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

Ioulia MatskevitchDagger §parallel , Carsten A. WagnerDagger §**, Carola StegenDagger Dagger , Stefan BröerDagger Dagger , Birgitta NollDagger , Teut Risler§§, H. Moo Kwon¶¶, Joseph S. Handler¶¶, Siegfried WaldeggerDagger , Andreas E. BuschDagger , and Florian LangDagger ||

From the Departments of Dagger  Physiology, Dagger Dagger  Biochemistry, and §§ Internal Medicine, University of Tübingen, Tübingen 72076, Germany, the ¶¶ Division of Nephrology, Department of Internal Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the § Sechenov Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma -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 gamma -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 = beta -alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km 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 Km 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 22Na+-, 36Cl--, and 14C-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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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+-coupled 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-11 for betaine/GABA (7-10), 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+- and Cl--coupled transporters for neurotransmitters, amino acids, and osmolytes. Four different GABA transporters have been cloned (7, 10, 15-18) 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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 [14C]GABA, 22NaCl, and H36Cl (NEN Life Science Products, Brussels, Belgium) and [14C]betaine (Biotrend, Cologne, Germany). BGT-1-expressing oocytes and noninjected control oocytes were washed twice with ice-cold OR2+ buffer (calcium-containing oocyte Ringer OR2+: 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, 1 mM CaCl2, 5 mM HEPES, pH 7.8 (28)). Uptake was initiated by addition of 100 µl of OR2+ buffer supplemented with 1 mM GABA and one of the radioactive substrates [14C]GABA, [14C]betaine (final specific activity 63.0 Bq/nmol), or 22NaCl (final specific activity 21.9 Bq/nmol). Chloride uptake was studied in transport buffer in which unlabeled NaCl was replaced by H36Cl (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 µl of supplemented OR2+ buffer. Uptake was stopped after 30 min or 1 h by washing the oocytes with ice-cold OR2+ buffer. Single oocytes were lysed by adding 200 µl 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 -50 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 CaCl2, 1 mM MgCl2 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, NO3-, or SO42-. 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 (OR2+ versus ND96) control experiments were performed, and currents and uptake were measured under both conditions. No significant difference was found.

Data are provided as means ± 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 < 0.05 were considered statistically significant.

    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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In current clamp studies superfusion of BGT-1 expressing Xenopus oocytes with 1 mM GABA or betaine caused a depolarization of 27.6 ± 2.0 mV (n = 5) and 24.2 ± 1.7 mV (n = 5), respectively. In the voltage clamp modus 1 mM GABA or betaine induced a mean inward current of -73.9 ± 3.8 nA and -75.3 ± 4.1 nA, respectively, as illustrated in Fig. 1A. In noninjected control oocytes GABA did not induce significant currents (0.9 ± 0.3 nA, n = 5), whereas betaine led to an inward current of -7.2 ± 1.5 nA (n = 5). The betaine-induced current in noninjected oocytes was independent of the presence of Na+ 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.


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Fig. 1.   Panel A, substrate-induced current in oocytes expressing BGT-1. Original tracings for GABA, betaine, DABA, beta -alanine, and proline (1 mM) are shown. Panel B, voltage dependence of GABA-induced currents (1 mM) expressed as a fraction of the current of GABA at -90 mV (I/Imax). Currents were measured during voltage ramps from -100 to +40 mV in the absence (control) and presence of GABA, and currents under GABA were subtracted from control currents.

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 = betaine > DABA > beta -alanine > proline > quinidine > glycine = putrescine > choline > sarcosine = tetraethylammonium = taurine = carnitine > betaine aldehyde = creatine (Table I; all substances were tested on the same oocytes, n = 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 = 5).

                              
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Table I
Substrates and specificity of BGT-1
Mean values ± 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 -50 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.

