From the Laboratoire de Neurobiologie, Unité
Mixte de Recherche 8544, Centre National de la Recherche Scientifique,
École Normale Supérieure, 46 rue d'Ulm, 75230 Paris cedex
05, France and the § Centro de Biología Molecular
"Severo Ochoa," Facultad de Ciencias, Universitad Autónoma de
Madrid, Consejo Superior de Investigaciones Científicas,
28049 Madrid, Spain
Received for publication, October 9, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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The neuronal (GlyT2) and glial (GlyT1) glycine
transporters, two members of the
Na+/Cl Na+/Cl Within this superfamily, the two glycine transporters (GlyT1 and GlyT2)
have distinctive properties. A well established difference concerns
their substrate specificity, because sarcosine is a substrate of GlyT1b
(1) but not of GlyT2a (2). Recently, other pharmacological differences
have been identified; the tricyclic antidepressant amoxapine and
ethanol both selectively inhibit GlyT2a (3, 4). Electrophysiological
characterization of the two transporters has revealed additional
features differentiating GlyT2a from GlyT1b: (i) a stoichiometry of 3 Na+/1 Cl Sequence comparison of EL12
in the Na+/Cl Transporter inhibition by MTSET required prolonged application and high
concentrations of this reagent, which suggests a low accessibility of
the reactive cysteine either because it is partially buried inside the
protein or because it is infrequently exposed to the surface (8). In
fact, the orientation of EL1 has been a matter of debate (8, 10-12).
The original topological model proposed for the We characterized under voltage clamp the effects of MTSET and
(2-sulfonatoethyl) methanethiosulfonate (MTSES) on the glycine uptake,
the glycine-evoked current, and the charge movement of the two wild
type glycine transporters, as well as on those of two point mutants
of the first amino acid of EL1, GlyT1b-C62A and GlyT2a-A223C, which
exchanged the MTSET reactivity. The difference in residue number for an
equivalent position in EL1 sequence is explained by the much longer N
terminus of GlyT2a (15). In addition, the GlyT2a-EL1 chimera resulting
from the mutation of the first three amino acids of GlyT2a-EL1 (AFQ) to
the GlyT1b sequence (CYR) was studied in detail because it influenced
MTSET reactivity and had a dramatic effect on the outward
Cl Site-directed Mutagenesis, RNA Synthesis, and
Expression--
Rat GlyT1b (gift from K. Smith, Synaptic Corporation
(16)) and rat GlyT2a (15) cDNAs were subcloned as indicated by
Supplisson and Bergman (1). Point mutations were performed using the
method of Kunkel (17). cRNAs were transcribed in vitro using
the T7 mMessage-mMachine kit (Ambion, Austin, TX), and 10-100 ng were injected into oocytes using a nanoliter injector (World Precision Instruments, Sarasota, FL). Xenopus laevis oocytes were
prepared and incubated as indicated by Roux and Supplisson (5).
Electrophysiology--
Two-electrode voltage clamp was performed
using a O-725A amplifier (Warner Instrument, Hamden, CT), a Digidata
1200 interface, and the pClamp7 software (Axon Instruments, Foster
City, CA). Glass electrodes were filled with a 3 M KCl
solution and had a resistance of 0.5-2 M Charge Movement--
Transient currents specific to GlyT1b or
GlyT2a were evoked in the absence of glycine by stepping the voltage at
various potentials. These currents are
Na+-dependent and capacitative in nature
because their time integrals estimated following the onset and return
from the voltage step are equal in amplitude but opposite in sign. For
GlyT1b, the charge movement was fitted with a single exponential
function of the current decay upon repolarization. For GlyT2a and
GlyT2a-EL1, the charge movement could not be fitted with a single
exponential and was quantified by integration of the transient currents
recorded during the membrane repolarization to the holding potential
(off current) after subtraction of the current recorded in
absence of Na+. The charge distribution as a function of
membrane potential was fitted with a Boltzmann distribution as shown
Equation 1.
