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Volume 16, Number 17,
Issue of September 1, 1996
pp. 5405-5414
Copyright ©1996 Society for Neuroscience
Ion Binding and Permeation at the GABA Transporter GAT1
Sela Mager1,
Nurit Kleinberger-Doron2,
Gilmor I. Keshet2,
Norman Davidson1,
Baruch I. Kanner2, and
Henry A. Lester1
1 Division of Biology, California Institute of
Technology, Pasadena, California 91125, and 2 Department of
Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem
91120, Israel
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
This study addresses the binding of ions and the permeation of
substrates during function of the GABA transporter GAT1. GAT1 was
expressed in Xenopus oocytes and studied
electrophysiologically as well as with [3H]GABA flux;
GAT1 was also expressed in mammalian cells and studied with
[3H]GABA and [3H]tiagabine binding. Voltage
jumps, Na+ and Cl concentration jumps, and
exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all
produce capacitive charge movements. Occlusive interactions among these
three types of perturbations show that they all measure the same
population of charges. The concentration dependences of the charge
movements reveal (1) that two Na+ ions interact with the
transporter even in the absence of GABA, and (2) that Cl
facilitates the binding of Na+. Comparison between the
charge movements and the transport-associated current shows that this
initial Na+-transporter interaction limits the overall
transport rate when [GABA] is saturating. However, two classes of
manipulation treatment with high-affinity uptake blockers and the W68L
mutation``lock'' Na+ onto the transporter by slowing or
preventing the subsequent events that release the substrates to the
intracellular medium. The Na+ substitutes Li+
and Cs+ do not support charge movements, but they can
permeate the transporter in an uncoupled manner. Our results (1)
support the hypothesis that efficient removal of synaptic transmitter
by the GABA transporter GAT1 depends on the previous binding of
Na+ and Cl, and (2) indicate the important
role of the conserved putative transmembrane domain 1 in interactions
with the permeant substrates.
Key words:
sodium;
chloride;
GAT1;
GABA;
transporter;
Xenopus oocyte
INTRODUCTION
This paper addresses the mechanism of GABA
transport by GAT1 (Guastella et al., 1990 ) and the implications for
efficient synaptic transmission. Within a few milliseconds after
release, the transmitter is removed from the extracellular space. As a
result, receptors begin to deactivate and transmitter cannot diffuse to
neighboring synapses (Isaacson et al., 1993; Lester et al., 1994). The
highly efficient enzyme acetylcholinesterase removes the transmitter at
nicotinic synapses, but at all other chemical synapses, ion-coupled
transporters play this role despite turnover rates approximately three
orders of magnitude lower than that of acetylcholinesterase and slower
than the decay rate of the synaptic event itself (Lester et al., 1994;
Wadiche et al., 1995).
The transport process is driven by the electrochemical potential of
Na+ and Cl (Kanner and Schuldiner, 1987;
Lester et al., 1994): two Na+ ions and one Cl
ion are transported for each GABA molecule. The cotransported ions must
bind to the transporter at some step during the cycle. Previous
experiments suggest that the ions can bind in the absence of GABA but,
interestingly, the interaction between Na+ and the
transporter requires several hundred millisecondsmany orders of
magnitude longer than required for Na+ permeation through
an ion channel (Mager et al., 1993; Cammack et al., 1994). The present
study attempts to understand the molecular nature of this interaction.
We address the specific suggestion that the binding of Na+
and Cl converts the transporter to a state with higher
affinity for GABA (tens of µM). This process allows the
transporter to initiate the steps leading to release of GABA into the
cytoplasm. Although we do not directly address the functional
importance of these mechanistic findings for synaptic transmission, we
extend earlier suggestions that a high-affinity binding state would
allow the transmitter to sequester the GABA that appears in the
synaptic cleft, preventing further receptor activation (Cammack et al.,
1994; Tong and Jahr, 1994; Wadiche et al., 1995).
We have studied the transporter with electrophysiological measurements
that illuminate individual steps in the transport cycle. Although one
cannot yet resolve the binding of one or two ions to a single
transporter protein, synchronized binding to many transporters
generates a measurable current. We synchronize the binding process by
rapidly changing the concentration of [Na+] or
[Cl], the concentration of a high-affinity blocker, or
the membrane potential (Mager et al., 1993; Galli et al., 1995; Wadiche
et al., 1995). We correlate our data with steady-state measurements of
[3H]GABA uptake and of the currents associated with the
entire transport cycle. We also study the poorly functional mu-
tant W68L.
Classical results show that only Na+ among monovalent
cations supports GABA flux (Kanner and Schuldiner, 1987; Lester et al.,
1994). Yet experience with other cation-binding proteins, such as ion
channels, leads one to expect that other monovalent cations would bind,
although presumably not as well as Na+ or with different
consequences. We find that Cs+ and Li+ permeate
through the transporter. However, in contrast to transport with
Na+, these monovalent cations do not pause while awaiting
GABA; they permeate in its absence. This uncoupled flux of
Cs+ or Li+ again emphasizes the unique nature
of the Na+-transporter interaction that prepares the
transporter to accept GABA.