As shown in Fig. 1B, the GABA-induced currents in BGT-1-expressing oocytes were voltage-dependent, increasing with hyperpolarization. In ND96 buffer the GABA-induced currents were strictly inwardly rectifying in the range between -90 and +40 mV (n = 5). The holding potential affected both the maximum velocity and the apparent Km of GABA- and betaine-induced currents. As shown in Fig. 2 the GABA concentration required for half-maximal current (apparent Km) increased from 0.009 ± 0.001 mM at -100 mV holding potential to 0.031 ± 0.004 mM at a more depolarized holding potential of -30 mV. On the other hand, a depolarization from -100 to -50 and -30 mV decreased the maximal current by 31 ± 2% and 51 ± 2%, respectively (Fig. 2A, n = 4). The apparent Km for betaine-induced currents was shifted significantly from 0.51 ± 0.04 mM at -80 mV holding potential to 2.1 ± 0.2 mM at -30 mV (n = 4). The maximal betaine-induced current was not significantly altered by depolarization (Fig. 2B).


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Fig. 2.   Kinetics of betaine- and GABA-induced currents. Panel A, voltage dependence of GABA-induced currents. All curves were obtained by fitting the data to the Hill equation: I = Imax * [substrate]n/[substrate]n + (Km)n, where n and [substrate] give the Hill coefficient and the substrate (GABA/betaine) concentration, respectively; Imax is the extrapolated maximal current; and Km is the apparent concentration needed for half-maximal current. All currents were normalized against the maximal current at 1 mM GABA at -90 mV holding potential. The Hill coefficients for the curves were not significantly different (-100 mV, 0.8 ± 0.1; -50 mV, 1.0 ± 0.1; -30 mV, 0.8 ± 0.1, n = 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 -80 mV holding potential and 5 mM betaine. The difference of the apparent Km for the different holding potentials was significant on the p < 0.05 level. The Hill coefficients for the curves were not significantly different (-80 mV, 1.1 ± 0.2; -50 mV, 1.0 ± 0.1; -30 mV, 0.6 ± 0.2).

The betaine-induced currents were strictly Na+- and partially Cl--dependent. The apparent Km for Na+ was 93.3 ± 3.9 mM and for Cl- 68.3 ± 0.8 mM (n = 5, Fig. 3, A and B). The Hill coefficient for Na+ was 2.5 ± 0.2 and for Cl- 1.7 ± 0.1. 


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Fig. 3.   Na+- and Cl- dependence of betaine (1 mM)-induced currents. All currents were normalized against the maximal current at 150 mM Na+/1 mM betaine and 150 mM Cl-/1 mM betaine, respectively.

Also, the GABA-induced currents were strictly Na+-dependent. In the absence of Na+, the addition of 1 mM GABA failed to induce any current. The GABA (1 mM)-induced current increased with the extracellular Na+ concentration (at 103.6 mM Cl- concentration) with a half-maximal current at 65.9 ± 8 mM Na+ concentration at -90 mV and at 89 ± 3 mM Na+ concentration at -50 mV (Fig. 4A, n = 4). Thus, hyperpolarization of the cell membrane increased the affinity of the carrier to Na+. The Hill coefficient for Na+ was 2.2 ± 0.4 at -50 mV and was not altered significantly by hyperpolarization to -90 mV. A decrease of ambient Na+ concentration decreased both maximal current and apparent affinity for GABA. The apparent Km for GABA increased slightly from 0.021 ± 0.002 mM at 130 mM Na+ in the superfusate to 0.056 ± 0.006 mM at 80 mM Na+ (Fig. 4B). The Hill coefficient, however, remained close to 1. To test whether GABA influences Na+ binding, the GABA concentration was decreased from 1 to 0.03 mM, which markedly shifted the apparent affinity for Na+ from 55 ± 2 mM to about 120 mM without affecting the Hill coefficient or the calculated Vmax value (Fig. 5, n = 5).


