The role of N-glycosylation in the stability, trafficking and
GABA-uptake of GABA-transporter 1
Terminal N-glycans facilitate efficient GABA-uptake activity
of the GABA transporter
Guoqiang Cai
1,2
, Petrus S. Salonikidis
3
, Jian Fei
1
, Wolfgang Schwarz
3
, Ralf Schu
¨
lein
4
,
Werner Reutter
2
and Hua Fan
2
1 Institute of Biochemistry and Cell Biology, SIBS, CAS, Shanghai, China
2 Institut fu
¨
r Molekularbiologie und Biochemie, CBF, Charite
´
Universita
¨
tsmedizin Berlin, Berlin-Dahlem, Germany
3 Max-Planck Institut fu
¨

Arnimallee 22, D-14195 Berlin-Dahlem,
Germany
Fax: +49 30 84451541
Tel: +49 30 84451544
E-mail: [email protected]
(Received 17 July 2004, revised 24 January
2005, accepted 2 February 2005)
doi:10.1111/j.1742-4658.2005.04595.x
Neurotransmitter transporters play a major role in achieving low concen-
trations of their respective transmitter in the synaptic cleft. The GABA
transporter GAT1 belongs to the family of Na
+
- and Cl
–
-coupled trans-
port proteins which possess 12 putative transmembrane domains and three
N-glycosylation sites in the extracellular loop between transmembrane
domain 3 and 4. To study the significance of N-glycosylation, green fluor-
escence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation
defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO
cells. Compared with the wild type, all N-glycosylation mutants showed
strongly reduced protein stability and trafficking to the plasma membrane,
which however were not affected by 1-deoxymannojirimycin (dMM). This
indicates that N-glycosylation, but not terminal trimming of the N-glycans
is involved in the attainment of a correctly folded and stable conformation
of GAT1. All N-glycosylation mutants were expressed on the plasma mem-
brane, but they displayed markedly reduced GABA-uptake activity. Also,
inhibition of oligosaccharide processing by dMM led to reduction of this
activity. Further experiments showed that both N-glycosylation mutations
and dMM reduced the V

information about the transport cycle [5–7].
Four subtypes of GABA transporters (GAT1–4)
have been found so far [8,9]. GABA transporter type
1 (GAT1) is a single polypeptide of about 67 kDa
with 12 putative transmembrane domains. Both
N- and C-termini are located in the cytoplasm. The
large extracellular loop between transmembrane
domains 3 and 4 contains three conserved N-glyco-
sylation sites (Asn176, Asn181 and Asn184). It has
been demonstrated that all three N-glycosylation sites
are used in vivo and that no additional sites are pre-
sent [10].
N-glycosylation is a major post-translational modi-
fication in eukaryotic cells. Recent results suggest
that this post-translational modification may influence
many of the physicochemical and biological proper-
ties of the proteins, such as protein folding, stability,
targeting, dynamics and ligand binding, as well as
cell-matrix and cell–cell interactions [11–16]. It has
been suggested that N-glycosylation is involved in
the regulation of the transport activity and surface
expression of neurotransmitter transporters [10,17].
Functional expression of the GABA transporter is
abolished by tunicamycin, a potent inhibitor of
N-glycosylation [18]. Experiments with HeLa trans-
fectants showed that removal of one or two glycosy-
lation sites by site-directed mutagenesis had little
effect on the expression of GABA-uptake activity.
However, removal of all three N-glycosylation sites
resulted in a reduction of GABA-uptake activity [10].

reduced GAT1-specific GABA-uptake activity. If all
three N-glycosylation sites were eliminated, a decreased
percentage of DDQ mutants was found on the cell
surface. However, the GABA-uptake activity could
hardly be detected in this mutant. Inhibition of
N-glycosylation processing by 1-deoxymannojirimycin
(dMM) affected neither the cell surface expression nor
stability of this protein, but it resulted in marked reduc-
tion of GABA-uptake activity. This suggests that
N-glycans, in particular terminal structures of N-gly-
cans, are involved in the GABA-uptake process of
GAT1. Finally, we found that deficiency of N-glycosy-
lation did not affect the affinity of GAT1 for GABA.
The observed reduction of GAT1-specific GABA-
uptake due to deficiency of N-glycans was attributed to
a reduced apparent affinity for extracellular Na
+
ions,
resulting in a reduction of the kinetics of the transport
cycle.
Results
Expression of GAT1/GFP fusion proteins
in CHO cells
cDNAs of GFP tagged wild type (NNN) and mutants
DND, DDN, DGN and DDQ were transfected into
CHO cells, which do not express endogenous GAT1
and GFP. Stable transfectants were selected by fluores-
cence activated cell sorting (FACS). Flow cytometry
analysis showed the expression of NNN and the
mutants on the surface of transfected CHO cells