MATERIALS AND METHODS
Oocytes. Wild-type (WT) GAT1 and W68L
(Kleinberger-Doron and Kanner, 1994) were transferred to pAMV-PA (Nowak
et al., 1995), which is optimized for oocyte expression. RNA was
synthesized in vitro with T7 polymerase and injected into
stage VI Xenopus oocytes (Quick and Lester, 1994). For rapid
solution changes, oocytes were held by pins in a chamber (volume, 100 µl) the volume of which was changed with a time constant of 700 msec
under the control of electrically operated valves and studied with a
two-electrode voltage-clamp circuit (Mager et al., 1993). We found that
oocytes expressing both WT and W68L GAT1 had increased linear
capacitive transients (time constant < 7 msec), presumably
because of increased membrane area, as observed for the  2 subunit of
voltage-gated Na+ channels (Isom et al., 1995). These
transients have not been included in our analyses. Normal Ringer
solution contains 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES. Equimolar
substitutions with other ions were made as described in the text.
GABA uptake in oocytes was measured by a 1 min incubation in 200 µl
of Ringer solution containing 100 µM GABA. Oocytes were
washed, dissolved in 1% SDS, and counted using liquid scintillation
spectrometry.
Mammalian cells. Cultured HeLa cells (Keynan et al., 1992)
were infected with recombinant vaccinia virus vTF7-3 (Fuerst et al.,
1986), which encodes the T7 RNA polymerase, and subsequently
transfected with pT7-GAT1 or the mutated transporter W68L
(Kleinberger-Doron and Kanner, 1994). [3H]GABA uptake was
measured in 35 mm dishes by a 10 min incubation in 42 nM
GABA (Keynan et al., 1992), followed by a wash with 2 ml of Ringer
solution.
For binding of [3H]tiagabine, cells were scraped from six
35 mm wells in 2.0 ml of ice-cold 150 mM LiCl containing 10 mM HEPES-Tris, pH 6; all further steps of the membrane
preparation were done at 4°C. A few grains of DNase were added, and
the cell suspension was disrupted using a Polytron homogenizer with two
exposures for 15 sec at setting 5. Cell extracts were pelleted and
resuspended in 9 ml of 1 M LiCl or NaCl containing 10 mM Tris-HEPES, pH 6.8. Ten microliters of a 10% ethanol
solution containing 2.5 pmol of [H3]tiagabine (39.8 Ci/mmol, gift of Dr. Peter Suzdak, Novo Nordisk) were added to 190 µl
of membrane preparation containing 40-50 µg of protein. After 30 min
at 30°C, the reactions were terminated on 1 ml spin columns
containing Sephadex-G-50-80 (Pharmacia, Uppsala, Sweden) pre-swollen
with 1 M LiCl containing 10 mM HEPES-Tris, pH
6.8 (Radian and Kanner, 1985). The recovered radioactivity was counted
using liquid scintillation spectrometry. In the presence of
Na+, membranes from cells expressing the WT transporter
attained equilibrium [3H]tiagabine binding over a time
course of several minutes, at a level of ~1 pmol/mg protein. In the
absence of Na+, ligand binding was lower (~0.25 pmol/mg
protein), reached equilibrium in <10 sec, and resembled levels for
membranes prepared from HeLa cells transfected with the vector
pBluescript (without the GAT1 cDNA insert) alone, regardless of the
presence of Na+. The Na+-independent binding,
therefore, represents nonspecific binding to the membranes and was been
subtracted from the value obtained in the presence of Na+.
Excess unlabeled GABA (10 mM) reduced
Na+-dependent tiagabine binding in the wild-type and the
W68L mutant to the nonspecific levels.
RESULTS
Charge movements linked to Na+ binding
The GABA transporter GAT1 was expressed in Xenopus
oocytes by cytoplasmic injection of cRNA. In the experiment shown in
Figure 1A, an oocyte expressing GAT1
generated a transient outward current when the bathing solution with
normal Na+ (96 mM) was switched to one
containing no Na+, replaced by
N-methyl-D-glucamine (NMDG). A similar but
inward transient current developed after re-addition of
Na+. These transient currents were absent in the presence
of the GABA uptake blocker SKF-100330A, as shown in Figure
1A, which allows a subtraction procedure that
isolates the transient currents (Fig. 1A,
bottom panel). The transient currents were also absent
in noninjected or water-injected oocytes (data not shown).
Fig. 1.
Na+ concentration jumps and voltage
jumps in a single oocyte expressing GAT1. A, The
membrane potential was held at  40 mV during perfusion with an NaCl
Ringer solution. At the indicated times, the perfusion was changed to
NMDG-Cl Ringer solution and returned to NaCl Ringer solution.
B, After an additional 5 sec, the membrane potential was
stepped to +60 mV and back to 40 mV. The oocyte was then perfused
with a Na+ Ringer solution containing 30 µM
SKF-100330A, and the protocol was repeated. The records in the presence
of SKF-100330A have been subtracted from those in the absence of the
drug so as to isolate GAT1 currents. C, Comparison of
charge movements for the Na+ concentration jumps (96 0 vs
096 mM Na+) at a holding potential of 40
mV. D, Charge movements during concentration jumps
(960 mM Na+) versus voltage jumps
(40+60 mV).
