From the Membrane Biology Program, Brigham and
Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115, ¶ Cecil B. Day Laboratory for
Neuromuscular Research, Massachusetts General Hospital, Harvard Medical
School, Charlestown, Massachusetts 02129, and ** Department of
Physiology, Institute of Basic Medical Sciences, University of Oslo,
N-0337 Oslo, Norway
Received for publication, May 4, 2000, and in revised form, August 29, 2000
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ABSTRACT |
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We have investigated the functional impact of a
naturally occurring mutation of the human glutamate transporter GLT1
(EAAT2), which had been detected in a patient with sporadic
amyotrophic lateral sclerosis. The mutation involves a substitution of
the putative N-linked glycosylation site asparagine 206 by
a serine residue (N206S) and results in reduced glycosylation of the
transporter and decreased uptake activity. Electrophysiological
analysis of N206S revealed a pronounced reduction in transport rate
compared with wild-type, but there was no alteration in the apparent
affinities for glutamate and sodium. In addition, no change in the
sensitivity for the specific transport inhibitor dihydrokainate was
observed. However, the decreased rate of transport was associated with
a reduction of the N206S transporter in the plasma membrane. Under ionic conditions, which favor the reverse operation mode of the transporter, N206S exhibited an increased reverse transport capacity. Furthermore, if coexpressed in the same cell, N206S manifested a
dominant negative effect on the wild-type GLT1 activity, whereas it did
not affect wild-type EAAC1. These findings provide evidence for
a role of the N-linked glycosylation in both cellular
trafficking and transport function. The resulting alteration in
glutamate clearance capacity likely contributes to excitotoxicity that
participates in motor neuron degeneration in amyotrophic lateral sclerosis.
Because synaptic glutamate is potentially neurotoxic, control of
extracellular glutamate levels at glutamatergic synapses is of critical
importance (1-4). Accumulation of extracellular glutamate to
neurotoxic levels is normally prevented by specialized transporters
located in the plasma membrane of neurons
and glial cells (5-9). Five different
isoforms of glutamate transporters, constituting a distinct family of
Na+-dependent carriers, have been identified:
GLAST (EAAT1), GLT1 (EAAT2), EAAC1 (EAAT3),
EAAT4, and EAAT5 (10-14). The different transporters of this family
share similar structural traits and exhibit different yet comparable
functions. Recent studies in several laboratories have provided
significant advances in the understanding of the structure and function
of these transporters (13, 15-25). These findings constitute a new
direction in our knowledge of ion coupling, stoichiometry, membrane
protein structure, and pathophysiological implications of transporters.
Dysfunction in glutamate homeostasis is implicated in a number of acute
and chronic neurodegenerative diseases (26). An abnormality in the
glutamate transport system has been reported in patients with
amyotrophic lateral sclerosis (ALS). ALS is a progressive neurological
disorder characterized by degeneration of upper and lower motor
neurons. Although the primary pathogenic trigger is unknown, evidence
is mounting to implicate a role for glutamate-mediated excitotoxicity
in the disorder. A decrease in glutamate transporter activity was
observed in synaptic preparations from motor and sensory cortex of
sporadic ALS patients (27), which was subsequently ascribed to reduced
levels of the glutamate transporter isoform GLT1, as assessed by
immunodetection techniques (28). An aberrant RNA editing process was
reported as a probable cause for loss of GLT1 in the sporadic form of
ALS (29). However, the involvement of aberrant splicing processes for
the GLT1 mRNA in ALS is still controversial (30-35).
Our recent investigations indicated that oxidative alterations of human
GLT1 caused by acquired reactivity of familial ALS-linked superoxide
dismutase 1 mutants lead to impaired glutamate uptake (36). Given the
accumulating evidence for an important role of GLT1 in both sporadic
and familial ALS, the question arises of whether mutations in GLT1
could be a cause or a risk factor in ALS pathogenesis.
Using single-strand conformation polymorphism analysis of genomic DNA,
we recently reported a mutation in the GLT1 gene associated with
sporadic ALS, which substitutes an asparagine for a serine at position
206 (37). We speculated that this mutation might be of functional
significance, because asparagine 206 is a putative glycosylation site
of GLT1 (12, 38-40). The experiments illustrated in this report
demonstrate the impact of this mutation (N206S) on the functional
properties of the human glutamate transporter GLT1.