polypeptide (Fig. 2C, lanes 4 and 5), indicating that
the 96 kDa polypeptide contains only N-glycans of
oligomannosidic type.
Fig. 1. Flow cytometry, fluorescence microscopy and immunofluo-
rescence microscopy of GFP-tagged GAT1 in transfected CHO
cells. (A) Flow cytometry of GFP-tagged GAT1 wild type and
mutants. The polyclonal anti-GAT1 IgG was used for immunostain-
ing. Visualization was performed with R-phycoerythrin-conjugated
goat anti-(rabbit IgG) Ig. NNN, GFP-tagged wild type GAT1. DND,
DDN, DGN and DDQ, GFP-tagged N-glycosylation mutants. (B)
Fluorescence microscopy of NNN. The fluorescence of GFP in
GFP ⁄ GAT-fusion protein (NNN) was detected. (C) Immunofluores-
cence microscopy of NNN. Anti-GAT1 polyclonal antibodies were
used for immunostaining after cell fixation and permeabilization.
Visualization was performed with R-phycoerythrin-conjugated goat
anti-(rabbit IgG) Ig.
Fig. 2. Protein expression and N-glycosylation processing of GFP-
tagged GAT1 in CHO cells. NNN stable transfected CHO cells were
incubated with and without dMM (1 m
M) for 72 h. The solubilized
protein of transfected cells (1 · 10
7
) was subjected to immunopre-
cipitation with anti-GFP Igs. Aliquots of each immunoprecipitate
were treated either with Endo H or PNGase F. The resulting mix-
ture and the other aliquots of the immunoprecipitate were analyzed
by SDS ⁄ PAGE (7.5%) and immunoblotting with anti-GAT1 pAb (A)
or anti-GFP mAb (B, C).
G. Cai et al. Role of N-glycosylation and N-glycan trimming of GAT1
FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS 1627

and mutants in a steady expression state were quanti-
fied. The distributions between cell surface and cell
interior of the mutants DGN, DND and DDN were
not significantly different from that of wild type NNN.
About 46 ± 4.7% is found on the cell surface. How-
ever, the percentage of the cell surface expression in
mutant DDQ which lacks all three N-glycosylation
sites was only 30 ± 4.4% in the steady expressed state
(Fig. 3B).
N-Glycosylation mutations result in reduction
of GABA-uptake activity
For quantitative measurement of the specific activity
of GABA-uptake, an aliquot of the stable CHO trans-
fectants was used for the GABA-uptake assay, and
another aliquot was used to determine the amount of
the membrane-expressed wild type or mutant proteins.
The GABA-uptake activities were normalized to the
same amount of cell surface proteins of wild type and
mutants. Compared with that of the wild type, the
GABA-uptake activities of the N-glycosylation
mutants were reduced significantly. Figure 3C shows
that the GABA-uptake activities of mutants with
double N-glycosylation mutations, DND, DGN and
Fig. 3. Determination of expression of GFP-tagged GAT1 mutants
on the surface of transfected CHO cells and measurement of
GABA-uptake by GFP-tagged GAT1 wild type and mutants in trans-
fected CHO cells. (A) Cell surface and intracellular expression of
GFP-tagged GAT1 wild type (NNN) and mutants (DGN, DND, DDN,
and DDQ) were analyzed by biotin labelling and Western blotting.
Anti-GAT1 serum or anti-GFP mAb MAB2510 were used for immu-