[View Larger Version of this Image (19K GIF file)]
The charge movement during the outward current is calculated as the
time integral of the transient current. This charge movement was nearly
equal and opposite to that during the inward current (average ratio of
0.885 for the 14 oocytes in Fig. 1C). Thus, these transient
currents are capacitive, rather than resistive; they result from ion
binding and dissociation rather than from ion permeation. The small
deviation from a ratio of 1 may be attributable to resistive leakage
currents (Cammack et al., 1994).
The interaction between ions and GAT1 can also be synchronized by
jumping the membrane potential (Fig. 1B) (Loo et
al., 1993; Mager et al., 1993). We compared charge movements during the
concentration-jump and voltage-jump procedures for synchronizing charge
movement. For both measurements, the experiment began with the membrane
clamped to 40 mV in normal Na+ Ringer solution. Either a
jump to zero Na+ (Fig. 1A) or a jump to
very positive voltage (+60 mV in the experiment of Fig.
1B) is expected to remove Na+ from the
transporters. In agreement with this view, the capacitive charge
movements were equal for these two types of perturbation (average ratio
of 1.01 for the 7 oocytes plotted in Fig. 1D). Thus, it is
likely that a common molecular event is responsible for Na+
binding and release during the concentration-jump and voltage-jump
procedures.
It is already thought that two Na+ ions accompany the
transport of a single GABA molecule (Kanner and Schuldiner, 1987).
[Na+] dependence of the concentration-jump charge
movements displayed a Hill coefficient (nH) near
2 (Fig. 2A), similar to the Hill
coefficient of the transport-associated current (Mager, 1993).
Therefore, we conclude that the charge movement arises either from a
highly cooperative binding of two Na+ ions within the
membrane dielectric or from independent binding followed by a
conformational change that moves charge(s) on the transporter within
the membrane. More rapid Na+ concentration jumps (Cammack
et al., 1994) may eventually allow a decision between these two
choices, but it is already clear that even this early event in the
transport cycle is much more likely to occur in the presence of two
bound Na+ ions than in the presence of a single
Na+ ion. We suggest that this early event is the step, or
one of the steps, that prepares the transporter to accept GABA.
Fig. 2.
Effect of [Na+] and membrane
potential on charge movements. A, Effect of
[Na+] on concentration-jump charge movements for the WT
( ) or W68L ( ). Membrane potential was held at 80 mV, and
[Na+] was increased in steps. The normalized charge
movement is plotted as a function of [Na+]. The original
data were fit to the Hill equation with Qmax
of 60 ± 8 and 99 ± 27 nC, an EC50 of 43 ± 6 and 2.7 ± 0.5 mM, and Hill coefficient
n of 1.8 ± 0.14 and 1.7 ± 0.13 for the WT (4 oocytes) and W68L (5 oocytes), respectively. B,
C, Effect of membrane potential and [Na+]
on the voltage-jump charge movements for oocytes expressing the WT
transporter (B) and W68L mutant (C). For
each oocyte, charge movement was measured during voltage jumps from a
holding potential of 40 mV to various voltages between 140 and +60
mV in various [Na+]. Charge movements were fit to a
Boltzmann distribution and shifted vertically so that the midpoints of
the curves at varying [Na+] lay on the same horizontal
line. [Na+] was 12, 24, 48, 77, and 96 mM in
B and 1, 3, 6, 12, 24, 48, 77, and 96 mM in
C. D, Fitted values for
V1/2 are plotted versus [Na+].
Data points are averaged from 3 oocytes for each transporter (SEM are
smaller than the size of the symbols). Lines are linear regression,
corresponding to V1/2 changes of 106 ± 5 and 101 ± 3 mV for the WT () and W68L (), respectively,
for a 10-fold change in [Na+]. E,
Dose-response relations for the transport-associated current at 80
mV. The data were fit to the Hill equation as follows. For the WT,
EC50 = 44 ± 1.8 mM,
nH = 1.8 ± 0.15; for the mutant,
EC50 = 6.76 ± 1.5 mM,
nH = 1.63 ± 0.07 (300 µM
and 3 mM GABA for WT and W68L, respectively; 3 oocytes
tested for each transporter).
[View Larger Version of this Image (35K GIF file)]
The W68L mutant: charge movements
The mutant W68L displays poor transport function
(Kleinberger-Doron and Kanner, 1994); our analysis of transport
function for this mutant will be presented in detail below. At this
point, we compare the first step in transport between W68L and WT
transporters by comparing the charge movements during
Na+-concentration jumps. Strikingly, W68L charge movements
occur at Na+ concentrations much lower than for WT; as
shown in Figure 2A, the EC50 was 2.7 mM versus 43 mM, respectively. On the other
hand, [Na+] dependence of the concentration-jump charge
movements displayed similar maximal charge movements
(Qmax) and Hill coefficients. Thus, a major
difference between WT and W68L is that the two Na+ ions
bind more tightly to the latter. We have found that the W68S mutant
also displays tighter Na+ binding (data not shown).
We compared the kinetics of voltage-jump charge movements for the
WT and W68L transporters (Fig. 3) and found that the
charge movements occur severalfold faster for the W68L transporter.
This difference persists even when the kinetics are corrected for
[Na+] dependence. For instance, at 140 mV charge
movements were half-complete at 12 mM for the WT and at 1 mM for W68L, and the corresponding time constants were
252 ± 11 and 56 ± 5 msec, respectively (mean ± SEM,
n = 9 and 4, respectively). Thus, the inefficient
transport for the W68L mutant cannot be explained by an abnormally slow
interaction between Na+ and the transporter; if anything,
this process (or the subsequent conformational change) proceeds more
quickly for the mutant.