Site-directed Mutagenesis--
The cDNA encoding the human
glutamate transporter GLT1 served as a template for site-directed
mutagenesis performed by the unique site elimination procedure using
the Transformer site-directed mutagenesis kit
(CLONTECH, Palo Alto, CA). The procedure used two
mutagenic primers: one primer (Mt-XhoI,
5'-CGGGCCCCCCCTGGAGGTCGACGGTA) served as a selection primer in the
mutagenesis reactions to eliminate the unique XhoI site in
the Bluescript II vector; a second mutated oligonucleotide (N206S,
5'-GACGAGGAGGCCAGCGCAACCAGCGCTGTTG) was designed
to replace the codon for Asn206 by Ser. The wild-type and
mutated human GLT1 cDNAs were subcloned either into the pTNLII
vector, for expression in Xenopus laevis oocytes, or into
pcDNA3 (Invitrogen, Carlsbad, CA), for expression in COS7 cells.
The presence of the mutated nucleotide sequence in the human GLT1
cDNA was verified by the dideoxy termination method, using the
Sequenase kit (version 2.0; US Biochemicals, Cleveland, OH).
cRNA Synthesis, Oocyte Injection, COS7 Transient Transfection,
and Electrophysiological Studies--
For oocyte injection, the
linearized pTNLII constructs were transcribed in vitro using
the mMESSAGE mMACHINE transcription kit (Ambion, Austin, TX).
Xenopus oocytes were enzymatically defolliculated with
collagenase (2 mg/ml, 2 h at 18 °C), and stage V-VI oocytes were injected with capped RNA (25 ng/50 nl or as indicated). Oocytes were used 2-4 days after injection. Two-electrode voltage clamp was
used to measure the glutamate transporter-mediated currents at room
temperature using the Clampator-1B device (Dagan, Minneapolis, MN)
interfaced to a personal computer by a Digidata 1200 analog-to-digital controller. Data were acquired with the pCLAMP 8.0 software (Axon Instruments, Foster City, CA). Microelectrodes were filled with 3 M KCl and had a tip resistance of 0.5-2 M Uptake Measurement--
Uptake was measured 2-4 days after
injection of cRNA in oocytes or 36-48 h after transfection in COS7
cells cultured in Petri dishes (60 mm diameter) or six-well plates.
ND96 buffer, containing 20 µM D-aspartate
([3H]D-aspartate, 10.5 Ci/mmol; isotopic
dilution, 1:20,000), was used to measure the uptake in oocytes for 30 min. Transporter-mediated efflux of radiolabeled substrate was
determined in oocytes previously injected with 23 nl of
D-aspartate solution
([3H]D-aspartate and 50 mM
D-aspartate in 50 mM potassium phosphate buffer, pH 7.4; isotopic dilution, 1:50) and by exposing them for
different times to the ND80 solution. After injection of the radioactive solution, oocytes were kept in ND96 containing 50 µM DHK for 1 h. The efflux was started after rapid
washing of oocytes in ND80 buffer. Incubation was carried out in 500 µl of ND80 at room temperature. Oocytes not expressing the
transporters were used to assess the nonspecific leak of
[3H]D-aspartate. This leak, which on average
represents ~60% of the total radioactivity released in the buffer at
a given time, was subtracted out from the efflux data. For assessment
of the oocyte radioactive content, oocytes were separately dissolved in
10% SDS, mixed with scintillation fluid, and counted for
radioactivity. The efflux rate constants were obtained by fitting the
data to the equation ln(1 Western Blot Analysis--
Oocytes were solubilized in ND96 and
1% Triton X-100 in presence of a mixture of protease inhibitors
(Complete; Roche Molecular Biochemicals), whereas transfected COS7
cells were lyzed with phosphate-buffered saline (PBS), 1% SDS, and
protease inhibitors. Proteins were resolved on SDS-polyacrylamide gel
electrophoresis (10%) under reducing condition, blotted onto a
polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), and
probed with a polyclonal antibody (Rb68518) raised against peptide
12-26 of the sequence of rat GLT1 (36). Protein bands were visualized by chemiluminescence using a horseradish peroxidase-conjugated secondary antibody.