cosylation processing of NNN was inhibited by 1-de-
oxymannojirimycin (dMM). Inhibition by dMM leads
to the formation of NNN molecules containing N-gly-
cans of oligomannosidic type. Figure 4A shows that
after treatment with dMM (1 mm) for 72 h, the
amount of plasma membrane NNN containing man-
nosidic N-glycans was in the same range as that of
NNN containing mature complex N-glycans without
treatment with dMM. However, the activity of GABA-
uptake was reduced to 37% after treatment with dMM
(Fig. 4B). This indicates that the terminal trimming of
N-oligosaccharides is not involved in the regulation of
plasma membrane trafficking of GAT1, but in the
regulation of GABA uptake.
As well as wild type, mutant DND, DGN and
DDN exhibited only one small band on SDS ⁄ PAGE
after treatment with dMM (data not shown), indica-
ting that, like wild type, they contain only mannosidic
N-glycans. The level of cell surface expression was sim-
ilar with and without dMM treatment for both wild
type and mutants (Figs 4A and 5A). However, their
GABA-uptake activity was reduced to half after treat-
ment with dMM (1 mm) for 48 h (Fig. 5B). Although
mutant DND, DGN and DDN contain only one
N-glycosylation site, deficiency of terminal trimming of
their N-oligosaccharides strongly affected their GABA-
uptake activity. These indicate that the terminal
structure of the oligosaccharides facilitate efficient
GABA-uptake activity of the GABA transporter.
Defective N-glycosylation results in reduction

but the terminal structure of the N-glycans is not.
Defective N-glycosylation reduces the trafficking
of GAT1 to the plasma membrane
In order to study the influence of N-glycosylation on
plasma membrane trafficking of GAT1, the distribu-
tion of wild type and mutants on the cell surface and
in the cell interior was kinetically analyzed by pulse-
chase experiments. Figure 7 shows that after a 40 min
chase, 34% of total wild type (NNN) proteins, whereas
only 18 and 12% of total mutant DDN and DDQ
proteins, respectively, were expressed on the plasma
membrane. After a 120-min chase, the membrane
expression of the NNN was increased to 50%, whereas
that of mutant DDN and DDQ was increased only to
40% and 15%, respectively. This result suggests that
deficiency of N-glycosylation impairs the plasma mem-
brane trafficking of GAT1.
Defective N-glycosylation or dMM treatment did
not increase the K
m
GABA values of GAT1
The above results show that both N-glycosylation
mutations and terminal structures of N-linked oligo-
saccharide side chains have a measurable effect on the
GABA-uptake activity of GAT1. To determine whe-
ther the N-linked oligosaccharide side chains of GAT1
influence the affinity of GAT1 for GABA, concentra-
tion dependencies were analyzed on the basis of the
Michaelis–Menten equation
V ¼ V

mutant DDN were reduced significantly. The V
max
GABA value of NNN without dMM is 1.21
pmolÆlgÆprotein
)1
Æmin
)1
, whereas the value for mutant
DDN was only 0.29 pmolÆlgÆprotein
)1
Æmin
)1
. After
treatment of NNN with dMM, the V
max
GABA value
of NNN containing mannose-rich N-glycans was
strongly reduced to 0.55 pmolÆlgÆprotein
)1
Æmin
)1
.
Although mutations at N-glycosylation sites, as well as
N-glycans of the oligomannosidic type reduced the
V
max
value of rate of GABA uptake markedly, the K
m
GABA values were not affected. The data in Fig. 8
were fitted with a common K

and mutants in transfected CHO cells. (A) CHO stable transfectants
were pulse-labelled with 3.7 · 10
6
Bq per dish [
35
S]methionine for
1 h and chased for 0 min, 40 min, 80 min, 120 min and 180 min.
Membrane biotinylation was performed after chase. After cell solu-
bilization, total GFP-tagged GAT1 wild type and mutant proteins
were immunoprecipitated with anti-GFP pAb and eluted with
100 lL sample buffer containing 0.5% SDS. The eluates were dilu-
ted with NaCl ⁄ P
i
buffer to 400 lL. The biotin-labelled membrane
proteins were isolated from the diluted eluates with streptavidin
beads. After removal of all membrane proteins, the intracellular pro-
teins were immunoprecipitated with anti-GFP antibodies. Both M
(membrane) and I (intracellular) precipitates were eluted and ana-
lyzed by SDS ⁄ PAGE. (B) The results of the pulse-chase experi-
ments were analyzed by phosphoimager scanning. The total
radioactivity of membrane and intracellular fractions obtained by
immunoprecipitation at each chase time were set at 100%. Each
value represents the mean ± SEM of membrane fractions derived
from three separate experiments.
G. Cai et al. Role of N-glycosylation and N-glycan trimming of GAT1
FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS 1631
after treatment with dMM may be caused by the
reduction in substrate translocation by GAT1 (turn-
over rate).
Defective N-glycosylation results in reduced

