From the Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem 91120, Israel
Received for publication, October 15, 2002, and in revised form, November 21, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sodium- and chloride-dependent
The neurotransmitter GAT-1 was purified to homogeneity in a form active upon reconstitution
(4) and subsequently cloned (5). The transporter catalyzes electrogenic
sodium/chloride/GABA cotransport with a stoichiometry of 2:1:1 (6-9).
It should be noted, however, that there still is some dispute regarding
this issue. In a recent report, it has been proposed that, during
sodium-coupled GABA transport, obligatory
Clout/Clin exchange takes place (10).
GAT-1, as well as the other members of the family, is predicted to have
12 transmembrane domains linked by hydrophilic loops with the amino and
carboxyl termini residing inside the cell (5). Studies on the serotonin
transporter SERT suggest that the theoretical topological model is
correct (11). Mutagenesis studies (in particular on GAT-1 and SERT, but
also on other members of the family) suggest the importance of
transmembrane domains I and III in the interaction of the transporters
with the neurotransmitters as well as with sodium and chloride
(12-16).
Using biochemical approaches, it has been shown that GAT-1 undergoes
extensive conformational changes upon interaction with its substrates
(17, 18). Electrophysiological measurements of GAT-1 have demonstrated
sodium-dependent GABA-induced inwardly rectifying currents
reflecting the electrogenic sodium- and chloride-coupled translocation
of GABA (7-9). Moreover, in the absence of GABA, but in the presence
of sodium, GAT-1 exhibits capacitative transient currents (8, 13, 14).
These transients are thought to reflect a charge-moving conformational
change that takes place after the binding of sodium and that enables
subsequent GABA binding (8, 13, 19). Remarkably, in the presence of
lithium, inwardly rectifying steady-state currents are observed in the
absence of GABA (13, 14). Such leak currents have also been observed in
many transporters for neurotransmitters (20-23) as well as in other
transporters such as proton-coupled metal transporters (24). In the
latter case, it has been suggested that this leak pathway may protect
cells from metal ion overload (24). However, the relationship of these
leak currents to coupled transport is poorly understood.
In the case of the uncoupled currents observed with the glutamate
transporters, it has been shown that the conformation of the
transporter mediating this process is distinct from the coupled process
(25-28). Recently, co-workers and I have obtained some evidence that
the same may be true for the leak currents mediated by GAT-1 (29). In
this study, I show that the leak mode of GAT-1 represents a unique
conformation of the transporter. Mutants at a water-accessible position
in the middle of the highly conserved transmembrane domain I that
appear to be locked in the leak mode are described. Moreover, other
determinants located in this transmembrane domain that control the
sodium-dependent transition of the leak to transport mode
have been identified here.
Generation and Subcloning of Mutants--
Mutations were made by
site-directed mutagenesis of wild-type GAT-1 in the vector pBluescript
SK( cRNA Transcription, Injection, and Oocyte
Preparation--
Capped runoff cRNA transcripts were made from
transporter constructs in pOG1 linearized with
SacII using mMessage mMachine (Ambion Inc.). Oocytes were
removed from anesthetized Xenopus laevis frogs
and treated with collagenase (type 1A, Sigma C-9891) until capillaries
were absent and injected with 50 nl of undiluted cRNA the same or the
next day. Oocytes were maintained at 18 °C in modified Barth's
saline (88 mM NaCl, 1 mM KCl, 1 mM
MgSO4, 2.4 mM NaHCO3, 1 mM CaCl2, 0.3 mM
Ca(NO3)2, and 10 mM HEPES, pH 7.5)
with freshly added 2 mM sodium pyruvic acid and 0.5 mM theophylline and supplemented with 10,000 units/liter
penicillin, 10 mg/liter streptomycin, and 50 mg/liter gentamycin.
Oocyte Electrophysiology--
Oocytes were placed in the
recording chamber; penetrated with two micropipettes (back-filled with
2 M KCl, with resistance varied between 0.5 and 2 megaohms); and voltage-clamped using GeneClamp 500 (Axon Instruments,
Inc.) and digitized using Digidata 1200A (Axon Instruments, Inc.), both
controlled with the pClamp6 suite (Axon Instruments, Inc.). Currents
were acquired with clampex6.03 and low pass-filtered at 10 kHz
every 0.5 ms. Oocytes were stepped from
In substitution experiments, sodium ions were replaced with equimolar
choline or lithium. The records shown in Figs. 2, 3, 6, and 7 are
typical and representative of results from four to eight oocytes. In
the other figures, the currents were normalized as indicated in the
legends to plot results of 3-13 oocytes as means ± S.E. Wherever
error bars are not visible, the error was smaller than the size of the
symbols. Analysis was performed with Clampfit Version 6.05 and Origin
Version 6.1 (Microcal).
Cell-surface Biotinylation--
Labeling of wild-type and mutant
transporters at the cell surface using sulfosuccinimidyl
2-(biotinamido)ethyl-1,3-dithiopropionate, SDS-PAGE, and Western blot
analysis was done as described by Stern-Bach et al. (32).