Fig. 3.
Kinetics of voltage-jump relaxations for WT and
W68L transporters. Top panels, Voltage-clamp currents
for jumps from 40 to +40 mV in various [Na+] were
measured ([Na+] in mM are shown for each
trace); GAT1 currents were isolated by subtraction of traces in the
presence of 10 µM NO-05-711. Single-exponential fits have
been superimposed on the traces. Bottom panels, Voltage
and [Na+] concentration of the time constants.
Asterisks denote data from traces in the top
panels.
[View Larger Version of this Image (33K GIF file)]
Interestingly, the data show that the time constants of charge
movements show a maximum as a function of membrane potential.
Comparison of Figures 2 and 3 shows that these maxima occur at
approximately the midpoint of the charge-voltage relation.
A formal description of the charge movements
We have measured the charge movements as they depend on both
Na+ (Fig. 2A) and membrane potential
(Fig. 2B,C) for both the WT and W68L transporter.
At this point, it is appropriate to present a formal synthesis of these
measurements. A modified Hill equation (Eq. 1) presents the effect of
[Na+] and the membrane potential (V)
on Na+ binding to the transporter (Fig.
2B-D):
![Q(V,Na)=<FR><NU>Q<SUB>max</SUB></NU><DE><FENCE>1+<FENCE>[Na]/K<SUB>Na</SUB> exp (−Vq&dgr;/kT)</FENCE><SUP>n<SUB>H</SUB></SUP></FENCE></DE></FR> .](/content/vol16/issue17/fulltext/5405/img001.gif)
|
(1)
|
In the above equation,
Q(V,Na) represents the
fraction of transporters without bound Na+, q is
the elementary charge,  is the electrical distance at which the two
Na+ bind, and KNa is a zero-voltage
intrinsic dissociation constant of the transporter for Na+.
Detailed physical interpretations of this equation are given in
Discussion. For the present, we point out the simple interpretation
that the charge movements occur if and only if two Na+ ions
bind simultaneously at the same electrical distance; however, basically
similar formalisms would allow for the possibilities either (1) that
some charge movement can occur in the absence of Na+
binding or (2) that the two ions bind at different electrical
distances.
The only parameter in Eq. 1 that differs significantly for WT and W68L
transporters is KNa (518 and 30 mM,
respectively; 3 oocytes in each case). The calculated electrical
distances, , were 0.63 ± 0.03 and 0.67 ± 0.02 for the WT
and the mutant, respectively; nH ranged from
1.66 to 1.45 (WT and W68L, respectively). Eq. 1 is also a Boltzmann
relation (Mager et al., 1993) with a midpoint potential of
V1/2 = kT/q ln
([Na]/KNa) and slope
s = kT/nHq. It is
equivalent, therefore, to conclude that Na+ binds more
tightly to the mutant at all potentials or that its binding is shifted
to more positive potentials.
The W68L mutant: substrate translocation
To extend the contrast between the WT and W68L transporters, we
measured GABA uptake and transport-associated current and compared
these parameters with the level of transporter expression as measured
either by binding of radiolabeled ligand or by charge movements.
Binding of the GABA analog [3H]tiagabine was measured on
membrane vesicle preparations from transfected HeLa cells and was
comparable with levels with WT membranes, although
[3H]tiagabine concentration dependence was not
systematically studied. However, the mutant transporters were severely
impaired in [3H]GABA transport when measured at 42 nM GABA (<5% of WT value). A similar picture emerged from
the oocyte experiments. The number of transporters per oocyte, measured
as Qmax in Eq. 1, was similar for the WT and
W68L transporter (80 ± 5 vs 105 ± 6 nC, 11 and 9 oocytes,
respectively). Yet oocytes expressing W68L showed very small
transport-associated current (~10% of WT at 100 µM
GABA) as well as low [3H]GABA transport (3.5% of WT at
100 µM GABA).
Both the oocyte and the mammalian cell experiments thus show that W68L
expresses at roughly WT levels, as measured by
[3H]tiagabine binding or charge movements, but functions
poorly. Measurements of transport-associated current in oocytes
expressing the WT and W68L transporters provide additional functional
kinetic parameters of the complete transport cycle. W68L showed a
16-fold higher EC50 for GABA compared with the WT
[298 ± 17 µM (n = 4) vs 18.6 ± 0.9 µM (n = 5), respectively, 80
mV]. Dose-response relations for the effect of [Na+] on
the transport-associated current (Fig. 2E) showed
for the WT an EC50 of 44 ± 1.8 mM,
nH = 1.8 ± 0.15 and for the mutant an
EC50 of 6.76 ± 1.5 mM,
nH = 1.63 ± 0.07 (300 µM and
3 mM GABA for WT and W68L, respectively; 80 mV; 3 oocytes
tested for each trans- porter). Thus, the shift to a higher
affinity for Na+, revealed by the concentration- and
voltage jumps, is also a characteristic of the complete transport
cycle.