Assessment of Cell Surface Expression by Biotinylation--
The
biotinylation reaction was performed following the protocol reported by
Daniels and Amara (41). Briefly, COS7 cells were grown near confluence
in six-well plates and transfected, and 36 h after transfection
they were incubated with biotinylation buffer containing 1 mg/ml
NHS-SS-biotin (Pierce). The cell extracts were assessed for
protein content and eventually adjusted to the same value. An aliquot
of the cell extracts was run on a gel to confirm equal protein
concentration. Cell extracts were then incubated with UltraLink
immobilized NeutrAvidin (Pierce) for immunoprecipitation of
biotinylated proteins. The immunoprecipitates were separated on
SDS-polyacrylamide gel electrophoresis and probed with anti-GLT1 antibody, and the biotinylated transporters were visualized by chemiluminescence.
Immunofluorescent Analysis--
COS7 cells were plated on 35-mm
Petri dishes and transfected with 3 µg of plasmid/dish containing
wild-type human GLT1, mutant N206S, or plasmid alone. Cells were fixed
in 4% paraformaldehyde for 15 min, rinsed three times, and incubated
1 h with blocking solution (PBS containing 5% normal goat serum,
10% bovine serum albumin, and 0.2% Triton X-100). The anti-GLT1
antibody was incubated overnight (1:500 dilution) in PBS, 1% bovine
serum albumin, and 0.1% Triton X-100 at 4 °C. The secondary
antibody (Cy3-conjugated goat anti-rabbit IgG; Molecular Probes,
Eugene, OR) was incubated for 1 h (1:1,000 dilution) in PBS, 1%
bovine serum albumin, and 0.1% Triton X-100. Cells were washed twice
with PBS and examined with a confocal microscope (Bio-Rad). Images
obtained with Bio-Rad software were then formatted in Adobe Photoshop.
Oocytes were embedded in Tissue-Tek and frozen, and 8-µm sections
were cut on a cryostat and mounted onto Superfrost Plus microscope
slides (Fisher). Sections were dried for 30 min at room temperature and then fixed in PBS and 4% paraformaldehyde for 10 min. After rinses in
PBS, sections were blocked in 2% normal donkey serum and 0.1% Triton
X-100 in PBS for 1 h and incubated overnight at 4 °C in the
same solution to which the anti-GLT1 antiserum (1:500) was added. Next,
the sections were rinsed, incubated for 1 h in Cy3-conjugated anti-rabbit serum (0.5%; Jackson ImmunoResearch) in PBS containing 1%
normal donkey serum, rinsed again, mounted in Vectashield (Vector Laboratories), and analyzed for immunofluorescence. Stained oocytes were examined with a Leica DM microscope connected to a video camera
and video-grabbing software.
Statistical Analysis--
Data are expressed as mean ± S.E. of at least three independent experiments.
N206S-human GLT1 Is a Less-glycosylated Transporter--
The amino
acid asparagine at position 206 resides in the third large
extracellular loop of the GLT1 carrier and is predicted to be
glycosylated in vivo (12). The N206S mutation of GLT1 found
in sporadic ALS was expected to suppress N-glycosylation (37). To verify the impact of this mutation on the glycosylation of the
transporter, we transiently transfected COS7 cells with cDNA
encoding either wild-type or N206S-human GLT1. Western blot analysis on
homogenates prepared from transfected cells showed that the apparent
molecular weight of N206S is reduced compared with that of wild type
(Fig. 1, inset), suggesting a
decrease in the extent of glycosylation. Our antibody, which was raised against the N-terminal region of the rat homologous to human GLT1 (amino acids 12-26; Ref. 36) recognized a band of ~70 kDa for wild
type and 60-65 kDa for N206S. The human GLT1 transporter has an
additional putative glycosylation site at position 216 (Fig.