For each determination, five oocytes expressing either the wild type or
the indicated mutant were used. One-eighth of the sample was retained
as the source of total protein, and the remainder was treated with
streptavidin beads to recover the biotinylated proteins. The GAT-1
protein was detected with an affinity-purified antibody to an epitope
located in the carboxyl terminus of GAT-1 (residues 571-586),
horseradish peroxidase-conjugated secondary antibody, and ECL as
described (33).
[3H]GABA Transport in HeLa Cells--
HeLa cells
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum, 200 units/ml penicillin, 200 µg/ml
streptomycin, and 2 mM glutamine. Infection with
recombinant vaccinia T7 virus vTF7-3 (34) and subsequent transfection
with plasmid DNA, as well as GABA transport (35), were done as
published previously.
Characterization of Currents Mediated by the G63S and G63C
Mutants--
Of the members of the sodium- and
chloride-dependent neurotransmitter transporter family, the
biogenic amine transporters possess a conserved aspartate residue in
the middle of transmembrane domain I. All other members of the
transporter family, including GAT-1, have a glycine at the
corresponding position (Gly63 in GAT-1). In the study of
the structure/function relationships in transmembrane domain I of
GAT-1, I mutated Gly63 to aspartate as well as to serine,
cysteine, and alanine. This led to the complete loss of sodium- and
chloride-dependent [3H]GABA uptake, as
observed upon expression in HeLa cells (Fig. 1). A similar loss of function was
observed with the sodium-dependent GABA-induced transport
currents in X. laevis oocytes as shown here for the G63C and
G63S mutants (Fig. 2, A,
C, and E). Remarkably, however, the two mutants
exhibited robust GABA-independent inwardly rectifying leak currents in
lithium medium (Fig. 2, D and F). The currents of
both of these mutants were similar in magnitude and voltage dependence
to that mediated by wild-type GAT-1 (Fig. 2B) and were not
influenced by GABA (data not shown). The kinetics (but not the
steady-state level) of the currents observed in lithium were dependent
on the nature of the substitution at position 63. In the G63S (but not
G63C) mutant, the onset of these currents was markedly slowed down
(Fig. 2, B, D, and F). Results similar to those obtained with G63C were obtained when Gly63 was
replaced by alanine, but no lithium leak currents were observed when
aspartate was substituted for Gly63 (data not shown).
Even though position 63 is located in the middle of transmembrane
domain I, it is possible that this conserved domain lines an aqueous
pathway through the transporter, and a cysteine residue introduced at
this position is accessible to hydrophilic membrane-impermeant sulfhydryl reagents. Consistent with this idea, the
lithium-dependent currents of G63C (but not of G63S) were
almost completely abolished by positively charged MTSET as well as by
negatively charged (2-sulfonatoethyl) methanethiosulfonate (Fig.
3) (data not shown). A conserved cysteine (Cys74 in GAT-1) located in the first extracellular loop is
the only endogenous cysteine that is externally accessible in GAT-1 and most members of this family (36-38). To exclude the possibility that
the sensitivity of the lithium-dependent currents observed with G63C is due to an increased accessibility of Cys74,
the G63C and G63S mutants were subcloned into a construct in which
Cys74 was replaced by alanine (C74A). The currents of this
latter mutant were very similar to those mediated by the wild type
(data not shown). The lithium-dependent currents of the
G63C/C74A mutant (Fig. 3A) were almost completely abolished
by pretreatment with MTSET (Fig. 3B). On the other hand,
no effect of MTSET on the lithium-dependent currents of the
G63S/C74A mutant was observed (Fig. 3, C and D).
Similarly, 2-sulfonatoethyl methanethiosulfonate also inhibited these
currents of G63C/C74A, but not of G63S/C74A (data not shown). It is of
interest to note that the difference in the kinetics of the onset of
the currents observed with G63C and G63S (Fig. 2, D and
F) was also observed in the mutants constructed in the C74A
background (Fig. 3, A and C).
Effects of Sodium on the Lithium Leak Currents Mediated by the Wild
Type--
The absence of [3H]GABA transport and
GABA-induced currents in the G63S and G63C mutants indicated that they
were confined to the leak current mode. Further evidence that the leak
currents represent a distinct mode of the transporter is presented in
Fig. 4. The effect of sodium on the
lithium currents of the wild type was measured at varying lithium
levels. The lithium-dependent current did not saturate with
increasing lithium concentrations, at least not up to 90 mM
(Fig. 4). Sodium at a concentration as low as 1 mM
inhibited the current at all lithium concentrations tested. However,
the inhibition by sodium was larger at higher lithium concentrations
(Fig. 4). Thus, these effects are not due to a competition between
lithium and sodium, but rather sodium appears to act at a distinct site
to suppress the leak mode. As sodium is required for coupled transport,
an important question is whether the sodium-induced conformational
changes of the transport cycle are related to the suppression of the
leakage mode. To address this, mutants with decreased and increased
apparent sodium affinities were analyzed.