A measure of the turnover rate is the ratio between the
transport-associated current and the charge movement for an individual
oocyte (Mager et al., 1993). This parameter was 15-fold lower for W68L
than for the WT [0.56 ± 0.01 sec1
(n = 6) vs 8.4 ± 0.4 sec1
(n = 8), respectively, 80 mV]. Furthermore, for W68L
some of this small Na+ flux is not directly coupled to GABA
flux, because the ratio of total charge transported to
[3H]GABA uptake (Kavanaugh et al., 1992) was 5.6 ± 0.7 versus 2.0 ± 0.1 for W68L and WT, respectively (100 µM GABA; 3 batches of oocytes; 5 oocytes per
measurement). Taking these corrections into account, we calculate that
the turnover rate for GABA is ~4 sec1 for the WT and
~0.1 sec1 for W68L at 80 mV and normal Ringer
solution.
The tighter and faster binding of Na+, looser binding of
GABA, and lower turnover rate for W68L all suggest that this mutant is
blocked at a point in the transport cycle after the binding of
Na+ and before that of GABA. Analysis of the W68L mutation,
therefore, supports the view that Na+ binds before GABA. In
this case, however, the Na+-transporter interaction does
not limit the rate of transport. Because W68L binds GABA poorly, its
bound Na+ remains for extended periods facing the
extracellular solution or sequestered within the transporter, and the
substrates are only slowly released to the intracellular side.
The Na+ dependence for charge movements and for
steady-state currents are similar
According to our hypothesis, the major rate-limiting requirement
for Na+ occurs before the binding of GABA. We believe that
the charge movements reflect this process. As a consequence, one
expects the external Na+ concentration to affect the entire
transport cycle only through its effect on the charge movements. The
entire transport cycle is monitored via transport-associated currents
in the presence of GABA. In agreement with the hypothesis, Figure 2,
A and E, shows that the charge movements and
transport-associated currents for the WT transporter have nearly equal
EC50 values (43 and 44 mM, respectively) and
Hill coefficients (1.63 and 1.8, respectively) under conditions
of saturat- ing GABA.
Although the W68L mutant displays much lower EC50 values,
the values for the charge movements and transport-associated currents
are still comparable with each other (6.8 and 2.7 mM,
respectively). Possibly, these two values would be even closer without
the complications of very small transport-associated currents, finite
intracellular [Na+], and lack of GABA saturation even at
concentrations of 3 mM. It is already clear, however, that
the Na+-transporter interaction dominates transport under
appropriate conditions for the W68L transporter as well as for
the WT .
Cl increases affinity for Na+
Cl is required for transport by members of the GAT1
superfamily and is cotransported with Na+ and the organic
substrate (Keynan and Kanner, 1988; Lester et al., 1994). For the WT
transporter, in the presence of 96 mM Na+, the
removal and addition of external Cl generated outward and
inward transient currents, respectively (Fig.
4A,B). This is
opposite to the expected direction for membrane exit and entry of an
anion. In the absence of Na+, Cl
concentration jumps generated no transient currents. Evidently,
Cl facilitates Na+ binding (or the subsequent
conformational change), because removal of Cl results in
at least partial reversal of this step. Charge movement during
Na+-concentration jumps was also recorded in zero external
Cl. This charge movement was reduced by 66 ± 3%
compared with that observed at 96 mM Cl (wild-type;
n = 5; 60 mV). Thus, Cl seems to
facilitate Na+ binding to the transporter, but
Cl is not absolutely required.
Fig. 4.
Cl concentration jumps.
Membrane potential was held at 60 mV at various [Na+];
for the indicated time, Cl in the perfusion medium was
replaced by acetate. For the WT (A), the removal of
Cl in the presence of 96 mM Cl
results in an outward current, and the addition of Cl
results in an inward current. At 24 mM [Na+],
only small currents developed. In the absence of Na+,
Cl concentration jumps evoked no charge movement. For the
W68L mutant (B), Cl concentration jumps
were smaller in 96 mM Na+ than in 24 mM Na+. No transient current developed in the
absence of Na+.
[View Larger Version of this Image (12K GIF file)]
For the W68L transporter, larger transient currents developed in 24 mM Na+ than in 96 mM
Na+ during Cl-concentration jumps (Fig.
4B). The W68L transporter has a high affinity for
Na+; therefore, at 96 mM Na+, the
transporter is fully occupied by Na+ even in the absence of
Cl. However, at lower [Na+], the decrease
in affinity for Na+ attributable to the removal of
Cl affects the Na+ occupancy of the
transporter.
These data show that Cl-concentration jumps fail to
generate charge movements in the absence of Na+. We
therefore reexamined voltage-jump relaxations in the presence and
absence of Cl. Omitting Cl produces shifts
along the voltage axis in the Q versus V relation
(Mager et al., 1993), as though KNa changes in
Eq. 1, but Qmax changed by <3% for both
wild-type (n = 6, 120 mM Na+)
and W68L (n = 9, 16 mM Na+).
This result may arise because Cl cannot bind in the
absence of Na+ and/or because the charge movements do not
involve a movement of Cl within the membrane dielectric.
Together with the data that transport is enhanced by Cl,
these experiments support the hypothesis that Cl and
Na+ binding prepares the transporter to bind GABA.
Li+ and Cs+ induce
channel-like currents
GABA transport by GAT1 is absolutely dependent on the presence of
Na+ and cannot be driven by other metal or organic ions
(Kanner and Schuldiner, 1987). Yet Figure 5 shows that
we found electrophysiological signals attributable to interactions
between Cs+ or Li+ and GAT1. In the absence of
GABA and Na+, steady inward currents developed when either
Cs+ or Li+ was applied to oocytes (Fig.
