na ϩ and had a low affinity for betaine (data not shown). Thus, for further kinetic analysis mostly GABA was used; and if betaine was used, betaine-induced currents in noninjected oocytes were subtracted from the currents obtained from BGT-1-injected oocytes.
To test the selectivity of BGT-1 the current elicited by a 1 mM concentration of different substrates was measured (Table I). The rank order of magnitude of induced currents was GABA ϭ betaine Ͼ DABA Ͼ ␤-alanine Ͼ proline Ͼ quinidine Ͼ glycine ϭ putrescine Ͼ choline Ͼ sarcosine ϭ tetraethylammonium ϭ taurine ϭ carnitine Ͼ betaine aldehyde ϭ creatine (Table I; all substances were tested on the same oocytes, n ϭ 5). Among those substrates, only betaine induced currents in noninjected oocytes (see above). Serotonin, histamine, noradrenaline, dopamine, and L-DOPA did not induce any currents in BGT-1-or noninjected oocytes (data not shown, n ϭ 5).
As shown in Fig. 1B, the GABA-induced currents in BGT-1expressing oocytes were voltage-dependent, increasing with hyperpolarization. In ND96 buffer the GABA-induced currents were strictly inwardly rectifying in the range between Ϫ90 and ϩ40 mV (n ϭ 5). The holding potential affected both the maximum velocity and the apparent K m of GABA-and betaineinduced currents. As shown in Fig. 2 the GABA concentration required for half-maximal current (apparent K m ) increased from 0.009 Ϯ 0.001 mM at Ϫ100 mV holding potential to give the Hill coefficient and the substrate (GABA/betaine) concentration, respectively; I max is the extrapolated maximal current; and K m is the apparent concentration needed for half-maximal current. All currents were normalized against the maximal current at 1 mM GABA at Ϫ90 mV holding potential. The Hill coefficients for the curves were not significantly different (Ϫ100 mV, 0.8 Ϯ 0.1; Ϫ50 mV, 1.0 Ϯ 0.1; Ϫ30 mV, 0.8 Ϯ 0.1, n ϭ 5). Panel B, voltage dependence of betaine-induced current. The curves were fitted applying the Hill equation, and all data were normalized against the maximal current at Ϫ80 mV holding potential and 5 mM betaine. The difference of the apparent K m for the different holding potentials was significant on the p Ͻ 0.05 level. The Hill coefficients for the curves were not significantly different (Ϫ80 mV, 1. 0.031 Ϯ 0.004 mM at a more depolarized holding potential of Ϫ30 mV. On the other hand, a depolarization from Ϫ100 to Ϫ50 and Ϫ30 mV decreased the maximal current by 31 Ϯ 2% and 51 Ϯ 2%, respectively ( Fig. 2A, n ϭ 4). The apparent K m for betaine-induced currents was shifted significantly from 0.51 Ϯ 0.04 mM at Ϫ80 mV holding potential to 2.1 Ϯ 0.2 mM at Ϫ30 mV (n ϭ 4). The maximal betaine-induced current was not significantly altered by depolarization (Fig. 2B).
The betaine-induced currents were strictly Na ϩ -and partially Cl Ϫ -dependent. The apparent K m for Na ϩ was 93.3 Ϯ 3.9 mM and for Cl Ϫ 68.3 Ϯ 0.8 mM (n ϭ 5, Fig. 3, A and B). The Hill coefficient for Na ϩ was 2.5 Ϯ 0.2 and for Cl Ϫ 1.7 Ϯ 0.1.
Also, the GABA-induced currents were strictly Na ϩ -dependent. In the absence of Na ϩ , the addition of 1 mM GABA failed to induce any current. The GABA (1 mM)-induced current increased with the extracellular Na ϩ concentration (at 103.6 mM Cl Ϫ concentration) with a half-maximal current at 65.9 Ϯ 8 mM Na ϩ concentration at Ϫ90 mV and at 89 Ϯ 3 mM Na ϩ concentration at Ϫ50 mV (Fig. 4A, n ϭ 4). Thus, hyperpolarization of the cell membrane increased the affinity of the carrier to Na ϩ . The Hill coefficient for Na ϩ was 2.2 Ϯ 0.4 at Ϫ50 mV and was not altered significantly by hyperpolarization to Ϫ90 mV. A decrease of ambient Na ϩ concentration decreased both maximal current and apparent affinity for GABA. The apparent K m for GABA increased slightly from 0.021 Ϯ 0.002 mM at 130 mM Na ϩ in the superfusate to 0.056 Ϯ 0.006 mM at 80 mM Na ϩ (Fig.  4B). The Hill coefficient, however, remained close to 1. To test whether GABA influences Na ϩ binding, the GABA concentration was decreased from 1 to 0.03 mM, which markedly shifted the apparent affinity for Na ϩ from 55 Ϯ 2 mM to about 120 mM without affecting the Hill coefficient or the calculated V max value (Fig. 5, n ϭ 5).