Effect of Sodium on Currents Mediated by
Y60T--
Tyr60 of GAT-1, also located in transmembrane
domain I, is unique among the GABA transporters. In the three other
GABA transporters (GAT-2-4), a glutamate residue occupies the
equivalent position of Tyr60. Interestingly, when the Y60E
mutant was expressed in HeLa cells, no sodium- and
chloride-dependent [3H]GABA uptake could be
detected, and the same was true for the threonine substitution mutant,
but the Y60C mutant exhibited significant activity (Fig. 1). Upon
expression of Y60E in X. laevis oocytes, no
sodium-dependent GABA-induced transport currents could be
detected, and the same was true for the lithium leak currents (data not shown). In contrast to Y60E, the Y60T mutant exhibited robust lithium
leak currents, ranging from
Even though the GABA transport currents of the mutant were of small
magnitude, their size was large enough to determine the sodium
dependence of this process (Fig. 5C). More than 5-fold higher sodium concentrations were needed to half-maximally activate the
GABA currents of Y60T compared with those of the wild type (Fig.
5C). It was not possible to determine the exact shift of the
apparent sodium affinity in the mutant because, even at 96 mM sodium, the GABA-induced current was not yet saturated
(Fig. 5C). In parallel with the decreased apparent affinity
of Y60T for sodium, 28.8 mM sodium inhibited the lithium
leak current by 26 ± 2% (n = 4). In contrast,
the IC50 for sodium in the same batch of oocytes expressing
the wild type was 1.1 ± 0.1 mM (n = 3) (Fig. 5D).
Effect of Sodium on Currents Mediated by
R69K--
Arg69, also located in transmembrane domain I of
GAT-1, is fully conserved in the entire family of sodium- and
chloride-dependent neurotransmitter transporters. Mutation
of this residue, even to a conserved one such as lysine (R69K), leads
to defective [3H]GABA uptake (12). Indeed, no
steady-state GABA currents were observed in this mutant even though
transients were present (Fig. 6A). Even at 5 mM
GABA, no currents could be observed, but the transients were somewhat
larger (data not shown). Furthermore, the mutant exhibited significant
lithium-dependent steady-state currents (Fig.
6B).
The observation that transient currents could be isolated by GABA (Fig.
6A) indicated that, even though GABA was not transported, it
could still bind to the R69K mutant. This was further substantiated by
the fact that, in sodium medium, the non-transportable GABA analog
SKF100330A could isolate the sodium-dependent transient currents mediated by R69K (Fig.
7A). In contrast to the
corresponding currents mediated by the wild type (Fig. 7B),
R69K exhibited transients in the "on" phase only when jumping from
a holding potential of Characterization of Potassium Leak Currents--
It has been found
that GAT-1 also mediates leak currents in cesium, even though they are
much smaller than those in the presence of lithium (13). Interestingly,
GAT-1 also mediated potassium leak currents, which, at Cell-surface Expression--
The transients mediated by R69K were
smaller than those mediated by the wild type (Fig. 7). To investigate
the possibility that this may be due to decreased cell-surface
expression, oocytes expressing wild-type and mutant transporters were
treated with the impermeant biotinylation reagent sulfosuccinimidyl
2-(biotinamido)ethyl-1,3-dithiopropionate. Upon solubilization of the
membrane proteins with detergent, the biotinylated proteins were
isolated using streptavidin beads. After SDS-PAGE of total
(unfractionated) and biotinylated proteins, followed by Western blot
analysis using affinity-purified antibody against GAT-1, the
transporter bands were visualized by ECL. In the case of the wild type,
major bands of ~50 and ~55-60 kDa in the total and biotinylated
samples were observed, respectively (Fig.
10). The latter apparently represents
the fully processed form of the GAT-1 monomer (33). The mobility of the
upper band in the biotinylated sample of the wild type was compatible
with that of a dimeric aggregate of the transporter. The expression of
R69K in the total sample was similar to that of the wild type. On the
other hand, much lower levels of the mutant transporter were detected
in the biotinylated sample; and besides a small amount of the
~55-60-kDa band, bands of lower mobility were detected (Fig. 10).
The expression level and pattern of the biotinylated G63C mutant
were similar to those of R69K (Fig. 10). Here, two sets of oocytes
expressing G63C were processed in parallel to get an impression of the
experimental variability. In contrast to R69K and G63C, the expression
level and pattern of the Y60T mutant were very similar to those of the
wild type (Fig. 10).
Mutation of Gly63, located in the conserved
transmembrane domain I of GAT-1, to serine or cysteine resulted in
defective GABA transport and transport currents (Figs. 1 and 2).
The G63S and G63C mutants did not even exhibit
sodium-dependent transient currents (data not shown), but
exhibited lithium leak currents, which were at least as large as those
exhibited by the wild type (Fig. 2). Therefore, these mutants
appear to be locked in a conformation that mediates the leak current of
GAT-1.
The model depicted in Fig.
11 describes how the leak mode of GAT-1
may relate to its transport cycle. For reasons of simplicity, the role
of chloride is not indicated. In step 1, sodium binds to the
outward-facing form of the transporter (oT). This enables subsequent GABA binding (step 2), followed by the
translocation step (step 3). Release of sodium and GABA to
the inside (step 4) yields the inward-facing form of the
unloaded transporter (iT); and upon reorientation to
the outside (step 5), a new transport cycle can commence.
