Functional Characterization of the Betaine/
-Aminobutyric Acid
Transporter BGT-1 Expressed in Xenopus Oocytes*
Ioulia
Matskevitch
§¶
,
Carsten A.
Wagner
§¶**,
Carola
Stegen
,
Stefan
Bröer
,
Birgitta
Noll
,
Teut
Risler§§,
H. Moo
Kwon¶¶,
Joseph S.
Handler¶¶,
Siegfried
Waldegger
,
Andreas E.
Busch
, and
Florian
Lang
||
From the Departments of
Physiology,

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 |
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 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 |
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 |
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 |
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,
-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.
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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 >
-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.
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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).
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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.
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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.
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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.
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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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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.
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 |
DISCUSSION |
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,
-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,
-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.
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,
-aminobutyric acid;
GAT, GABA transporter;
DABA, diaminobutyric acid.
 |
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