As illustrated in Fig. 6, GABA-induced currents were also partially Cl Ϫ -dependent. Reduction of extracellular Cl Ϫ from 150 to 0 mM reduced the GABA-induced current by about 80% at Ϫ90 mV and by 90% at Ϫ50 mV (Fig. 6, A and B). Replacing Cl Ϫ with anions other than gluconate (NO 3 Ϫ and SO 4 2Ϫ ) showed similar results. Varying the extracellular Cl Ϫ concentration revealed that the half-maximal current induced by 1 mM GABA was reached at 78 Ϯ 4 mM Cl Ϫ concentration at Ϫ90 mV and at about 114 Ϯ 5 mM Cl Ϫ concentration at Ϫ50 mV. For fitting the curves an offset (Cl Ϫ -independent GABA-induced current) of 0.2 and 0.08 for Ϫ90 and Ϫ50 mV, respectively, was assumed. The apparent K m for Cl Ϫ at a holding potential of Ϫ50 mV could only be estimated because saturating conditions could not be reached under experimental conditions (n ϭ 5). The apparent Cl Ϫ affinity of the carrier was decreased by depolarization. The Hill coefficient for Cl Ϫ was close to 1 irrespective of the holding potential. Furthermore, a reduction of the extracellular Cl Ϫ FIG. 4. Na ؉ dependence of GABA-induced currents in BGT-1expressing oocytes. Panel A, GABA-induced currents are Na ϩ -dependent, and the apparent K m for Na ϩ is voltage-dependent. The apparent affinity for Na ϩ was significantly shifted from 89 Ϯ 3 mM at Ϫ50 mV to 66 Ϯ 8 mM at Ϫ90 mV holding potential. The Hill coefficient was 2.2 Ϯ 0.5 for Ϫ50 mV and 1.8 Ϯ 0.2 for Ϫ90 mV. All data were normalized against the maximal current at 150 mM Na ϩ and 1 mM GABA, and the Hill equation was used as described above. Panel B, effect of Na ϩ on the apparent K m for GABA. Reduction of Na ϩ from 150 to 80 mM in the superfusate significantly increases the apparent K m from 0.021 Ϯ 0.002 mM to 0.056 Ϯ 0.006 mM. Curves were obtained by fitting the data to the Hill equation. The Hill coefficient was 1.5 Ϯ 0.2 for 130 mM Na ϩ and 1.1 Ϯ 0.1 for 80 mM Na ϩ . Inset, Lineweaver-Burk plot for the same data. concentration from 130 to 50 mM resulted in a slight increase of the apparent K m value for GABA from 0.033 Ϯ 0.001 to 0.049 Ϯ 0.003 mM, pointing to a small but significant decrease of GABA affinity (Fig. 6C, n ϭ 5). The maximum velocity of GABA uptake decreased significantly with decreasing Cl Ϫ concentration.
To examine the mutual interaction of Na ϩ and Cl Ϫ we performed Na ϩ and Cl Ϫ kinetics at different Cl Ϫ and Na ϩ concentrations, respectively (Fig. 7). The apparent K m for Na ϩ was shifted from 54 Ϯ 3 mM at 150 mM Cl Ϫ to 82 Ϯ 7 mM at 80 mM Cl Ϫ . The Hill coefficient was 2.5 Ϯ 0.2 for both extracellular Cl Ϫ concentrations (Fig. 7A, n ϭ 5). On the other hand, a decrease of the ambient Na ϩ concentration from 150 to 80 mM turned the apparent K m for Cl Ϫ from 72 Ϯ 3 mM (Hill coefficient of 1.1 Ϯ 0.2) into a nonsaturable chloride concentration dependence (Fig. 7C, n ϭ 5). A summary of the kinetic constants derived from these experiments is shown in Table II. To determine further the Na ϩ and Cl Ϫ dependence and coupling ratio we carried out uptake studies with radioactive [ 14 C]GABA, 22 Na ϩ , and 36 Cl Ϫ . The uptake of GABA was linear The apparent K m for Cl Ϫ was voltage-dependent and increased from 78 Ϯ 4 mM to 114 Ϯ 5 mM with depolarization of the holding potential from Ϫ90 to Ϫ50 mV. The apparent K m for Ϫ50 mV is only an estimation but clearly indicates a significant shift. Curves were obtained by fitting the data to the Hill equation considering an offset (Cl Ϫ -independent GABA-induced current) of 0.2 and 0.07 for Ϫ90 and Ϫ50 mV, respectively. All data were normalized against the maximal current at 150 mM Cl Ϫ and Ϫ90 mV holding potential. The Hill coefficient for Cl Ϫ was 1.1 Ϯ 0.1 and 1.0 Ϯ 0.1 for Ϫ90 and Ϫ50 mV holding potential, respectively. Panel C, Cl Ϫ influenced the apparent K m for GABA. Reduction of Cl Ϫ significantly increased the apparent K m for GABA from 0.033 Ϯ 0.001 mM (130 mM Cl Ϫ ) to 0.049 Ϯ 0.003 mM (80 mM Cl Ϫ ). Curves were obtained by fitting the data to the Hill equation. All data were normalized against the maximal current at 130 mM Cl Ϫ and 1 mM GABA. The Hill coefficient for GABA was 1.02 Ϯ 0.05 for 130 mM Cl Ϫ and 1.09 Ϯ 0.07 for 50 mM Cl Ϫ . Inset, Lineweaver-Burk plot of the same data.