The leak mode of the transporter (][T) can be
generated from the unloaded outward- and/or inward-facing form of the
transporter upon hyperpolarization (Fig. 5). Sodium inhibited the
lithium currents mediated by the GAT-1 transporter (Fig. 4), and this
is in accordance with a study that was published during the preparation
of this manuscript (39). Moreover, sodium also inhibited the potassium
leak currents mediated by GAT-1 (Fig. 9). The inhibition of the leak
currents by sodium was noncompetitive (Fig. 4). This indicates that
sodium binds at a distinct site from the permeating ions, bringing it
into a different conformation. Because sodium induces the initial steps
of the transport cycle (Fig. 11), a simple explanation is that these
critical steps represent the conformational change resulting in the
inhibition of the leak currents. Supporting this explanation are
observations on mutants at two other positions, also located in
transmembrane domain I. A decreased (Fig. 5C) or an
increased (Fig. 7A) apparent affinity for sodium was
paralleled by the ability of sodium to inhibit the leak currents (Figs.
5D, 8, and 9).
-aminobutyric acid (GABA) transporter is essential for synaptic
transmission by this neurotransmitter. GAT-1 expressed in
Xenopus laevis oocytes exhibits
sodium-dependent GABA-induced inward currents reflecting
electrogenic sodium-coupled transport. In lithium-containing medium,
GAT-1 mediates GABA-independent currents, the relationship of which to
the physiological transport process is poorly understood. In this
study, mutants are described that appear to be locked in this cation
leak mode. When Gly63, located in the middle of the highly
conserved transmembrane domain I, was mutated to serine or
cysteine, sodium-dependent GABA currents were abolished.
Strikingly, these mutants exhibited robust inward currents in lithium-
as well as potassium-containing media. Membrane-impermeant sulfhydryl
reagents inhibited these currents of the cysteine but not of the serine
mutant, indicating that this position was accessible to the external
aqueous medium. The cation leak currents mediated by wild-type
GAT-1 were inhibited by low millimolar sodium concentrations in a
noncompetitive manner. Mutations at other positions of transmembrane
domain I increased or decreased the apparent sodium affinity, as
monitored by the sodium-dependent steady-state GABA
currents or transient currents. In parallel, the ability of
sodium to inhibit the cation leak currents was increased or decreased,
respectively. Thus, transmembrane domain I of GAT-1 contains
determinants controlling both sodium-coupled GABA flux and the cation
leak pathway as well as the interconversion of these distinct modes.
Our observations suggest the possibility that the permeation pathway in
both modes shares common structural elements.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid
(GABA)1 is a major inhibitory
neurotransmitter in the central nervous system. Its synaptic action,
like that of most other neurotransmitters, is terminated by
sodium-coupled transport. Many neurotransmitters are removed from the
synaptic cleft by sodium- and chloride-dependent neurotransmitter transporters (for a review, see Refs. 1 and 2). These
transporters form a large family and include transporters for biogenic
amines and amino acids. Besides GAT-1, the first member of the family
to be identified, it contains three other GABA transporters. One of the
best examples of the importance of these neurotransmitter transporters
comes from studies of dopamine transporter knockout mice; the decay of
extracellular dopamine in brain slices of such mice is ~100 times
longer than normal (3).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (Stratagene) according to the method of Kunkel et al.
(30) as described (31). Briefly, the parent DNA was used to transform
Escherichia coli CJ236
(dut
,ung
).
From one of the transformants, single-stranded uracil-containing DNA
was isolated upon growth in uridine-containing medium according to the
standard protocol from Stratagene using helper phage R408. This yields
the sense strand; and consequently, mutagenic primers were designed to
be antisense. Restriction sites ClaI and AvrII were used to subclone the mutations into the construct containing wild-type GAT-1 in pOG1 (14). The latter is an oocyte
expression vector that contains a 5'-untranslated Xenopus
-globin sequence and a 3'-poly(A) signal. The coding and noncoding
strands were sequenced between the above two restriction sites.
160 to +40 mV in 25-mV
increments, using
25 mV as the holding potential unless stated
otherwise in the figure legends. Each potential was clamped for 500 ms.
The membrane potential was measured relative to an extracellular
Ag/AgCl electrode in the recording chamber. The recording solution
contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,
and 5 mM HEPES, pH 7.4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
[in a new window]
Fig. 1.
Transport activity of replacement mutants at
positions 60 and 63. [3H]GABA uptake of HeLa cells
expressing the wild type (WT) or the indicated mutants in
the vector pBluescript SK( ) was performed as described under
"Experimental Procedures." Uptake values from cells transfected
with the vector alone have been subtracted. The data shown are the
means ± S.E. and are shown as percent of wild-type uptake.
View larger version (19K):
[in a new window]
Fig. 2.
GABA-induced and lithium currents mediated by
wild-type, G63S, and G63C transporters. A, C, and
E, currents recorded in sodium medium in the absence of GABA
during 500-ms voltage jumps from 160 to +40 mV (steps of +25 mV) have
been subtracted from currents in the same medium containing 1 mM GABA. The prepulse potential was
25 mV. B,
D, and F, currents recorded in choline medium
have been subtracted from those in lithium medium using the same
oocytes as in A, C, and E,
respectively, with the same voltage jump protocol. The dotted
lines indicate 0 current. Representative oocytes are shown.