Glycine Uptake Assays--
Oocytes (n = 5-15)
expressing wild type glycine transporters or various EL1 constructs
were incubated for 10 min in 5 ml of choline chloride Ringer
solution in the presence or the absence of 2 mM MTSET. Then
oocytes were incubated for 10 min in control NaCl Ringer solution or in
choline chloride Ringer solution containing 5 µM
[U-14C]glycine (specific activity, 103 µCi/µmol;
Amersham Pharmacia Biotech). Oocytes were washed three times in
ice-cold NaCl Ringer solution and placed individually in vials
containing 500 µl of SDS (2%). The radioactivity was measured on a
Kontron liquid scintillation counter.
Charge Coupling--
Oocytes were voltage clamped at Transfer of EL1 Confers MTSET Sensitivity from GlyT1b to
GlyT2a--
The glycine transporters GlyT1b and GlyT2a, expressed in
Xenopus oocytes, showed differential sensitivities to MTSET.
The glycine-evoked current (IT) (Fig.
1a) and the transient currents (Fig. 1b) recorded in the absence of glycine in
GlyT1b+ oocytes were strongly and irreversibly reduced
following a 5-min superfusion with choline chloride Ringer solution
containing 1 mM MTSET. In contrast, similar applications of
MTSET affected neither IT (Fig. 1c)
nor the transient currents nor the outward rectifying leak conductance
(Fig. 1d) recorded in GlyT2a+ oocytes. To test
whether these differences could result from the presence (GlyT1b) or
the absence (GlyT2a) of a cysteine in the first extracellular loop
(EL1), as suggested by results obtained on GAT-1 (8) and SERT (9), we
applied MTSET both on the point mutant GlyT2a-A223C (Fig. 1,
e and f) and on the GlyT2a-EL1 chimera (Fig. 1,
g and h) in which the AFQ sequence of GlyT2a was
replaced by the GlyT1b sequence CYR (Table
I for sequence comparison). With
GlyT2a-A223C and GlyT2a-EL1, application of MTSET reduced
IT (Fig. 1, e and g), the
transient currents, and the outwardly rectifying leak conductance (Fig.
1, f and h). However, the inhibition of
GlyT2a-EL1 was partial, as for GlyT1b, whereas GlyT2a-A223C was fully
inhibited under the same conditions.
MTSET and Leak Current--
MTSET did not change the leak current
in oocytes expressing either GlyT1b (Fig. 1b) or GlyT2a
(Fig. 1d), but it affected the leak current of GlyT2a-A223C
and GlyT2a-EL1. Like GlyT2a, both of these constructs exhibit an
outwardly rectifying "leak" conductance at depolarized potentials
that is particularly large in the case of GlyT2a-EL1. Although
this outward leak conductance was not changed by MTSET in GlyT2a (Fig.
1d), it was suppressed in both GlyT2a-A223C and GlyT2a-EL1
(Fig. 1, f and h). In contrast, we observed after
MTSET treatment a limited increase in the inward steady state leak
current at hyperpolarized potential for these two constructs (Fig. 1,
f and h).
MTSET Blocks the Uptake Current and the Charge Movement in the Same
Proportion--
A 5-min application of MTSET reduced the charge
movement of GlyT1b (Fig. 2a),
GlyT2a-EL1 (Fig. 2e), and GlyT2a-A223C (Fig. 2g)
but not that of GlyT2a (Fig. 2c). The reduction in
Qmax (Table II), which is
proportional to the number of functional transporters present in the
plasma membrane (see "Experimental Procedures"), indicates that the
loss of transport activity observed after cysteine modification by
MTSET results from the hindrance of a kinetic step, which precedes
substrate binding and translocation because the charge movement of
glycine transporters corresponds to Na+- and
voltage-dependent transitions that occur in the absence of
glycine (5). The Na+ dependence of the transient currents
for oocytes expressing GlyT1b, GlyT2a, GlyT2a-El1, and GlyT2a-A223C is
shown in Fig. 3 with the unsubstracted
off currents recorded during the repolarization step to the
holding potential in NaCl Ringer solution, in choline chloride Ringer
solution, and after MTSET application.