2A). If treated with the
N-glycosidase enzyme PNGase F (Fig. 2B),
wild type and N206S originated a band of the same apparent molecular
weight and with lower electrophoretic mobility, suggesting that the
mutant GLT1 is indeed a partially glycosylated transporter and that the
wild-type GLT1 has both the N206 and N216 sites glycosylated in
vivo. A similar shift in electrophoretic mobility was observed by
Danbolt and collaborators (40) when the purified rat GLT1 protein was
treated with N-glycosydase.
Transport Properties of the Glycosylation Mutant--
The impact
of N206S on the transport activity was first assessed by measuring
[3H]D-aspartate uptake in transiently
transfected COS7 cells. The expression of the wild type resulted in a
300-fold increase in uptake over mock-transfected cells. Expression of
N206S resulted in an ~50% reduction in uptake compared with the wild
type (Fig. 1). Treatment of COS7 expressing the transporters with
tunicamycin, a drug that interferes with the glycosylation pathways of
proteins, caused a reduction in glutamate uptake (Fig. 2C)
for both wild type and N206S. The reduction in activity was paralleled
by a defect in glycosylation (Fig. 2D).
We also expressed mutant and wild-type human GLT1 in Xenopus
oocytes. As seen in COS7 cells, N206S expressed in oocytes exhibited a
lower apparent molecular weight compared with the wild type (Fig.
3A), and the uptake of
glutamate was ~40% less than in wild type (Fig. 3B). Wild
type and N206S showed the same apparent affinity for
L-glutamate when determined by measuring glutamate-evoked uptake currents for a wide range of membrane potentials ( N206S Results in Impaired Plasma Membrane
Expression--
Reduction in maximal transport activity may be
attributable to reductions in the catalytic activity, biosynthesis, or
plasma membrane expression of the transporter. Indirect
immunofluorescence, in combination with confocal laser-scanning
microscopy of transfected COS7 cells, showed diffuse membrane labeling
for wild-type human GLT1 (Fig.
4A), a significant decrease in
plasma membrane and an increase in cytoplasmic staining for N206S (Fig.
4A). No staining was observed for mock-transfected cells
(data not shown). Immunofluorescence analysis was also performed in
Xenopus oocytes. Injection of cRNA for wild type caused
robust expression of the transporter in the plasma membrane with
virtually no intracellular staining (Fig. 4A). Conversely,
the N206S mutant resulted in a marked increase in the cytoplasmic
punctate immunoreactivity, in parallel with a decrease in the plasma
membrane expression. Water-injected oocytes exhibited negative labeling
for both plasma membrane and cytoplasmic compartments (data not
shown).
In addition, diminished plasma membrane expression of N206S was
confirmed by selective labeling of the transporters at the cell surface
by a biotinylation reaction. Compared with wild type, the decrease of
the biotinylated N206S mutant at the plasma membrane of transfected
COS7 cells is evident for both the oligomeric and monomeric forms of
the transporter (Fig. 4B).
These findings strongly suggest that N206S is either targeted
inefficiently to the cell surface or removed from the plasma membrane
at a higher rate than wild type.
Glutamate Efflux in the N206S Mutant--
Glutamate transporters
are capable of running in both forward and reverse modes, and
the overall direction is determined by the transmembrane
electrochemical gradients of the transporter substrates and coupling
ions. In GLT1-expressing oocytes, reverse transport of glutamate can be
triggered by depolarized membrane potentials (Vm
This behavior of N206S was confirmed by radiotracer measurements. We
injected an equal amount of radiolabeled D-aspartate into
oocytes expressing wild type and N206S to normalize the intracellular content of radioactivity. Incubation of the oocytes for 15 or 30 min in
efflux buffer (ND80) caused a release of radioactivity that was
statistically greater for N206S than for wild type (Fig. 5D), despite lower expression of N206S in the plasma
membrane. The rate constant of the efflux process was 0.52 × 10
Another approach we followed to evaluate the efflux capacity of the
N206S implied manipulation of the intracellular concentrations of
Na+ and glutamate. A similar approach was used for glycine
transporters 1b and 2a expressed in oocytes (43). Oocytes expressing
wild-type or N206S-human GLT1 were injected with a solution containing
MSG (23 nl, 1 M), thereby increasing the intracellular
concentrations of Na+ and glutamate to ~40 mM
(assuming an internal oocyte volume of 0.5 µl). Fig.