WT, wild type.
View larger version (14K):
[in a new window]
Fig. 3.
Effect of MTSET on lithium-induced currents
mediated by G63C/C74A and G63S/C74A. Lithium leak currents were
recorded exactly as described in the legend to Fig. 2 on oocytes
expressing G63C/C74A and G63S/C74A before (A and
C) and after (B and D) treatment with
0.25 mM MTSET for 2 min on the same oocytes. Immediately
before the treatment, the perfusion with sodium medium was stopped, and
MTSET was added directly to the bath. After 2 min of incubation and a
washout of 1 min, the records shown in B and D
were obtained.
View larger version (12K):
[in a new window]
Fig. 4.
Leak currents at varying lithium
concentrations. Lithium-dependent steady-state currents
(obtained by subtracting the response in sodium) at 160 mV were
determined in the presence or absence of 1 mM sodium at
28.8, 57.6, and 86.4 mM lithium. Choline was used to bring
the cation concentration to 96 mM. The results were
normalized to the net current in 86.4 mM lithium and are
averages from four different oocytes. The S.E. is smaller than the size
of the symbols.
1195 to
2791 nA (n = 6) at
160 mV (Fig. 5A). The
same was true for oocytes expressing Y60C; but in contrast to G63C,
these currents mediated by Y60C could not be blocked by MTSET (data not
shown). The size of the lithium-dependent currents of Y60T
was even larger than those of wild-type GAT-1 (Fig. 5B):
485 to
2081 nA (n = 13). Sodium- and
chloride-dependent [3H]GABA uptake was
typically measured at very low GABA concentrations. However, at a
saturating concentration of 1 mM, Y60T exhibited significant GABA transport currents (Fig. 5A), ranging from
31.6 to
90.8 nA at
160 mV (n = 6). In the
corresponding oocytes expressing the wild type, the GABA transport
currents ranged from
342 to
1544 nA (n = 13). The
voltage dependence of the currents of the mutant and wild type was
similar (Fig. 5, A and B), but it is obvious that
the ratio of the lithium leak current to the GABA current differed
dramatically between Y60T and the wild type.
View larger version (20K):
[in a new window]
Fig. 5.
Effects of sodium on GABA-induced and
lithium-dependent currents mediated by Y60T mutant and
wild-type GAT-1. Lithium-induced steady-state currents and
GABA-induced currents in sodium-containing medium (both obtained by
subtracting the response in sodium) in Y60T (A) and the wild
type (WT) (B) were averaged over 40 ms at each of
the potentials shown (the interval between 430 and 470 ms after the
onset of the voltage jump). The results were normalized to the
GABA-induced current at 160 mV and are the averages of six oocytes
from four different batches (Y60T) and 13 oocytes from seven different
batches (wild type). The S.E. is indicated and, in most cases, is
smaller than the size of the symbols. The GABA-induced currents ranged
from
342 to
1544 nA, and the lithium-induced currents ranged from
485 to
2081 nA both at
160 mV for the wild type. The
corresponding values for Y60T were
31.6 to
90.8 nA and
1195 to
2791 nA for GABA- and lithium-induced currents, respectively. Net
GABA-induced currents (C) and lithium-dependent
currents (D) at steady state at
160 mV were recorded at
the indicated sodium concentrations. The results were normalized to the
values for the wild type and mutant obtained at 96 mM
sodium (C) or at 0 sodium (D). The lithium
concentration in D was 67.2 mM, and choline was
used to maintain iso-osmolarity. The data are averages from four
oocytes of the wild type and Y60T in A and three (wild type)
and four (Y60T) oocytes in D.
View larger version (13K):
[in a new window]
Fig. 6.
Net currents mediated by mutant R69K.
GABA-induced currents in sodium medium (A) and
lithium-dependent currents obtained by subtraction of the
response in sodium (B) are shown using voltage jumps from a
holding potential of 25 mV to potentials ranging from
140 to +50 mV
in 20 steps with increments of +10 mV.
25 mV to more positive potentials of up to +50
mV, and the opposite was true in the "off" phase (Fig.
7A). The sodium-dependent transients were
observed only when Arg69 was changed to lysine, but not
when the substitution was with glutamine or cysteine (data not shown).
The transients observed with R69K were capacitative; the transient
currents were of the same magnitude as those obtained when jumping back
to the holding potential (data not shown), and they increased when the
potential was jumped to values more positive than +50 mV (data not
shown). An attractive explanation is that, at a holding potential of
25 mV, all the transporters are already in the sodium-bound state, and the sodium is released by jumps to positive potentials, thereby giving rise to the outward transient current seen in Fig.
7A. Indeed, when the external sodium concentration was
progressively decreased, an inward transient became apparent (data not
shown). In parallel with the increased apparent affinity of R69K for
sodium, this cation inhibited the lithium-dependent
currents of R69K at lower concentrations (IC50 = 0.3 ± 0.1 mM, n = 4) compared with the wild
type expressed in the same batch of oocytes (IC50 = 1.0 ± 0.1 mM, n = 3) (Fig.