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Fig. 4.   Na+ dependence of GABA-induced currents in BGT-1-expressing oocytes. Panel A, GABA-induced currents are Na+-dependent, and the apparent Km for Na+ is voltage-dependent. The apparent affinity for Na+ was significantly shifted from 89 ± 3 mM at -50 mV to 66 ± 8 mM at -90 mV holding potential. The Hill coefficient was 2.2 ± 0.5 for -50 mV and 1.8 ± 0.2 for -90 mV. All data were normalized against the maximal current at 150 mM Na+ and 1 mM GABA, and the Hill equation was used as described above. Panel B, effect of Na+ on the apparent Km for GABA. Reduction of Na+ from 150 to 80 mM in the superfusate significantly increases the apparent Km from 0.021 ± 0.002 mM to 0.056 ± 0.006 mM. Curves were obtained by fitting the data to the Hill equation. The Hill coefficient was 1.5 ± 0.2 for 130 mM Na+ and 1.1 ± 0.1 for 80 mM Na+. Inset, Lineweaver-Burk plot for the same data.


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Fig. 5.   GABA influences Na+ binding. Panel A, Na+ kinetics with 1 mM or 0.03 mM GABA. The apparent Km for Na+ was shifted from 55 ± 2 mM under 1 mM GABA to about 120 mM under 0.03 mM GABA. Experiments were carried out at a holding potential of -80 mV (n = 5). Both curves were obtained by fitting the data to the Hill equation. The Hill coefficient was about 2 for both conditions. The calculated Vmax value for 1 and 0.03 mM GABA was 1.1 ± 0.1. Panel B, Lineweaver-Burk plot for the same data.

As illustrated in Fig. 6, GABA-induced currents were also partially Cl--dependent. Reduction of extracellular Cl- from 150 to 0 mM reduced the GABA-induced current by about 80% at -90 mV and by 90% at -50 mV (Fig. 6, A and B). Replacing Cl- with anions other than gluconate (NO3- and SO42-) showed similar results. Varying the extracellular Cl- concentration revealed that the half-maximal current induced by 1 mM GABA was reached at 78 ± 4 mM Cl- concentration at -90 mV and at about 114 ± 5 mM Cl- concentration at -50 mV. For fitting the curves an offset (Cl--independent GABA-induced current) of 0.2 and 0.08 for -90 and -50 mV, respectively, was assumed. The apparent Km for Cl- at a holding potential of -50 mV could only be estimated because saturating conditions could not be reached under experimental conditions (n = 5). The apparent Cl- affinity of the carrier was decreased by depolarization. The Hill coefficient for Cl- was close to 1 irrespective of the holding potential. Furthermore, a reduction of the extracellular Cl- concentration from 130 to 50 mM resulted in a slight increase of the apparent Km value for GABA from 0.033 ± 0.001 to 0.049 ± 0.003 mM, pointing to a small but significant decrease of GABA affinity (Fig. 6C, n = 5). The maximum velocity of GABA uptake decreased significantly with decreasing Cl- concentration.


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Fig. 6.   Cl- dependence of GABA-induced current. Panel A, original tracing showing GABA-induced currents at -90 mV in the presence (150 mM) and the absence (0 mM) of Cl-. The start of a 30-s superfusion period is indicated by the arrows. Panel B, GABA-induced currents were a curvilinear function of Cl- concentration in the superfusate. The apparent Km for Cl- was voltage-dependent and increased from 78 ± 4 mM to 114 ± 5 mM with depolarization of the holding potential from -90 to -50 mV. The apparent Km for -50 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--independent GABA-induced current) of 0.2 and 0.07 for -90 and -50 mV, respectively. All data were normalized against the maximal current at 150 mM Cl- and -90 mV holding potential. The Hill coefficient for Cl- was 1.1 ± 0.1 and 1.0 ± 0.1 for -90 and -50 mV holding potential, respectively. Panel C, Cl- influenced the apparent Km for GABA. Reduction of Cl- significantly increased the apparent Km for GABA from 0.033 ± 0.001 mM (130 mM Cl-) to 0.049 ± 0.003 mM (80 mM Cl-). Curves were obtained by fitting the data to the Hill equation. All data were normalized against the maximal current at 130 mM Cl- and 1 mM GABA. The Hill coefficient for GABA was 1.02 ± 0.05 for 130 mM Cl- and 1.09 ± 0.07 for 50 mM Cl-. Inset, Lineweaver-Burk plot of the same data.