In the potential range from
As expected for an inactivation process, the glycine EC50
was not changed by MTSET application for the two transporters tested, GlyT1b (22.5 ± 1.10 µM (n = 7) and
19.5 ± 1.4 µM (n = 3) before and
after MTSET application, respectively) and GlyT2a-EL1 (24.1 ± 3.1 µM (n = 3) and 23.2 ± 1.8 µM (n = 3) before and after MTSET application, respectively).
EL1 Sequence Influences the Cysteine Accessibility--
To compare
the accessibility of the EL1 cysteine for the various sensitive
constructs, we studied the time course of the MTSET inhibition. The
progressive decrease in uptake current plotted as a function of the
cumulative exposure to MTSET is shown in Fig.
4. The decay in current had time
constants of 3.8 and 3.9 min for GlyT1b and GlyT2a-EL1, respectively,
but was 5-fold faster with GlyT2a-A223C, with a time constant of 0.67 min. The difference in the time constants indicates that in glycine
transporters, the accessibility of the EL1 cysteine to the
extracellular solution and its reactivity to MTSET are strongly
influenced by the next two residues that are modified in GlyT2a (FQ
versus YR in GlyT1b).
To confirm that EL1 was fully responsible for GlyT1b sensitivity to
MTSET, we constructed the reverse chimera GlyT1b-EL1. For this
transporter, no change in the amplitude of glycine-evoked current
was observed for period of incubation up to 20 min (Fig. 4).
MTSET Has the Same Effect on Glycine Uptake and
Glycine-evoked Current--
To verify that the reduction of the
glycine-evoked current by MTSET was reflecting a comparable change in
the influx rate of glycine in oocytes expressing sensitive glycine
transporters, we repeated the MTSET inactivation protocol using tracer
flux of glycine. A summary of these uptake experiments is shown in Fig.
5A that confirms the large
reduction of the Na+-dependent glycine uptake
with oocytes expressing GlyT1b, GlyT2a-EL1, and GlyT2a-A223C. No
significant inhibition by MTSET is observed with GlyT2a, GlyT1b-EL1,
and the point mutant GlyT1b-C62A.
Finally, we checked the charge coupling of each transporter by applying
radiolabeled glycine under voltage clamp conditions as described by
Roux and Supplisson (5). The results shown in Fig. 5B
indicate that the specific charge coupling of the wild type
transporters (1 and ~2 for GlyT1b and GlyT2a, respectively) are not
affected by EL1 sequence modification and that glycine-evoked current
is proportional to glycine uptake.
Na+, Cl Voltage Dependence of MTSET and MTSES Reactivity on EL1
Cysteine--
GlyT1b oocytes were perfused with 1 mM MTSET
for 5 min in choline chloride Ringer solution at holding potentials
between
The inhibition of GlyT2a-EL1 by MTSET or MTSES was not
voltage-dependent (Fig. 7B). In addition, the
potency of MTSES on GlyT2a-EL1 and GlyT2a-A223C (Fig. 7C)
was much lower than that of MTSET, as expected from their reported
relative reactivity (18). This contrasts with the fact that the two
reagents inhibited GlyT1b with comparable potencies (Fig.
7A).
Inactivation of Glycine Transporters by MTSET Can Be Reversed with
DTE--
Covalent modification of cysteine by MTSET produced an
inactivation of sensitive glycine transporters that each contain a cysteine in their EL1 sequence. This inactivation persisted after complete wash-out of MTSET (Fig.
8B). We tested whether the
application of a reducing agent like DTE may reverse the alkylation by
MTS reagents, as reported for the glucose transporter SGLT1 (19). The
data shown in Fig. 8 indicate that superfusion with DTE (5 min, 10 mM in choline chloride Ringer solution) restored partially or totally the glycine-evoked current, with a more dramatic effect with
GlyT2a-A223C. In addition, the small increase in leak current observed
with GlyT2a-EL1 (Fig. 1h) and GlyT2a-A223C (Figs.
1f and 8, A and B) was reversed by
DTE.