FIG. 7. Mutual interaction of Na ؉ and Cl ؊ concentration on GABA-induced current. Panel A, effect of Cl Ϫ on the apparent K m of Na ϩ . Reduction of Cl Ϫ in the superfusate from 150 to 80 mM significantly decreased the affinity for Na ϩ . The apparent K m was increased from 54 Ϯ 3 mM (150 mM Cl Ϫ ) to 82 Ϯ 7 mM under 80 mM Cl Ϫ . The Hill coefficient, however, remained close to 2 for both conditions (1.8 Ϯ 0.4 for 150 mM Cl Ϫ and 2.5 Ϯ 0.3 for 80 mM Cl Ϫ ). Curves were obtained by fitting the data to the Hill equation. All data were normalized against the maximal current at 150 mM Na ϩ and 150 mM Cl Ϫ . Panel B, Lineweaver-Burk plot for the same data. Panel C, influence of Na ϩ on the affinity for Cl Ϫ . Reduction of Na ϩ from 130 to 80 mM significantly shifted the apparent K m for Cl Ϫ from 72 Ϯ 3 to an estimated apparent K m of about 220 mM. The Hill coefficient was 1.2 Ϯ 0.1 for 150 mM Na ϩ . Curves were obtained by fitting the data to the Hill equation considering an offset of 0.1. All data were normalized against the maximal current at 150 mM Cl Ϫ and 150 mM Na ϩ . Panel D, Lineweaver-Burk plot of the GABA concentration dependence. over a time period of 30 min (Fig. 8). In agreement with GABAinduced currents, the uptake of labeled GABA was completely Na ϩ -dependent (n ϭ 7) but only partially Cl Ϫ -dependent (n ϭ 7) (Table III). The cotransport stoichiometry in relation to GABA was 1:3.1 for Na ϩ and 1:1.3 for Cl Ϫ (Table IV). The addition of 10 mM betaine to the incubation buffer resulted in a substrate-stimulated 22 Na uptake of 5.2 Ϯ 1.2 nmol/h and a substrate-stimulated 36 Cl uptake of 3.3 Ϯ 0.7 nmol/h, reflecting a ratio of 3 Na ϩ to 1.9 Cl Ϫ . Because of the low specific activity of available tracer, the betaine uptake could not be evaluated properly in these experiments. Taken together, these data suggest a coupling ratio of 3:1:1 or 3:2:1 for Na ϩ , Cl Ϫ , and organic substrate, respectively. This conclusion is supported further by the comparison of flux and current in the same batch of oocytes. In those oocytes currents of 29 Ϯ 9 nA (n ϭ 7) and 59 Ϯ 6 nA (n ϭ 7) have been determined for the Na ϩ /GABA and the Cl Ϫ /GABA flux measurements, respectively. Electrophysiological experiments under conditions mimicking the flux experi-ments revealed that during current clamp, the addition of 1 mM GABA led to an initial depolarization (by 13.6 Ϯ 1.0 mV, n ϭ 9) followed by slow recovery of cell membrane potential (by 14.6 Ϯ 1.1 mV, n ϭ 9). At voltage clamp conditions, the GABA (1 mM)-induced current decreased by 12 Ϯ 3% (n ϭ 4). Correcting the measured current at Ϫ50 mV holding potential for altered voltage and current yielded a value of 1.0 Ϯ 0.2 and 1.0 Ϯ 0.1 nmol/h net charge transfer/nmol/h organic substrate transported (Table III). The uncorrected values amount to 1.8 Ϯ 0.3 and 1.7 Ϯ 0.2.