8).
View larger version (13K):
[in a new window]
Fig. 7.
Sodium-dependent transient
currents mediated by R69K mutant (A) and wild-type
(B) GAT-1. Currents in sodium medium supplemented
with 30 µM SKF100330A were subtracted from those obtained
in the absence of the blocker. The same voltage jump protocol as
described in the legend to Fig. 6 was used. WT, wild-type
GAT-1.
View larger version (12K):
[in a new window]
Fig. 8.
Effects of sodium on the lithium leak
currents of wild-type and R69K mutant GAT-1.
Lithium-dependent currents obtained by subtracting the
response in sodium at 160 mV were normalized to the values for the
wild type or mutant and recorded at the indicated sodium
concentrations. The lithium concentration was 86.4 mM, and
choline was used to maintain iso-osmolarity. The data are averages from
three (wild type) and four oocytes (R69K).
160 mV, were
62.8 ± 3.8% (n = 6) of those observed in lithium
(Fig. 9A). Such currents were
not observed in non-injected oocytes (data not shown). The potassium
currents had a similar voltage dependence compared with the currents
observed in lithium (data not shown); and as with the lithium currents, they were also inhibited by sodium ions (Fig. 9B). The
potassium currents mediated by R69K were more sensitive to sodium than
those mediated by the wild type (Fig. 9B). In Y60T, the
sensitivity of the potassium currents to sodium was markedly reduced
(Fig. 9B), as in the case of the lithium currents (Fig.
5D). Importantly, the ratio of the potassium to lithium
currents was different in wild type, R69K, and Y60T (Fig.
9A). The same was also true for the G63S and G63C mutants,
in which this ratio was 0.95 ± 0.10 and 0.93 ± 0.11, respectively (n = 3). As for the lithium currents, the
potassium currents mediated by G63C/C74A (but not by G63S/C74A) were
inhibited by treatment with MTSET (data not shown).
View larger version (14K):
[in a new window]
Fig. 9.
Comparison between lithium and potassium leak
currents. The ratio of steady-state potassium- and lithium-induced
currents obtained by subtraction of the response in sodium at 160 mV
is plotted for the wild-type (WT), R69K, and Y60T
(A). The potassium current in the presence of 2 or 10 mM sodium (white and black bars,
respectively) was normalized to that in the absence of sodium
(gray bars; choline substitution) at
160 mV and is
depicted for the wild type and the two mutants (B).
View larger version (77K):
[in a new window]
Fig. 10.
Cell-surface biotinylation of the wild type
and mutants. Oocytes expressing the wild type (WT) and
the indicated mutants were labeled and processed as described under
"Experimental Procedures." The first lane (M) depicts
the positions of the prestained molecular mass standards. The next six
lanes show the total samples, followed by six lanes of the
corresponding biotinylated samples. The first lanes in both groups are
total and biotinylated samples of uninjected oocytes
(Uninj.). Two independent groups of oocytes expressing G63C
were processed in parallel and are shown next to each other. All
samples were separated on the same SDS gel, transferred to
nitrocellulose, and detected as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
[in a new window]
Fig. 11.
Conversion of an uncoupled mode of GAT-1 to
its coupled form is mediated by sodium ions. The leak pathway
(][T) can be opened in the absence of
extracellular sodium by hyperpolarization ( V) of the
unloaded outward-facing (oT) and/or inward-facing
(iT) transporter. Binding of extracellular sodium
converts the transporter into the sodium-loaded outward-facing form of
the transporter (step 1). Subsequent GABA binding
(step 2) is followed by translocation (step 3)
and release of sodium and GABA to the inside of the cell (step
4). Upon reorientation of the binding sites to the outside and
binding of extracellular sodium, the transport cycle is completed
(step 5). For simplicity, chloride is omitted from the
scheme.
The model depicted in Fig. 11 is also in harmony with the observations that the GABA currents began to saturate at negative potentials, whereas this was not the case for the lithium leak currents (Fig. 5B). The GABA currents reflect electrogenic sodium-coupled GABA translocation involving steps 1-5. Therefore, increasingly negative potentials stimulate the translocation of positive charges that accompany GABA transport. However, at very negative potentials, a voltage-independent step apparently becomes rate-limiting for transport. It has been suggested that this is a voltage-independent interaction between GABA and the transporter (8). The leak currents reflect only the voltage-dependent gating of the unloaded transporter. They do not require steps 1-4, one or perhaps more of which involve voltage-independent interactions.
In choline medium, no inwardly rectifying leak currents were observed. Moreover, the lithium currents did not saturate with increasing concentrations of this cation (Fig. 4). Thus, it appears that only small cations (but not the larger choline) pass through the permeation pathway when this is activated by hyperpolarization.