6A shows a representative
recording from an oocyte expressing wild-type human GLT1. Intracellular
injection of MSG evoked an outward current that was partly blocked by a
saturating concentration (1 mM) of DHK (Fig. 6A,
#2). This concentration of DHK fully inhibited the forward
uptake current evoked by application of 100 µM glutamate
in both wild-type and N206S-human GLT1 (Fig. 3) without inducing a
current per se (recording not shown). The expected
alteration of the ionic gradients for Na+ and glutamate was
confirmed by the decreased extent of the forward uptake current evoked
by application of extracellular glutamate in normal sodium solution
(ND96; Fig. 6A, #1). We therefore attribute the
DHK-sensitive component of the outward current to the current evoked by
the reverse operation of the transporter. The remaining outward current
was 29.8 ± 8 nA for wild type (n = 8) and
24.9 ± 8 nA (n = 6) for N206S and was
statistically not different from the one measured in control oocytes
(data not shown). It was, therefore, not further investigated.
The DHK-sensitive outward current was compared between wild type- and
N206S-expressing oocytes. ND80 solution (20 mM
K+ and 80 mM Na+; see
"Experimental Procedures") was perfused after intracellular injection of MSG (Fig. 6B). For appropriate comparison, the
expression level of wild-type human GLT1 was scaled down to match the
forward glutamate uptake current of N206S. Under these conditions, the outward current blocked by DHK (reverse transport current) was much
higher (>2-fold) for the N206S transporter compared with wild type
(Fig. 6B). Moreover, the reverse transport current was 46.7 ± 3.2% (n = 8) of the forward uptake
current in wild type and 285 ± 20% (n = 6) of
the forward uptake current in N206S (Fig. 6C).
N206S causes selective down-regulation of wild-type human GLT1
activity We have analyzed the functional characteristics and potential
pathophysiological impact of a naturally occurring mutation of the
human glutamate transporter GLT1. The mutation involves the
substitution of one of the two putative glycosylation sites, asparagine
206 to serine, and was identified in a patient with sporadic ALS (37).
On the basis of Western blot analysis of homogenates prepared from
transiently transfected COS7 cells and Xenopus oocytes, our
data provide evidence for a defect in glycosylation in the GLT1 mutant
N206S and document a 5-10-kDa reduction in apparent molecular
mass. Furthermore, our analysis of the impact of the N206S
mutation on the functional properties of the GLT1 carrier indicates
that this mutation does not affect the affinities for glutamate,
Na+, and the nontransported inhibitor DHK (Fig. 3). These
findings are consistent with previous observations reported for GLAST, the other known glial glutamate transporter isoform (44), indicating that the N-linked glycosylation is not involved in substrate
recognition and Na+ coupling. However, we found substantial
functional differences between wild-type and mutant transporter in
terms of maximal transport capacity and the propensity to reverse the
mode of operation. Oocytes and COS7 cells expressing N206S exhibit an
~50% reduction in Vmax. This reduced
transport rate parallels an increase in immunoreactivity for GLT1 in
the cytoplasmic compartment and a concurrent decrease in
immunoreactivity on the plasma membrane of the cells.
A defect in plasma membrane targeting was also reported for other
glycosylation-deficient transporters (45, 46). The cytoplasmic retention of N206S may be caused by a slower rate of insertion in the
plasma membrane from the Golgi apparatus. Alternatively, there may be
increased instability of the newly synthesized protein, caused by
aberrant folding of the glycosylation-deficient mutant, resulting in
its accelerated retrieval from the plasma membrane and degradation.
These alterations in membrane targeting or turnover of GLT1, and the
resulting reduction in glutamate clearing capacity from the synaptic
cleft, may significantly contribute to the increased susceptibility of
neurons, in particular motor neurons, to excitotoxic insults. Evidence
supporting a crucial role of GLT1 in preventing glutamate
excitotoxicity comes from studies of GLT1-knockout mice, which are
considerably more vulnerable to excitotoxic insults than control mice
and exhibit exacerbation of brain injury (47).