To examine the mutual interaction of Na+ and Cl- we performed Na+ and Cl- kinetics at different Cl- and Na+ concentrations, respectively (Fig. 7). The apparent Km for Na+ was shifted from 54 ± 3 mM at 150 mM Cl- to 82 ± 7 mM at 80 mM Cl-. The Hill coefficient was 2.5 ± 0.2 for both extracellular Cl- concentrations (Fig. 7A, n = 5). On the other hand, a decrease of the ambient Na+ concentration from 150 to 80 mM turned the apparent Km for Cl- from 72 ± 3 mM (Hill coefficient of 1.1 ± 0.2) into a nonsaturable chloride concentration dependence (Fig. 7C, n = 5). A summary of the kinetic constants derived from these experiments is shown in Table II.


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Fig. 7.   Mutual interaction of Na+ and Cl- concentration on GABA-induced current. Panel A, effect of Cl- on the apparent Km of Na+. Reduction of Cl- in the superfusate from 150 to 80 mM significantly decreased the affinity for Na+. The apparent Km was increased from 54 ± 3 mM (150 mM Cl-) to 82 ± 7 mM under 80 mM Cl-. The Hill coefficient, however, remained close to 2 for both conditions (1.8 ± 0.4 for 150 mM Cl- and 2.5 ± 0.3 for 80 mM Cl-). Curves were obtained by fitting the data to the Hill equation. All data were normalized against the maximal current at 150 mM Na+ and 150 mM Cl-. Panel B, Lineweaver-Burk plot for the same data. Panel C, influence of Na+ on the affinity for Cl-. Reduction of Na+ from 130 to 80 mM significantly shifted the apparent Km for Cl- from 72 ± 3 to an estimated apparent Km of about 220 mM. The Hill coefficient was 1.2 ± 0.1 for 150 mM Na+. 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- and 150 mM Na+. Panel D, Lineweaver-Burk plot of the GABA concentration dependence.

                              
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Table II
Summary of kinetic constants

To determine further the Na+ and Cl- dependence and coupling ratio we carried out uptake studies with radioactive [14C]GABA, 22Na+, and 36Cl-. The uptake of GABA was linear over a time period of 30 min (Fig. 8). In agreement with GABA-induced currents, the uptake of labeled GABA was completely Na+-dependent (n = 7) but only partially Cl--dependent (n = 7) (Table III). The cotransport stoichiometry in relation to GABA was 1:3.1 for Na+ and 1:1.3 for Cl- (Table IV). The addition of 10 mM betaine to the incubation buffer resulted in a substrate-stimulated 22Na uptake of 5.2 ± 1.2 nmol/h and a substrate-stimulated 36Cl uptake of 3.3 ± 0.7 nmol/h, reflecting a ratio of 3 Na+ to 1.9 Cl-. 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+, Cl-, 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 ± 9 nA (n = 7) and 59 ± 6 nA (n = 7) have been determined for the Na+/GABA and the Cl-/GABA flux measurements, respectively. Electrophysiological experiments under conditions mimicking the flux experiments revealed that during current clamp, the addition of 1 mM GABA led to an initial depolarization (by 13.6 ± 1.0 mV, n = 9) followed by slow recovery of cell membrane potential (by 14.6 ± 1.1 mV, n = 9). At voltage clamp conditions, the GABA (1 mM)-induced current decreased by 12 ± 3% (n = 4). Correcting the measured current at -50 mV holding potential for altered voltage and current yielded a value of 1.0 ± 0.2 and 1.0 ± 0.1 nmol/h net charge transfer/nmol/h organic substrate transported (Table III). The uncorrected values amount to 1.8 ± 0.3 and 1.7 ± 0.2. 


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Fig. 8.   Uptake of [14C]GABA by BGT-1-expressing oocytes. [14C]GABA uptake in BGT-1-expressing oocytes (closed circles) and noninjected oocytes (closed squares) is shown over a time period of 30 min. The difference of both experiments is shown as the net uptake (open circles).