The topological organization of the
Na+/Cl Glycine transporters are good candidates for the substituted cysteine
accessibility method because one of them (GlyT2a) is totally
insensitive to MTS reagents in uptake assays and voltage clamp studies,
thus allowing further structure/function characterization without the
need for a cysteine-less mutant (18). Sequence comparison reveals that
GlyT2a lacks the well conserved cysteine in EL1 that is present in
GlyT1b and to which MTSET sensitivity was attributed in GAT-1 (8) and
SERT (9). This cysteine substitution (A223C) is the first of three
consecutive differences between GlyT1b (CYR) and GlyT2a (AFQ) within a
stretch of 25 otherwise conserved amino acids in the transmembrane
segment 1 and 2 region (Table I), which is known to be critical for
function (27-30).
We decided to probe EL1 accessibility and function in glycine
transporters combining glycine uptake assay and transport current recordings. We have shown previously that charge and glycine uptake are
precisely correlated in oocytes expressing GlyT1b (+1 elementary charge
(e)/glycine) and GlyT2a (+~2 e/glycine) (5) and
that the two transporters differ notably in their pre-steady state kinetics and leak current. The interest of a dual approach was reinforced by the fact that conflicting results have been reported on
GAT-1 concerning MTSET accessibility (8, 10, 14).
We report here that the first amino acid of EL1 (Cys62) in
GlyT1b, which is also found in GAT-1 (Cys74), SERT
(Cys109), and DAT (Cys90), is accessible to
extracellularly applied MTSET and MTSES. All transporter-associated
currents (pre-steady state and uptake) were blocked similarly after
cysteine modification by MTS reagents. In contrast, we found that none
of the electrophysiological properties (glycine-evoked current, charge
movement, and leak current) of the wild type GlyT2a were affected by
MTSET. Introduction of a cysteine (A223C), either by point mutation or
by replacing EL1 with the GlyT1b sequence, conferred MTS reagent sensitivity.
Previous results on the cysteines of
Na+/Cl The rate of inactivation was influenced by the two other nonconserved
residues of EL1. Differences in reactivity may be attributed to the
presence (GlyT1b, GlyT2a-EL1) or the absence (GlyT2a-A223C) of the
positive charge of an arginine in position n + 2. GAT-1, which has a lysine in the equivalent position, is inhibited by MTSET
with a time course (8) comparable with GlyT1b and GlyT2a-EL1. This
positive charge may also be responsible for the similar rate of
inhibition by MTSET and MTSES on GlyT1b, which contrasts with the
>10-fold difference in reactivity in solution between these two
compounds (18). Comparable changes in reactivity ratio have been
observed in the case of the CFTR channel (32), for which residues
located deep inside the pore (Thr351 and
Gln353) show a higher reactivity for MTSES than MTSET. In
this channel, the arginine at position 352 is considered to be a key
residue for anion selectivity (32). In the wild type GlyT1b,
Arg64 may facilitate the access to Cys62 for
the negatively charged but less reactive MTSES.
The orientation of the arginine, in addition to its charge, plays a
role in controlling the access to EL1 cysteine, because GlyT2a-EL1 was
much less sensitive to MTSES than to MTSET (by a factor of ~5, as
estimated from the inhibition after 5 min). In addition, position 225 of GlyT2a (an arginine in GlyT2a-EL1) is not accessible to
extracellularly applied MTSET.3 Together,
these results suggest that EL1 has a different conformation in GlyT1b
and GlyT2a.
Another difference between the two transporters was that membrane
potential influenced the MTS inhibition of GlyT1b but not of GlyT2a-EL1
in the absence of external Na+. Because the voltage
dependence of MTSET and MTSES inhibition of GlyT1b were similar, a role
of the reagent charge could be ruled out. Some
voltage-dependent transition in the transporter must favor
EL1 cysteine exposure at negative potentials. This transition is
unlikely to correspond to extracellular Cl The presence of both Na+ and Cl-dependent
neurotransmitter transporter superfamily, differ by many aspects,
such as substrate specificity and Na+ coupling. We have
characterized under voltage clamp their reactivity toward the membrane
impermeant sulfhydryl reagent
[2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). In
Xenopus oocytes expressing GlyT1b, application of MTSET
reduced to the same extent the Na+-dependent
charge movement, the glycine-evoked current, and the glycine uptake,
indicating a complete inactivation of the transporters following
cysteine modification. In contrast, this compound had no detectable
effect on the glycine uptake and the glycine-evoked current of GlyT2a.