5A,B). Similar signals were not
observed in noninjected oocytes. The Li+ currents were
fourfold larger for W68L than for the WT transporter (157 ± 7 and
41 ± 8 nA, respectively) despite the similar charge movements for
Na+-concentration jumps. This shows again that W68L seems
to favor uncoupled fluxes of Li+ or Cs+. When
the concentration jumps were performed between Na+ (rather
than NMDG) and Li+ or Cs+, the records showed
both binding current and permeation current (data not shown). LiCl
produced 5.8 ± 0.6 and 2.4 ± 0.2 times larger steady-state
current than did LiAc for the WT and W68L, respectively (8 and 7 cells,
respectively; Fig. 5C,D), again
suggesting that Cl generally enhances the interaction
between cations and the transporter.
Fig. 5.
Cs+ and Li+ leakage
current. Holding potential, 60 mV. A,
B, Oocytes were first superfused with NMDG · Cl
Ringer. At the indicated time, the perfusion was changed to CsCl or
LiCl Ringer. Cs+ and Li+ generated a steady
inward current. C, D, Effect of
Cl on the Li+ leakage current. Oocytes were
first superfused with NMDG · Cl Ringer. At the indicated time, the
perfusion was changed to either LiCl or Li acetate
(LiAc) Ringer. For all panels, GAT1-specific current was
isolated as follows. After each series of changes pictured, oocytes
were perfused for 10 sec with 30 µM SKF-100330A in 96 mM NaCl to block GAT1 currents. The ionic concentration
changes were then repeated. The SKF-blocked record was then subtracted
from the original record. The subtraction procedure yielded flat traces
in similar protocols applied to uninjected oocytes.
[View Larger Version of this Image (13K GIF file)]
Tiagabine analogs induce a Na+
``lock-in'' current
The GABA uptake inhibitors SKF-89976A (Mager et al., 1993),
NO-05-711 (Novo Pharmaceutical; data not shown) and SKF-100330A (Fig.
1) all eliminate both concentration-jump and voltage-jump charge
movements. This effect could arise either because the drug prevents
Na+ binding or because it leads to Na+
occlusion by the transporter. Figure 6 shows that in the
presence of Na+, application of SKF-100330A stimulates
inward current. We now present evidence that this current is
attributable to Na+ binding induced by the inhibitor,
lending support to an occlusion mechanism. The current depended on the
presence of Na+ (Fig.
6A,B). However, at higher
[Na+], the charge movements (integral of the current over
time) decreased for the WT (126 mM Na+) and
almost disappeared for the mutant (96 mM Na+).
Thus, the inhibitor generates no current if it binds to transporters
that are already occupied by Na+. Furthermore, the charge
movement induced by application of the inhibitor is equal to the charge
movement induced by voltage jumps to high negative potential (140 mV,
Fig. 6C) or by concentration jumps to higher
[Na+] (data not shown). For each of three procedures
(increased [Na+], hyperpolarization, and application of
inhibitor), therefore, charge movements occur by increasing
Na+ occupancy of the transporter.
Fig. 6.
Inhibitor-induced Na+ ``lock-in''
current. A, B, Oocytes were perfused with
a Ringer solution containing the specified [Na+] at a
holding potential of 60 mV. At the indicated time, SKF-00330A (30 µM) was added to the perfusion medium. C,
Comparison of charge movements for inhibitor applications as in
A and B (x-axis) and for
voltage jumps from the holding potential to 140 mV
(y-axis). Open and closed
symbols each represent an oocyte expressing WT or W68L GAT1,
respectively. D, Effect of [Na+] on the
charge movements obtained by integrating the traces in A
and B.
[View Larger Version of this Image (28K GIF file)]
DISCUSSION
The data strongly support the idea that, in normal physiological
solution, GAT1 is fully occupied by two Na+ ions and one
Cl ion and that this occupation prepares the transporter
to bind GABA immediately, with the result that transport is initiated.
The charge movements during concentration- and voltage-jump experiments
show that WT GAT1 binds two Na+ ions and undergoes a
subsequent event, probably a conformational change. These
Na+-dependent charge movements occur at lower
[Na+] if Cl is present, and vice-versa, as
though Na+ and Cl bind to a common state
associated with completion of the charge movements. When GABA is
present, these ion-transporter interactions continue to dominate the
entire transport cycle, as shown by the similar Na+
dependence of the charge movements and the transport-associated
currents (EC50 of 43 and 44 mM, respectively),
by their similar Hill coefficients (1.66 and 1.8, respectively), and by
their similar voltage dependence (equivalent charges of 1.04 and 0.89, respectively; Mager et al., 1993). Thus, the transport process has
already undergone its rate-limiting steps when a GABA molecule binds
(Cammack et al., 1994).