The role of Gly63 in GABA transport is as yet unclear. In the related serotonin transporter SERT, an aspartate residue occupies the position equivalent to Gly63 of GAT-1. This aspartate residue, which is conserved in all biogenic amine transporters of this family, has been implicated in the binding of the amine group of the substrate and also influences the apparent affinity of SERT for the two cosubstrates, sodium and chloride (16). It is tempting to speculate that, in the amino acid transporter members of the family, the carboxyl group of the transported amino acid fulfills a role played by the carboxyl group of the conserved aspartate in the biogenic amine transporters. If, indeed, Gly63 is part of the binding pocket for the carboxyl group of GABA, it is easy to see that even small changes at this position impair GABA transport. As sodium-dependent transients are also defective in the G63S and G63C mutants, the leak currents mediated by these mutants are their only functional property. The presence of these leak currents indicates that the conformation of the outward- and/or inward-facing unloaded transporter is not affected by the mutations at position 63. Thus, even though the leak currents do not have an obvious physiological function, they can be an important tool for structure/function studies in GAT-1.
The lithium leak currents and the transient currents
mediated by R69K are usually smaller than those mediated by the wild type (Figs. 6 and 7). This indicates a lower cell-surface expression of
this mutant, and this is, in fact, supported by surface biotinylation experiments (Fig. 10). It is not clear why bands of lower mobility are
observed in the biotinylated samples of R69K (and also of G63C)
than in those of the wild type. One possibility is that, due to the
lower expression levels at the cell surface, during the processing of
the samples, these mutant transporters are more prone to proteolysis
and subsequent aggregation. Whatever the reason, the important point
for this study is that the expression level is high enough to test the
hypothesis that an increased apparent affinity of sodium binding to the
transporter can be correlated with an increased potency of sodium to
inhibit the leak currents (Figs. 7-9). In R69K, GABA has lost the
ability to induce a transport current (Fig. 6A) as well as
to catalyze [3H]GABA uptake (12). Nevertheless, GABA can
bind inefficiently to the R69K transporters, as judged by its ability
to partially isolate the transient currents at high concentrations
(Fig. 6A). A possible role of Arg69 could be to
interact with the carboxyl group of GABA. This would be reminiscent of
the role of Arg447 of the glutamate transporter EAAC-1 in
binding the -carboxyl group of glutamate (40). In the other amino
acid transporters, this conserved arginine could play a similar role.
Determinants located in the highly conserved transmembrane domain I
influence the affinity of the transporter for sodium (Figs. 5C and 7),
the ability of sodium to inhibit the leak currents (Figs.
5D, 8, and 9B), the potassium/lithium selectivity
of the leak currents (Fig. 9A), and the interconversion of
the leak to transport mode (Figs. 2 and 3). Consistent with the
possibility of a direct involvement of at least one amino acid residue
of transmembrane domain I in permeation is the fact that the position of Gly63 located in the middle of this domain appears to be
accessible via an aqueous pathway to the extracellular medium (Fig. 3).
The simultaneous effects of the mutations on GABA transport and the leak mode suggest that the permeation pathway of these two modes may
share common structural elements.
![]() |
ACKNOWLEDGEMENTS |
---|
I thank Lars Borre and Elia Zomot for stimulating discussions and for help in preparing the figures. Thanks are also due to Nir Melamed and Ester Bennett for the preparation of mutants and cRNA, to Annie Bendahan for performing the biotinylation experiments, and to Beryl Levene for expert secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Israel Science Foundation Grant 150/00-16.1, by the Federal Ministry of Education, Science, Research, and Technology of Germany and its International Bureau at the Deutsches Zentrum für Luft und Raumfahrt, and by the Bernard Katz Minerva Center for Cellular Biophysics.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Hadassah Medical School, Hebrew University, P. O. Box 12272, Jerusalem
91120, Israel. Tel.: 972-2-675-8506; Fax: 972-2-675-7379; E-mail:
kannerb@cc.huji.ac.il.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M210525200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GABA, -aminobutyric acid;
MTSET, 2-(trimethylammonium)ethyl
methanethiosulfonate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Kanner, B. I.
(1994)
J. Exp. Biol.
196,
237-249 |
2. | Nelson, N. (1998) J. Neurochem. 71, 1785-1803[Medline] [Order article via Infotrieve] |
3. | Giros, B., Jaber, M., Jones, S. R., Wightman, R. M., and Caron, M. G. (1996) Nature 379, 606-612[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Radian, R.,
Bendahan, A.,
and Kanner, B. I.
(1986)
J. Biol. Chem.
261,
15437-15441 |
5. | Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M. C., Davidson, N., Lester, H. A., and Kanner, B. I. (1990) Science 249, 1303-1306[Medline] [Order article via Infotrieve] |
6. | Keynan, S., and Kanner, B. I. (1988) Biochemistry 27, 12-17[Medline] [Order article via Infotrieve] |
7. |
Kavanaugh, M. P.,
Arriza, J. L.,
North, R. A.,
and Amara, S. G.
(1992)
J. Biol. Chem.
267,
22007-22009 |
8. | Mager, S., Naeve, J., Quick, M., Labarca, C., Davidson, N., and Lester, H. A. (1993) Neuron 10, 177-188[Medline] [Order article via Infotrieve] |
9. |
Lu, C. C.,
and Hilgemann, D. W.