The sensitivities to MTSET of the two transporters can be permutated by
suppressing a cysteine (C62A) in the first extracellular loop (EL1) of
GlyT1b and introducing one at the equivalent position in GlyT2a, either
by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1
and GlyT2a-EL1) resulting in AFQ
CYR modification. Inactivation by
MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or
GlyT1b, suggesting that the arginine in position +2 reduced the
cysteine reactivity. Protection assays indicate that EL1 cysteines are
less accessible in the presence of all co-transported substrates:
Na+, Cl
, and glycine. Application of
dithioerythritol reverses the inactivation by MTSET of the
sensitive transporters. Together, these results indicate that EL1
conformation differs between GlyT1b and GlyT2a and is modified by
substrate binding and translocation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-coupled transporters control the
extracellular and intracellular concentrations of neurotransmitters
like bioamines,
-aminobutyric acid, and glycine. Through the
thermodynamically coupled uptake of Na+/Cl
,
and the transmitter, transporters help terminate synaptic
transmission, keeping a low extracellular concentration of
neurotransmitter, and limiting the spill-over of neurotransmitter
between neighboring synapses. At some presynaptic boutons, their
concentrative power may also be required for an efficient supply of
neurotransmitter to the low affinity vesicular transporters.
/glycine compared with only 2 Na+/1 Cl
/glycine for GlyT1b (resulting in a
charge to glycine flux ratio of ~2 for GlyT2a versus 1 for
GlyT1b) (5); (ii) a reduced capacity for reverse uptake (5); (iii) an
uncoupled, outwardly rectifying Cl
conductance; and (iv) a biexponential
transient current recorded in the absence of glycine.1
The amino acids involved in these differences between
GlyT1b and GlyT2a have not been identified yet.
-coupled transporter
superfamily shows that GlyT2a together with PROT (6) and
ATB0,+ (7) are the only members that lack a conserved
cysteine in EL1 that has been identified in GAT-1
(Cys74; Ref. 8) and SERT (Cys109; Ref.
9) as a primary target for the membrane-impermeant sulfhydryl reagent
MTSET. Mutation in GlyT2a EL1 induced an increase in the outwardly
rectifying leak current characteristic of this
transporter,1 which further suggested that the conformation
of this loop may differ between GlyT1b and GlyT2a. To assess this
issue, we tested the reactivity to sulfhydryl reagents of GlyT1b and GlyT2a.
-aminobutyric acid
transporter GAT-1 (13) predicted cytoplasmic N and C termini and 12 transmembrane segments linked by six extracellular loops and five
intracellular loops. In accordance to this model, most positions in EL1
were found to be accessible to extracellularly applied MTSET when
mutated to cysteine in GAT-1 (8). In addition, MTSET dramatically
increased the oocyte leak current (8). However, inhibition by MTSET was not detected in uptake experiments in GAT-1 transfected mammalian cells
(10), whereas the inactivation produced by the membrane permeant
(2-aminoethyl)methane thiosulfonate was attributed to cysteine 399 located in intracellular loop 4 (14).
current specific of GlyT2a.1
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. The typical extracellular
recording Ringer solution contained 100 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.2 adjusted with KOH. In some experiments,
choline and gluconate were substituted for Na+ and
Cl
, respectively. Agar bridges (equilibrated in a 3 M potassium gluconate solution) were used when required.
Salts and glycine were purchased from Sigma. Aliquots of
dithioerythritol (300 mM, Sigma), MTSET, and MTSES (100 mM, Toronto Research Chemical Co., Toronto, Canada) were
prepared in ice-cold water and kept at
20 °C. They were thawed and
diluted in the appropriate solution just prior to perfusion (<10 s).
Data are given as the means ± S.E. with the number of experiments.
where V1/2 is the half-distribution
potential, z
(Eq. 1)
is the equivalent charge per transporter
moving through the membrane electric field, and R,
T, and F have their usual meanings.
Qmax is the total charge movement and is defined as Qmax = z
F
NT, where NT is the
number of transporters/oocyte.