DISCUSSION
This is the first demonstration that BGT-1 conveys electrogenic transport of betaine and GABA together with Na ϩ and Cl Ϫ . The substrate-induced currents in BGT-1-expressing oocytes parallel several transport properties defined previously (7)(8)(9)(10). In these studies it was observed that transport of betaine and GABA was inhibited by quinine, ␤-alanine, and DABA (7)(8)(9)(10). In agreement with these studies we found a significantly higher K m value for betaine compared with the value for GABA. According to the present results quinine, ␤-alanine, and DABA are not only inhibitors of GABA transport (7) but are substrates. The substrate recognition by BGT-1 mostly relies on the presence of an amino group. Methylated amines are accepted as well but with lower affinity, whereas guanidino groups are hardly recognized. A second negatively charged group is preferred but does not seem to be necessary. The substrate recognition also seems to be flexible with respect to the distance between both groups.
As shown previously (7-10), the substrate-induced current requires the presence of extracellular Na ϩ and Cl Ϫ . The Hill  coefficients and comparison of the tracer fluxes clearly indicate a coupling ratio for 1 GABA or betaine with 3 Na ϩ ions/transport cycle. For each GABA transported the carrier transports one or two Cl Ϫ ions. The data do not allow a clear discrimination between these two transport ratios. The Hill coefficient for Na ϩ above 2 is compatible with the binding of 3 Na ϩ . A variable stoichiometry has been described for another member of the GABA transporter family, GAT-1 (21). An earlier report showed a coupling ratio of GABA:Na ϩ :Cl Ϫ of 1:3:2 for the human betaine/GABA transporter (8). However, a nonsaturating GABA concentration was used and no saturation reached, both of which may influence the determination of the Hill coefficient for Cl Ϫ (8). Also, nontransporter Na ϩ or Cl Ϫ currents or fluxes in BGT-1-expressing oocytes could influence the calculated stoichiometry but were not significantly different from noninjected control oocytes in our experiments (Table III). The characteristics of the GABA/betaine transporter BGT-1 differ significantly from those of the well characterized neuronal GABA transporter GAT-1. First, no currents or radioactive fluxes of Na ϩ and Cl Ϫ are observed in the absence of GABA, although Na ϩ and Cl Ϫ are present (21,22). Second, GABA is cotransported with 3 Na ϩ instead of 2 (14, 18 -20). The maximal amplitude and apparent K m of GABA-and betaine-induced currents were voltage-dependent but not the Hill coefficients for GABA, Na ϩ , and Cl Ϫ . The currents were strictly inwardly rectifying as described for the GAT family (14,19,20,29). Depolarization of the cell membrane or a decrease of the GABA concentration strongly decreased the apparent Na ϩ affinity. Presumably the Na ϩ binding site is within the cell membrane and senses part of the electrical field. Hyperpolarization of the cell membrane favors entry of Na ϩ and thus increases the Na ϩ concentration at the binding site. We have tried to deduce the binding order from the effects of substrate and ion concentration on the maximum velocity (30). Because binding usually is not rate-limiting for transport, the uptake velocity should be dictated by the number of fully occupied transporters. In an ordered binding mechanism maximum velocity can therefore be reached with saturating concentrations of the last binding substrate, even in the presence of subsaturating concentrations of earlier binding substrates (30). The maximum velocity of GABA uptake decreases with decreasing Na ϩ and Cl Ϫ concentrations. On the other hand, the maximum transport current can still be reached at subsaturating GABA concentrations (see Fig. 5B). This is indicated by the intersection of the straight lines on the y axis in Fig. 5B. We therefore tentatively suggest that GABA binds prior to Na ϩ . The significant Cl Ϫ -independent GABA transport that was visible in flux and electrophysiological studies can be explained best by assuming a limited translocation of the GABA⅐3Na ϩ transporter complex. Alternatively, cotransport of the replacement anions gluconate, NO 3 Ϫ or SO 4 2Ϫ has to be assumed. However, in the face of the similar current in the presence of these completely different anions, this possibility is considered unlikely. It is very likely that binding of Cl Ϫ should occur after binding of Na ϩ . Based on the presented data we propose a transport model of ordered binding ( Fig. 9) in which GABA or betaine binds first to the extracellular side of the transporter. Na ϩ binding occurs after GABA/betaine but before Cl Ϫ binding. Cl Ϫ facilitates the translocation of the transporter, but there is a substantial transport rate even in the absence of extracellular Cl Ϫ .