(1999)
J. Gen. Physiol.
114,
429-444 |
10. |
Loo, D. D. F.,
Eskandari, S.,
Boorer, K. J.,
Sarkar, H. K.,
and Wright, E. M.
(2000)
J. Biol. Chem.
275,
37414-37422 |
11. |
Chen, J. G.,
Liu-Chen, S.,
and Rudnick, G.
(1998)
J. Biol. Chem.
273,
12675-12681 |
12. |
Pantanowitz, S.,
Bendahan, A.,
and Kanner, B. I.
(1993)
J. Biol. Chem.
268,
3222-3225 |
13. | Mager, S., Kleinberger-Doron, N., Keshet, G. I., Davidson, N., Kanner, B. I., and Lester, H. A. (1996) J. Neurosci. 16, 5404-5414 |
14. |
Bismuth, Y.,
Kavanaugh, M. P.,
and Kanner, B. I.
(1997)
J. Biol. Chem.
272,
16096-16102 |
15. |
Chen, J. G.,
Sachpatzidis, A.,
and Rudnick, G.
(1997)
J. Biol. Chem.
272,
28321-28327 |
16. |
Barker, E. L.,
Moore, K. R.,
Rakshan, F.,
and Blakely, R. D.
(1999)
J. Neurosci.
19,
4705-4717 |
17. | Mabjeesh, N. J., and Kanner, B. I. (1993) Biochemistry 32, 8540-8546[Medline] [Order article via Infotrieve] |
18. |
Golovanevsky, V.,
and Kanner, B. I.
(1999)
J. Biol. Chem.
274,
23020-23026 |
19. |
Hilgemann, D. W.,
and Lu, C. C.
(1999)
J. Gen. Physiol.
114,
459-474 |
20. | Mager, S., Min, C., Henry, D. J., Chavkin, C., Hoffman, B. J., Davidson, N., and Lester, H. A. (1994) Neuron 12, 845-859[Medline] [Order article via Infotrieve] |
21. |
Sonders, M. S.,
Zhu, S.-J.,
Zahnisher, N. R.,
Kavanaugh, M. P.,
and Amara, S. G.
(1997)
J. Neurosci.
17,
960-974 |
22. | Fairman, W. A., Vandenberg, R. J., Arriza, J. L., Kavanaugh, M. P., and Amara, S. G. (1995) Nature 375, 599-603[CrossRef][Medline] [Order article via Infotrieve] |
23. | Wadiche, J. I., and Kavanaugh, M. P. (1995) Neuron 15, 721-728[Medline] [Order article via Infotrieve] |
24. |
Chen, X.-Z.,
Peng, J.-B.,
Cohen, A.,
Nelson, H.,
Nelson, N.,
and Hediger, M. A.
(1999)
J. Biol. Chem.
274,
35089-35094 |
25. |
Borre, L.,
and Kanner, B. I.
(2001)
J. Biol. Chem.
276,
40396-40401 |
26. |
Seal, R. P.,
Shigeri, Y.,
Eliasof, S.,
Leighton, B. H.,
and Amara, S. G.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
15324-15329 |
27. |
Ryan, R. M.,
and Vandenberg, R. J.
(2002)
J. Biol. Chem.
277,
13494-13500 |
28. |
Borre, L.,
Kavanaugh, M. P.,
and Kanner, B. I.
(2002)
J. Biol. Chem.
277,
13501-13507 |
29. |
MacAulay, N.,
Bendahan, A.,
Loland, C. J.,
Zeuthen, T.,
Kanner, B. I.,
and Gether, U.
(2001)
J. Biol. Chem.
276,
40476-40485 |
30. | Kunkel, T. A., Roberts, J. D., and Zarkour, R. A. (1987) Methods Enzymol. 154, 367-383[Medline] [Order article via Infotrieve] |
31. |
Kleinberger-Doron, N.,
and Kanner, B. I.
(1994)
J. Biol. Chem.
269,
3063-3067 |
32. | Stern-Bach, Y., Bettler, B., Hartley, M., Sheppard, P. O., O'Hara, P. J., and Heinemann, S. F. (1994) Neuron 13, 1345-1357[Medline] [Order article via Infotrieve] |
33. |
Bennett, E. R., Su, H.,
and Kanner, B. I.
(2000)
J. Biol. Chem.
275,
34106-34113 |
34. | Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8122-8126[Abstract] |
35. | Keynan, S., Suh, H.-J., Kanner, B. I., and Rudnick, G. (1992) Biochemistry 31, 1974-1979[Medline] [Order article via Infotrieve] |
36. | Wang, J. B., Moriaki, A., and Uhl, G. R. (1995) J. Neurochem. 64, 1416-1419[Medline] [Order article via Infotrieve] |
37. | Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Bennett, E. R.,
and Kanner, B. I.
(1997)
J. Biol. Chem.
272,
1203-1210 |
39. |
MacAulay, N.,
Zeuthen, T.,
and Gether, U.
(2002)
J. Physiol. (Lond.)
544,
447-458 |
40. |
Bendahan, A.,
Armon, A.,
Madani, N.,
Kavanaugh, M. P.,
and Kanner, B. I.
(2000)
J. Biol. Chem.
275,
37436-37442 |