40 mV and
perfused for 60-90 s with a solution containing 25 µM
[U-14C] glycine and 75 µM cold glycine.
After complete wash-out and after the current returned to the base-line
level, oocytes were transferred in 5 ml of ice-cold Ringer solution,
washed twice, then lysed in vials containing 500 µl of SDS (2%), and
measured for radioactivity. The charge coupling was determined as the
ratio between the glycine-evoked current integral and the glycine
uptake as described by Roux and Supplisson (5).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Glycine-evoked and glycine-independent
currents prior to and after MTSET application for GlyT1b, GlyT2a,
GlyT2a-A223C, and GlyT2a-EL1. a, c,
e, and g, current evoked by 200 µM
glycine in NaCl Ringer solution (gray bar) at 40 mV prior
to (thin line) and after (thick line) MTSET
application (1 mM MTSET in choline chloride Ringer solution
for 5 min,
40 mV holding potential). The horizontal and
vertical scale bars are 10 s and 75 nA, respectively.
b, d, f, and h, currents
evoked by voltage jumps to potentials between +50 and
130 mV from a
holding potential of
40 mV in NaCl Ringer solution prior to and after
MTSET application (same conditions as above). Horizontal and
vertical scale bars are 50 ms and 250 nA,
respectively.
Sequence alignment of the TM1-TM2 region of wild type and mutant
glycine transporters.
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Fig. 2.
Q-Vm and
IT-Vm
relationships for GlyT1b (a and b),
GlyT2a (c and d), GlyT2a-EL1
(e and f), and GlyT2a-A223C
(g and h) before ( ) and after
(
) MTSET application (1 mM MTSET). Charge was
measured in NaCl Ringer solution, and IT was
measured in NaCl Ringer solution and 200 µM glycine. For
each oocyte, data were normalized to the Qmax
determined prior to MTSET application (n = 4-5).
Percentage of reduction of Qmax and Imax
induced by 1 mM MTSET for 5 min in oocytes expressing wild type and
EL1 mutant of glycine
transporter
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Fig. 3.
Na+-dependent charge
movement. Unsubtracted off currents for GlyT1b, GlyT2a,
GlyT2a-EL1, and GlyT2a-A223C recorded during the repolarization
step to the holding potential ( 40 mV) from +50 to
130 mV, in the
presence of NaCl and choline chloride and after exposure to MTSET (1 mM).
140 to +50 mV, the
IT-voltage relationships of GlyT1b, GlyT2a, and
GlyT2a-A223C (Fig. 2, b, d, and g)
were quasi-linear, whereas that of GlyT2a-EL1 was inwardly rectifying
(Fig. 2f). For comparison, IT values
were normalized to the Qmax determined before MTSET
treatment. The transport currents were reduced in the same proportion
at all potentials by MTSET for GlyT1b, GlyT2a-EL1, and GlyT2a-A223C
transporters, as shown in Fig. 2 (b, f, and
h). For each oocyte, the percentage of the remaining
transport current was subsequently calculated as the mean of the
experimental values between
140 and 0 mV (every 10 mV). Values
obtained at voltages above 0 mV were ignored because of their small
amplitudes (especially for GlyT2a-EL1). The reduction in
IT after MTSET treatment was comparable with the
reduction observed in Qmax as indicated in Table II.
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Fig. 4.
Time course of MTSET inhibition. Plot of
the glycine-evoked current for GlyT1b ( ), GlyT1b-EL1 (
),
GlyT2a-EL1 (
), and GlyT2a-A223C (
) (200 µM glycine
in NaCl Ringer solution) as the function of the time of exposure to 1 mM MTSET in choline chloride Ringer solution (
40 mV
holding potential; n = 3-5). Solid lines
are fits to the data with single exponential functions.
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Fig. 5.
Effect of MTSET on glycine uptake.
A, glycine uptake was performed as described under
"Experimental Procedures" with oocytes expressing GlyT1b,
GlyT1b-EL1, GlyT1b-C62A, GlyT2a, GlyT2a-EL1, or GlyT2a-A223C. Oocytes
were pretreated for 10 min in choline chloride Ringer solution with or
without MTSET (2 mM). Oocytes were incubated for 10 min in
5 ml of control NaCl Ringer solution or choline chloride Ringer
solution containing 5 µM [U-14C]glycine.