concentration reveals that muta-
tion of the two N-glycosylation sites reduced the
apparent affinity from 24 m
)1
to about 8 m
)1
. The
transient currents in the absence of GABA were ana-
lyzed for jumps in potential to the holding potential of
)30 mV. The kinetics of the reaction step associated
with the extracellular Na
+
binding was slowed down
by both mutations (Fig. 9B). All the rate constants
slightly increased with increasing Na
+
concentration,
Fig. 8. Kinetic analysis of GABA-uptake by GFP-tagged GAT1 wild
type (NNN) with and without dMM and N-glycosylation mutant
(DDN). Kinetic analysis of GABA-uptake by GFP-tagged GAT1 wild
type (NNN) with and without dMM and N-glycosylation mutant
(DDN). GABA-uptake assays of wild type NNN pre-incubated with
and without dMM (1 mM) and mutant DDN were performed with
different GABA concentrations. All values presented were calcula-
ted after subtraction of the mock values. The data were fitted by a
Michealis–Menten equation with a common K
m
value of 4.1 lM
and V
max

Discussion
There is increasing evidence that cotranslational N-gly-
cosylation crucially influences the three-dimensional
structure, the biological half-life and intracellular traf-
ficking of proteins. It is also essential for many recog-
nition processes [13,14,16]. Previous studies showed
that the mutation of N-glycosylation sites resulted in a
reduction of GABA-uptake activity by GAT1 [7,10].
However, the possibility that this reduction in function
results from a decrease in the number of GABA trans-
porters per cell was not excluded. In order to clarify
whether N-glycans are directly involved in the GABA-
uptake process and whether the modulation of N-gly-
cosylation influences the biochemical properties of this
protein, quantitative and kinetic analysis of GABA
transport expression and activity was performed using
stable CHO transfectants. Both our own and commer-
cially available anti-GAT1 antibodies were unsuitable
for the quantitative analysis of GAT1, as they bind
very weakly to this protein. Therefore, wild type
GAT1 and N-glycosylation defective mutants were
tagged with GFP, which has been reported not to
influence the intracellular distribution of GAT1; more-
over, the tag does not modulate the relevant functions
of GAT1 [20]. For the quantitative analysis of GABA
transport activity, the cell surface expression of GAT1
wild type and N-glycosylation mutants was determined
by cell surface biotinylation and the resulting values
were used for normalization. This is a well established
method for the quantitative analysis of cell surface

trafficking of N-glycosylation mutants was delayed,
and ⁄ or partly inhibited due to retention of some
mutant protein in the ER, followed by digestion.
The transfectants of GAT1 wild type (NNN) and
mutant DGN, DND and DDN exhibited in SDS ⁄
PAGE (Fig. 3A) two intracellular bands, whereas only
one large band was found in the plasma membrane
fraction. However, treatment of the transfectants with
dMM, which inhibits N-glycosylation processing,
resulted in only small band in the SDS ⁄ PAGE for
both the wild type (NNN) (Figs 2C and 4A) and
mutant DNG, DND and DDN (data not shown). The
mutant DDQ, which does not possess any N-glycosyla-
tion site, expressed only one N-glycan-free band of
90 kDa in both the intracellular and the plasma mem-
brane compartments (Fig. 3A). The 108 kDa large
band of NNN was Endo H resistant, whereas digestion
with PNGase F converted it to a 90 kDa N-glycan free
polypeptide (Fig. 2C), indicating a mature N-glycan of
complex type. However, the 96 kDa small band of
NNN was converted to a 90 kDa polypeptide after
either Endo H- or PNGase F-digestion (Fig. 2B,C),
indicating an N-glycan of the mannosidic type. The
N-glycosylation mutants DGN, DDN and DND
exhibited a reduced molecular mass in accordance with
the absence of N-glycans at the two eliminated N-gyl-
cosylation sites in those proteins. This suggests that
the mutants DGN, DND and DDN, as well as wild
type NNN, were N-glycosylated in CHO cells and
their N-glycans were processed before they arrived at