                              
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Table III
Ion dependence of GABA transport in BGT-1-expressing oocytes
Uptake of [14C]GABA (1 mM) was determined under different ionic conditions. Groups of seven oocytes were washed, and subsequently uptake activity was determined in ND96, Na+-free, or Cl--free ND96 buffer. Uptake was terminated by washing the oocytes four times in ice-cold ND96 buffer. The uptake activity of noninjected oocytes was determined under identical conditions.

                              
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Table IV
Uptake of [14C]GABA, 32Na+, and 36Cl- by BGT-1-expressing Xenopus oocytes
All data are given in nmol/h/oocytes calculated from the respective tracer fluxes. The charge transfer was calculated from GABA-induced currents considering that 1 nA corresponds to 36 pmol/h/oocyte. The corresponding data are indicated by an asterisk (*). For a second set of experiments similar data were obtained (not shown); n = seven oocytes for each experiment.


    DISCUSSION
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INTRODUCTION
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This is the first demonstration that BGT-1 conveys electrogenic transport of betaine and GABA together with Na+ and Cl-. The substrate-induced currents in BGT-1-expressing oocytes parallel several transport properties defined previously (7-10). In these studies it was observed that transport of betaine and GABA was inhibited by quinine, beta -alanine, and DABA (7-10). In agreement with these studies we found a significantly higher Km value for betaine compared with the value for GABA. According to the present results quinine, beta -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+ and Cl-. The Hill coefficients and comparison of the tracer fluxes clearly indicate a coupling ratio for 1 GABA or betaine with 3 Na+ ions/transport cycle. For each GABA transported the carrier transports one or two Cl- ions. The data do not allow a clear discrimination between these two transport ratios. The Hill coefficient for Na+ above 2 is compatible with the binding of 3 Na+. 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+:Cl- 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- (8). Also, nontransporter Na+ or Cl- 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+ and Cl- are observed in the absence of GABA, although Na+ and Cl- are present (21, 22). Second, GABA is cotransported with 3 Na+ instead of 2 (14, 18-20). The maximal amplitude and apparent Km of GABA- and betaine-induced currents were voltage-dependent but not the Hill coefficients for GABA, Na+, and Cl-. 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+ affinity. Presumably the Na+ binding site is within the cell membrane and senses part of the electrical field. Hyperpolarization of the cell membrane favors entry of Na+ and thus increases the Na+ 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+ and Cl- 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+. The significant Cl--independent GABA transport that was visible in flux and electrophysiological studies can be explained best by assuming a limited translocation of the GABA·3Na+ transporter complex. Alternatively, cotransport of the replacement anions gluconate, NO3- or SO42- 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- should occur after binding of Na+. 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+ binding occurs after GABA/betaine but before Cl- binding. Cl- facilitates the translocation of the transporter, but there is a substantial transport rate even in the absence of extracellular Cl-.


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Fig. 9.   Proposed model for ordered binding of GABA/betaine, Na+, and Cl- from the exterior side of the membrane to the BGT-1 transporter. GABA/betaine binds first to the transporter, and Na+ binding occurs before Cl- binding. However, for the translocation of the transporter to the interior side of the membrane (dotted line), Cl- is not required. About 20% of the GABA/betaine-induced current and uptake is Cl--independent. Full transport is only evoked after Cl- binding. The coupling ratio of GABA/betaine to Na+ and Cl- may be either 1:3:2 or 1:3:1. The order of substrate release at the interior side is completely hypothetical and not known so far.


    FOOTNOTES

* 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. The 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.

parallel Supported by European Molecular Biology Organization and DAAD fellowships.

** Supported by Fellowship Fö. 01KS9602 from the Federal Ministry of Education, Science, Research, and Technology and by the Interdisciplinary Clinical Research Center, Tübingen, Germany.

|| To whom correspondence should be addressed: Institute of Physiology I, University of Tübingen, Gmelinstrasse 5, 72076 Tübingen, Germany. Tel.: 49-7071-297-2194; Fax: 49-7071-295-618; E-mail: florian.lang{at}uni-tuebingen.de.

    ABBREVIATIONS

The abbreviations used are: BGT-1, betaine/GABA transporter; GABA, gamma -aminobutyric acid; GAT, GABA transporter; DABA, diaminobutyric acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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