The transient currents in response to concentration jumps are
linked to ion binding and dissociation
In response to rapid application of Na+,
Cl, or SKF-100330A, oocytes expressing the GABA
transporter generate transient inward current. We believe that these
transient currents occur at the GABA transporter because no similar
transient current appeared in uninjected oocytes and because the
current was blocked by various specific, high-affinity uptake
inhibitors. We also have several reasons to believe that these
transient currents are attributable to ion binding at the transporter
(and within the membrane) rather than to complete permeation through
the membrane. Equal and opposite charge movements (integral of the
transient current over time) occurred when [Na+] or
[Cl] was reduced back to the initial concentration
(Figs. 1C, 4A). Such charge
conservation, which is formally modeled as a capacitance, is a typical
characteristic of a reversible binding/dissociation reaction within the
membrane dielectric, or of a conformational change in a membrane
protein, but not of ion permeation. Most important, the charge movement
for concentration jumps was nearly equal to that for voltage jumps
under conditions designed to produce similar endpoints of ionic
concentration and membrane potential (Fig. 1D).
Previous studies show that the voltage-jump charge movements are also
capacitive currents, closely related to binding and dissociation (Mager
et al., 1993).
We wish to point out that we have noted briefer (time constant < 6 msec) components of voltage-jump relaxations at GAT1 (S. Mager and H. Lester, unpublished observations). These components may correspond to
the voltage-dependent capacitances observed by Lu et al. (1996) and
presumably arise from events faster than those considered here.
Molecular nature of the event that produces charge movements
The charge movements depend on [Na+] with a Hill
coefficient near 2 (Fig. 2A), providing the
strongest evidence yet that these charge movements are much more likely
to occur in the presence of two bound Na+ ions than in the
presence of a single bound Na+ ion. What is the event that
produces charge movement? In one plausible mechanistic view, the two
Na+ ions reach a binding site in the transporter that is
functionally within the membrane dielectric, followed by a
conformational change that greatly increases binding affinity but does
not itself move charge. In a second plausible view, the Na+
ions do not initially bind in a voltage-dependent manner; instead, the
conformational change is the voltage-dependent step because it moves
charge within the membrane electric field. Within the context of either
interpretation, Eq. 1 implies rather tight coupling between the binding
and the conformational change, in the sense that binding occurs with
higher affinity as the conformational change becomes more probable, and
vice-versa. Because of this tight coupling, the overall binding of
Na+ would be expected to display a Hill coefficient of 2 even in the absence of cooperative interactions during the microscopic
binding events. We suggest, therefore, that a conformational change
occurs even before GABA binds, and that this conformational change
prepares the transporter to bind GABA with high affinity.
We are impressed by the parallel between the charge movements and the
conformational changes thought to govern ion channel gating. Both
events occur on time scales of tens to hundreds of milliseconds. Rates
for both events display a maximum with voltage, as though transitions
are governed by forward and reverse rate constants with opposite signs
of voltage dependence (in further support for such a model, the minimum
rate occurs at the midpoint of the charge-voltage relation). Both
events can be shifted in voltage dependence, but not in overall
amplitude, by changes in ionic concentrations. These similarities would
be expected from theories such as the alternating-access models of
transporter function. On the other hand, the conformational change need
not be global; recent simulations suggest that small changes in
substrate interactions within a channel-like lumen can explain coupled
transport (Su et al., 1996). Our data contrast with results for the
Na/K pump, in which the slow charge movements recorded for
Na+ transport show a monotonic increase in rate with
hyperpolarization, and the actual electrogenic events are much faster
(submicrosecond) ion-binding reactions (Hilgemann, 1994).
It is at first surprising that Cl jumps produce charge
movements opposite to the direction expected for an anion moving within
the membrane dielectric. We conclude that Cl favors the
binding of Na+, directly at the Na+ binding
site(s), allosterically by modifying the Na+ binding site,
or allosterically by facilitating the conformational change that
produces the charge movements. The latter possibility is quite
attractive because the actual amplitude of charge moved, as measured by
Qmax, does not change in zero Cl,
although it occurs at different voltages. Cl also binds
at the intracellular face of the transporter (Lu et al., 1996).
Inhibitor-induced current
That an inward charge movement results from application of a
high-affinity transport inhibitor (Fig. 6) provides direct support for
suggestions that nonsubstrate GABA inhibitors, as well as some
monoamine transporter inhibitors, act by analogy with transition-state
analog inhibitors of enzymes: they lock the transport protein at an
intermediate step in the transport cycle (Rudnick and Clark, 1993;
Lester et al., 1994). Another analogy concerns the action of cardiac
glycosides on the Na+ pump: they lock in two
Na+ ions (Jorgensen and Andersen, 1988; Sturmer and Apell,
1992). In support of the ``lock-in'' idea, direct binding experiments
show that a tiagabine analog binds more tightly to GABA transporters in
the presence of Na+ (Braestrup et al., 1990). In our view,
tiagabine and its analogs bind at least partially to the GABA binding
site itself, after the conformational change associated with charge
transfer. By freezing the transporter at this point, tiagabine and its
analogs apparently prevent Na+ from dissociating to the
intracellular solution, thereby increasing its affinity. Importantly,
there were equal charge movements for inhibitor application and for
voltage jumps that completely forced Na+ onto the
transporter, suggesting that the inhibitor locks both Na+
ions into their normal binding sites. Because tiagabine and its analogs
produce neurotransmitter transporters with tightly bound
Na+, these inhibitors may prove important in future
experiments that seek to reveal structural properties of transporters
at atomic scale.