edly reduced GABA-uptake activity (Fig. 3C) as well
as a GAT1-mediated current in CHO cells (Fig. 9A).
This is in accordance with our previous work using the
expression system of the Xenopus oocyte [7]. In order
to exclude the possibility that the reduction in function
in the mutants could be due to a reduction in the num-
ber of GABA transporters per cell, values for the
transport activity were normalized for the surface pro-
tein of these mutants. Double N-glycosylation mutants
showed a marked reduction of GABA-uptake activity
of 60–40% of that of the wild type. GAT-mediated
GABA transport activity could hardly be detected in
the mutant lacking all three N-glycosylation sites
(DDQ), despite the fact that this protein was expressed
on the surface of CHO cells (Fig. 3A). The N-glyco-
sylation processing inhibitor 1-deoxymannojirimycin
(dMM) also strongly inhibited GABA-uptake (Figs 4B
and 5B), although the amount of cell surface expres-
sion and the intracellular trafficking of GAT1 were
not affected by dMM (Figs 4A and 5A). This indicates
that the observed reduction of GABA-uptake activity
is a result of a deficiency of N-glycans. The possibility
that the reduced GAT1 activity could be due to a gen-
eral effect of the inhibitor on other glycoproteins
required for GAT1 activity is very unlikely. It has been
demonstrated that GAT transport function can be
reconstructed in liposomes and that no other pro-
teins are needed for GABA-uptake activity [30]. Our
results suggest that N-glycans, in particular their
terminal structure, are involved in the GABA-uptake

electrogenic substrate transport. Voltage-clamp experi-
ments suggest that deficient N-glycosylation reduces
the affinity of GAT1 for Na
+
[7]. The present work
revealed that the reduced transport activity can at least
partially be attributed to a reduced apparent affinity of
GAT1 for extracellular Na
+
and slowed kinetics of
the transport cycle (Fig. 9). This was observed in both
wild type and mutants after inhibition with dMM. As
the GABA transport process is driven by the gradient
of Na
+
, it is reasonable to deduce that the affinity of
GAT1 for Na
+
determines the turnover rate of GABA
transport. As the data presented in Fig. 9 are for a sin-
gle, functional transporter expressed on the cell sur-
face, the reduced GABA-uptake cannot be due to
reduced cell surface expression of transporters. In this
event the oligosaccharides of GAT1 play a role in the
regulation of GABA-uptake by affecting the affinity
for sodium ions.
In conclusion, cotranslational N-glycosylation is
important for the correct folding of GAT1 to a func-
tional conformation. Defective N-glycosylation leads
to decreased protein stability and disturbed intracellu-

(Clontech, Heidelberg, Germany) containing the cDNA
encoding the red-shifted GFP-variant. In this construct,
the cDNAs of GAT1 wild type or mutants were ligated
with cDNA of GFP with an identical reading frame, which
was confirmed by sequence analysis.
Preparation of polyclonal anti-GAT
and anti-GFP sera
Four different oligopeptides: LPWKQCDNPWNTDR
(159–172), MHQMTDGLDKPGQIRC(197–211), DEYPR-
LLRNRRELFC(409–423) and SEDIVRPENGPEQPQAC
(584–599), corresponding to sequences in the extracellular
and intracellular loops of GAT1, were synthesized and used
for immunization of rabbits. Specificity of the antiserum
was verified by immunoblotting with single or mixed
peptides. A polyclonal anti-GFP serum was prepared as
described previously [35].
Transfection of CHO cells and selection
of stable transfectants
In order to produce stable transfectans, each plasmid DNA
(2–4 lg) was transfected into 4 · 10
5
CHO cells using the
Eppendorf Multiporator and an appropriate Eppendorf
protocol (Wesseling-Berzdorf, Germany). Transfectants
were cultured in six-well plates in alpha-modified Eagle’s
medium (MEM alpha) containing 440 mgÆL
)1
l-glutamine
and 10% (v ⁄ v) fetal bovine serum for 2 days, then selected
with 400 mgÆL

at room temperature for
10 min. After permeabilization with 0.1% (v ⁄ v) Triton
X-100 in NaCl ⁄ P
i
at room temperature for 5 min, cells were
then extensively washed with NaCl ⁄ P
i
and blocked with 5%
bovine serum albumin and 0.1 m glycine in NaCl ⁄ P
i
for
30 min and washed with NaCl ⁄ P
i
again. Polyclonal antibod-
ies against GAT1 were used for immunostaining at room
temperature for 2 h. After further washing with NaCl ⁄ P
i
,
the cells were incubated with R-phycoerythrin conjugated
goat anti-(rabbit IgG) Ig (diluted 1 : 200) at room tempera-
ture for 1 h. The cells were extensively washed again with
NaCl ⁄ P
i
and then mounted with glycerol ⁄ NaCl ⁄ P
i
(10 : 1,
by volume) for fluorescence microscopy.
Immunoprecipitation and western blotting
analysis
Harvested cells were solubilized; followed by centrifugation