B, charge coupling was estimated as the ratio between the
time integral of the current evoked by the application (60-90 s) of 25 µM [U-14C]glycine with 75 µM
cold glycine and the simultaneous glycine uptake (see "Experimental
Procedures").
, and Glycine Limit EL1 Cysteine
Accessibility--
MTSET applications (1 mM for 5 min at
40 mV) were performed on GlyT1b, GlyT2a-EL1, and GlyT2-A223C in the
presence or the absence of the transporter substrates to see whether
these substrates protected against MTSET. As described for Fig.
6, inhibition by MTSET was reduced by the
joint presence of Na+ and Cl
and was further
reduced by glycine. Sarcosine, a specific substrate of GlyT1b,
protected this transporter to a similar extent as glycine but did not
protect GlyT2a-EL1. In all transporters tested, Na+,
Cl
, or glycine alone did not induce any protection (Fig.
6). This is in good agreement with the fact that neither the uptake
current nor the charge movement can be observed when one of the
co-transported ions is absent (Ref. 5).1 The best
protection was observed for GlyT2a-EL1, for which a quasi-complete
protection by NaCl and glycine was found.
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Fig. 6.
Protection assays. Normalized
glycine-evoked current after MTSET application (5 min, 1 mM, 40 mV) in the presence or the absence of the ionic or
organic substrates for GlyT1b, GlyT2a-EL1, and GlyT2a-A223C. For each
transporter, all conditions that are not significantly different
(p < 0.02) from our control conditions (choline
chloride (choline Cl)) are represented in light colors
(gray or white bars), whereas conditions that
correspond to a significant protection compared with control are
represented as black bars (n = 4-5).
n.d., not done.
120 and 0 mV. Inhibition increased with hyperpolarization,
from 62.3 ± 5.2% (n = 4) at 0 mV to 84.4 ± 0.9% (n = 4) at
120 mV (Fig. 7A). To test whether this
voltage dependence was due to a voltage-dependent transition of the transporter in the absence of Na+ or to
the positive charge of MTSET, we repeated the experiments using the
negatively charged MTSES. Fig. 7A shows that MTSES
inhibition of GlyT1b had the same sign of voltage dependence as MTSET,
with the inhibition increasing from 51.5 ± 1.3% at 0 mV to
79.0 ± 0.9% at
120 mV.
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Fig. 7.
Voltage dependence of the MTSET and MTSES
inhibition. Normalized GlyT1b (A) and GlyT2a-EL1
(B) remaining glycine-evoked currents (200 µM,
NaCl Ringer solution) after MTSET ( ) or MTSES (
) application as a
function of the membrane potential during MTS reagents application. MTS
reagents were applied for 5 min at 1 mM during voltage
clamp at the indicated membrane potential in choline chloride Ringer
solution. C, similar results for GlyT2a-A223C for a holding
potential of
40 mV (n = 4 or 5 for all
conditions).
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Fig. 8.
Glycine transporter inactivation by MTSET is
reversed by DTE. A, representative traces of glycine
(100 µM) evoked current for GlyT1b (a-c) and
GlyT2a-A223C (d-f) recorded in control Ringer solution
(a and d), after 5-min applications of MTSET (1 mM) in choline chloride Ringer solution (b and
e), and after a 5-min application of DTE (10 mM)
in choline chloride Ringer solution (c and f).
The dotted line represents the initial base-line level.
B, representative experiment with GlyT2-A223C showing the
base-line current ( ) and the steady state glycine evoked current
(
) recorded every minute for a 10-s application of glycine (100 µM). No current were recorded during the application of
MTSET and DTE in choline chloride Ringer solution. C,
normalized glycine uptake current in control NaCl Ringer solution
(open bars), after a 5-min application of 1 mM
MTSET (black bars), or after a 5-min application of 10 mM DTE (gray bars).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
neurotransmitter transporter family
has now received good experimental support in favor of the original
model of 12 putative transmembrane segments proposed for GAT-1 (13).