Channel-like mode
The comparatively large currents produced by Cs+ and
Li+ are analogous to uncoupled Cs+ and
Li+ currents at other neurotransmitter transporters (Lester
et al., 1994; Mager et al., 1994). A partial explanation for the lack
of coupled transport by Cs+ and Li+ is that
these two ions permeate through the transporter but do not require the
binding of GABA. Uncoupled ion fluxes attributable to intracellular
Cs+ solutions may also explain that leakage currents were
high, that concentration-jump charge movements were not reversible, and
that Na+ concentration jumps produced no charge movements
in the study by Cammack et al. (1994). The Cs+ and
Li+ currents represent a contrast to the action of
tiagabine analogs, which allow no current at all. At this point, we do
not know whether the Cs+ and Li+ currents are
comprised of single-ion events or of clusters comparable in size with
single-channel events, but single-channel events do underlie some
leakage currents at both GAT1 (Cammack and Schwartz, 1996) and the
mammalian 5-HT transporter (Lin et al., 1995).
The W68L mutation
Trp68 is in the first putative transmembrane domain,
which is strikingly well conserved among members of the
Na+/Cl-coupled neurotransmitter transporter
superfamily. It appears that both WT and W68L transporters can bind
Na+ and Cl and can undergo the conformational
change that produces charge movements. The Na+-transporter
interaction and the Cl-transporter interaction dominate
the entire transport cycle, for W68L as well as for the WT. Only the
WT, however, can efficiently undergo the subsequent eventspresumably
including conformational changesthat release the substrates to the
intracellular solution. Alternatively, the intracellular
[Na+] may exceed the dissociation constant for the
interaction between intracellular Na+ and W68L, so that
Na+ is not efficiently released to the cytoplasm. Although
we are not yet ready to choose among models that specify the bindings,
dissociations, and conformational changes during transport (Su et al.,
1996), we can certainly point out that poor balance among these
processes leads to inefficient transport.
It would not be surprising if efficient transport requires that  electrons of aromatic groups (Trp68 in this case) interact
with permeant cations and/or with cationic moieties of the organic
substrate (Dougherty and Stauffer, 1990; Kleinberger-Doron and Kanner,
1994). The combination of high-resolution physiological measurements
and site-directed mutagenesis, which has been fruitfully applied to ion
channels in the past, can now be applied for identification of key
amino acid residues and domains within several families of
transporters. These families include neurotransmitter transporters as
studied here and by others (Lu and Hilgemann, 1995; Wadiche et al.,
1995), ion antiporters (Hilgemann et al., 1991), and ATPase pumps
(Gadsby et al., 1993; Hilgemann, 1994; Holmgren and Rakowski, 1994; Lu
and Hilgemann, 1995).
Transport function and synaptic transmission
GAT1 and its homologs play a key role in shaping the time course
and spatial extent of synaptic transmission, apparently by removing
GABA within a few milliseconds after its release from the presynaptic
terminal (for review, see Lester et al., 1994). The data presented here
provide additional support for the hypothesis that the cotransported
ions bind before GABA, inducing a state that then accepts GABA (Kanner,
1987; Cammack et al., 1994; Tong and Jahr, 1994; Wadiche et al., 1995).
From the dependence of charge movements on voltage and
[Na+] (Figs. 2, 3) (Mager et al., 1993), one may conclude
that the transporters undergo this conformational change with a time
constant of ~250 msec (80 mV, 96 mM NaCl, 20°C) and
eventually reach a steady-state distribution of >90% in the
GABA-receptive state. However, because these ion-binding steps and the
conformational change occur in the absence of GABA, the transporter can
then respond quickly to the appearance of GABA by binding the
transmitter, removing it from the receptors and preventing their
reactivation. Of course, individual transporter molecules would require
another ~250 msec before binding another GABA molecule; synaptic
transmission at intervals less than this might not be effectively
shaped by transporters.
A key unknown fact that bears on our hypothesis is the density of
transporter molecules near a GABA synapse. If this value is comparable
with that of acetylcholinesterase molecules at a nicotinic synapse
(>2000 molecules/µm2) or of GAT1 molecules in an
expressing oocyte (>10,000 molecules/µm2), then
transporters at a noncholinergic synapse can be as effective as
acetylcholinesterase despite the much lower turnover rate of
transporters.
Several other neurotransmitter transporters display functional
characteristics analogous to those of GAT1. The similarities include
Na+ specificity, Cl dependence, charge
movements (Galli et al., 1995; Wadiche et al., 1995), and positive
interactions between the binding of Na+ and of
high-affinity blockers (Rudnick and Clark, 1993). Our conceptual
framework, therefore, may explain transmitter removal at various
chemical synapses.
FOOTNOTES
Received April 26, 1996; revised June 14, 1996; accepted June 18, 1996.
This work was supported by grants from the National Institute of
Neurological Diseases and Stroke and the U.S./Israel Binational Science
Foundation, and by fellowships from the Lester Deutsch Foundation and
the Muscular Dystrophy Association (S.M.). We thank G. Rudnick for help
with the [3H]tiagabine binding assay, C. Armstrong and A. Finkelstein for discussion of charge movements and membrane
dielectrics, and J. Li and M. Nowak for comments on this
manuscript.
Correspondence should be addressed to Henry A. Lester, Division of
Biology 156-29, California Institute of Technology, Pasadena, CA
91125.
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