times, cells were solubilized. The expressed GAT1 ⁄ GFP
fusion proteins were immunoprecipitated with polyclonal
anti-GFP antiserum and analyzed by SDS ⁄ PAGE (7.5%).
Quantification of radio-labeled protein was carried out on a
PhosphorImager
TM
(Molecular Dynamics, Sunnyvale, CA,
USA) using iplabgel software. The total protein of the cell
surface and intracellular bands of each wild type or mutant
were set at 100%.
Endoglycosidase H treatment
Immunoprecipitates were eluted by boiling for 4 min in
buffer containing 0.4% SDS, 1% 2-mercaptoethanol and
40 mm EDTA. Endoglycosidase H (Endo H, Boehringer
Mannheim) treatment was performed with Endo H
(0.02 U ⁄ 80 lL) at 37 °C for 16 h in 50 mm sodium acetate
containing 0.5 lL protease inhibitor cocktail (Sigma) at
pH 5.5.
PNGase F treatment
Immunoprecipitates were eluted by boiling for 4 min in
buffer containing 0.5% (v ⁄ v) SDS, 50 mm 2-mercaptoetha-
nol. PNGase F (Roche) treatment was performed with
PNGase F (15 UÆ40 lL
)1
)at37°C 16 h in 500 mm
NaCl ⁄ P
i
containing 0.5% (w ⁄ v) Mega 10 and 0.5 lL pro-
tease inhibitor cocktail (Sigma) at pH 7.5.
Labelling of cell surface proteins with sulfo-NHS-

by boiling for 4 min in SDS sample buffer, and then ana-
lyzed by SDS ⁄ PAGE and western blotting. Either anti-
GFP mAb MAB2510 or anti-GAT1 pAb was used for the
immunostaining. The protein bands obtained in western
blotting were analyzed by phosphoimager scanning. The
total protein of the cell surface and intracellular bands of
each wild type or mutant were set at 100%.
Measurement of [
3
H]GABA uptake
To determine the transport activity, uptake of [
3
H]GABA
(Amersham-Pharmacia Biotech, Freiburg, Germany) was
measured in the presence of 128 mm external Na
+
and
10 lm total GABA. Cells incubated in 96-well tissue culture
plates were washed three times with wash buffer (128 mm
NaCl, 5.2 mm KCl, 2.1 mm CaCl
2
, 2.9 mm MgSO
4
,5mm
dextrose and 10 mm Hepes) and then incubated with
200 lL wash buffer containing 3.7 · 10
4
Bq [
3
H]GABA,

amounts of plasma membrane proteins of GAT1 or
mutants were analyzed by imager scanning of western blots.
The GABA-uptake activity was normalized with the same
amount of plasma membrane proteins of wild type and
mutants. The activity of GABA-uptake by NNN was set
at 100%. All other values were expressed relative to this
value.
Patch-clamp experiments
Voltage-clamp experiments were performed on CHO tran-
sient transfectants in the whole-cell patch-clamp configur-
ation. Steady-state and transient currents were measured in
response to rectangular voltage jumps from a holding
Role of N-glycosylation and N-glycan trimming of GAT1 G. Cai et al.
1636 FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS
potential of )30 mV to potentials of )100 or +40 mV,
using the EPC9 patch-clamp system and pulse software
(HEKA, Lambrecht, Germany). From transient charge
movements in the absence of GABA, the amount and volt-
age dependence of external Na
+
interaction with the trans-
porter can be determined [7]. From the time course of the
exponential current decline, the rate constant for a step
associated with extracellular Na
+
binding can be deter-
mined. In addition, the total amount of charge Q
max
moved
by the transporters and the effective valency of the charge

inschaft Bonn, the Sonnenfeld-Stiftung and the Fonds
der Chemischen Industrie, Frankfurt⁄ Main. The
cooperation between China and Germany was suppor-
ted on the basis of an agreement between the Max-
Planck Gesellschaft and the Chinese Academy of
Sciences. We are grateful to P Donner and A Becker
(Schering, AG) for the synthesis of peptides, to A
Niedergesa
¨
ss for production of anti-GFP sera, to Q
Gu and M Richter for technical assistance.
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