Nevertheless the precise organization of the first third of the
protein, which contains amino acids critical for function, is still
debated (8-12, 14, 20, 21). Recent insight into the localization of
the loops and the accessibility of membrane segments has been obtained
(8, 9, 12, 14) through the identification of solvent-accessible
residues using the substituted cysteine accessibility method (for
review, see Ref. 18). This method has been extensively applied to
determine the residues lining the pore of ionic channels and receptors
(22-25) and to map the ligand-binding crevice in G-protein-coupled
receptors (26).
-coupled transporters involved high
concentrations of MTS reagents (
1 mM) and prolonged applications (5-10 min in most cases) to achieve only partial inhibition (8-10, 12, 14). Similarly, the reduction of uptake current
of the wild type GlyT1b was only partial after a 5-min exposure to
MTSET (1 mM). This partial inhibition resulted from a loss
of function of the fraction of transporters that had a modified
cysteine. Complete inhibition of GlyT1b and GlyT2a-EL1 required
20
min of incubation with MTSET, whereas GlyT2a-A223C was fully inhibited
in about 4 min. Rate constants estimated from the time constants of the
inhibition (18) were in the order of 4.4 M
1
s
1 for GlyT1b and GlyT2a-EL1 and of 25 M
1 s
1 for GlyT2a-A223C. These
rate constants are many orders of magnitude slower than for the
reaction of MTSET with 2-mercaptoethanol in solution (18) or for
exposed cysteines near the entrance of the pore in acetylcholine
receptor (31). Nevertheless, these rate constants are in the range
reported for cysteines located near the cytoplasmic end of the
acetylcholine receptor pore in the absence of ligand (31). This
indicates that even in the more reactive GlyT2a-A223C the EL1 cysteine
is exposed only transiently to the extracellular solution or is located
in a diffusion-limiting crevice.
binding to the
transporter, because similar inhibition levels were achieved in choline
chloride and choline-gluconate solutions at
40 mV. Although glycine
transporters capacitative currents are
Na+-dependent as are those of other
Na+-coupled transporters, around 20% of GlyT1b charge
movement persists after Na+ removal (choline substitution)
from the extracellular solution.1 Such residual charge
movement is not observed in GlyT2a or GlyT2a-EL1, consistent with the
fact that no voltage dependence was observed for GlyT2a-EL1 inhibition
by MTSET or MTSES. GlyT1b probably has either endogenous charges or
ions bound to intracellular sites and can undergo
voltage-dependent transitions even in the absence of ions
bound to the extracellular sites.
in the
extracellular medium, which are required for the charge movement
observed in response to voltage jumps in the absence of glycine,
partially protected the three MTSET-sensitive transporters GlyT1b,
GlyT2a-A223C, and GlyT2a-EL1. Na+ or Cl
alone
(choline or gluconate substitution) did not protect, suggesting a
strong allosteric interaction between the Na+- and
Cl
-binding sites. The addition of glycine to
Na+ and Cl
further protected both GlyT1b and
GlyT2a-EL1. Altogether, these results indicate that the conformational
changes occurring in the transport cycle modify the EL1 cysteine
accessibility. In addition, the conformation of this loop appears to
differ between GlyT1b and GlyT2a, maybe as a consequence of the
structural constraints imposed by the extra Na+-binding
site of GlyT2a.
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ACKNOWLEDGEMENTS |
---|
We thank Philippe Ascher and Boris Barbour for critical comments and suggestions on the manuscript.
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FOOTNOTES |
---|
* This work was supported by the CNRS.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. Tel.: 331-44-32-38-94; Fax: 331-44-32-38-87; E-mail: supplis@wotan.ens.fr.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M009196200
1 M. J. Roux and S. Supplisson, unpublished observations.
3 B. López-Corcuera, R. Martínez-Maza, E. Núñez, A. Geerlings, and C. Aragón, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EL1, extracellular loop 1; MTS, methanethiosulfonate; MTSES, (2-sulfonatoethyl) methanethiosulfonate; MTSET, [2-(trimethylammonium)-ethyl]-methanethiosulfonate; DTE, dithioerythritol.
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