The sporadic ALS patient with the N206S mutation was heterozygous for
this mutation (37). Therefore, we became interested in exploring how
the simultaneous presence of wild-type and mutant GLT1 affects cellular
glutamate uptake capacity. We show that N206S has a substantial
dominant negative impact on wild-type activity. This may significantly
impair the capacity of GLT1 to mop up glutamate at synapses. However,
how the mutated transporter prevents the wild-type from being fully
active is not yet known. It is reasonable to speculate that the
efficient expression of the transporter in the plasma membrane requires
its assembly as a fully glycosylated oligomer in the endoplasmic
reticulum. Evidence in this direction came from Eskandari and
colleagues (48), who showed that one member of the glutamate
transporter family, the neuronal glutamate transporter EAAC1, assembles
in the plasma membrane as a pentamer made of identical subunits.
Therefore, it is possible that oligomers formed by incompletely
glycosylated monomers may be recognized as aberrant and may not be
targeted efficiently to the plasma membrane or, if correctly targeted, may be actively removed from the membrane and degraded. Data from the
literature suggest that, in vivo, GLT1 monomers may
self-associate to form homo-oligomers (49). Dominant-negative
interactions between transporter monomers produced by aberrant splicing
of the RNA have already been reported to account for a loss of
glutamate uptake mediated by GLT1 in sporadic ALS (29). The finding
that N206S does not exert this dominant negative effect on another glutamate transporter isoform, such as the neuronal subtype EAAC1, supports the concept that, whereas the different glutamate transporters are made up by homo-oligomers, subunits of two different transporter subtypes do not assemble together.
Glutamate transporters are capable of working in both forward and
reverse directions, depending on the intracellular and extracellular ionic concentrations. The normal electrochemical gradients of Na+, H+, and K+ ensure glutamate
uptake against a large concentration gradient (forward transport). In
pathological situations such as during brain ischemia or even during
intense and repetitive neuronal firing, there is depolarization of the
cell membranes caused by elevation of extracellular K+
concentration in the synaptic cleft and rundown of electrochemical gradients. This induces the glutamate transporters to reverse their
direction, allowing glutamate to exit the cells (reverse transport;
Refs. 42, 50, 51). An interesting feature of N206S is its marked
propensity to reverse transport operation relative to the forward
uptake (Figs. 5 and 6). This capability is less pronounced for the wild
type, both in terms of DHK-sensitive outward current and efflux of
radiolabeled transporter substrate. How can a mutation in one of the
glycosylation sites facilitate the reverse operation of the
transporter? Although we do not have an explanation for this yet, we
anticipate that the N206S mutation affects a specific step in the
glutamate transport cycle, which leads to an increase in reverse
transport. Unfortunately, the resolution of radiotracer-based flux
techniques in conjunction with the relatively low levels of expression
of N206S and the relatively slow solution exchange protocol typically
used in the two-electrode voltage clamp approach limit the
possibilities for dissecting these features of the transporter.
It is well established that both sporadic and familial ALS are
associated with impairment of the glutamate transport system mediated
by the glial glutamate transporter GLT1 (27-29, 36, 52, 53). The
combined evidence that a missense mutation of human GLT1 is present in
a patient with sporadic ALS, and that such a mutation affects both the
capacity of a cell to clear glutamate and the ability to release
glutamate, supports the concept that this mutation contributes to the
excitotoxicity that occurs in ALS. In addition, its analysis has
provided new insights into the role of N-glycosylation on
glutamate transporter function and trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Voltage step
commands were applied in 10-mV increments to generate current-voltage
curves; the current output was low pass filtered at 1 kHz; and the
signal was averaged three times before and after solution exchange.
Recordings were made from oocytes bathed with a solution that contained
96 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 2 mM KCl, and 5 mM HEPES-Na, pH 7.4 (ND96). For reverse transport
experiments, the solution contained 80 mM NaCl, 20 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 4 mM BaCl2,
and 5 mM HEPES-Na, pH 7.4 (ND80). Transporter-mediated efflux was determined by including the GLT1-nontransported inhibitor dihydrokainate (DHK, 1 mM) in the efflux buffer, and the
carrier-mediated efflux current was estimated by subtracting the
current evoked in ND80 to that in the presence of ND80 and DHK. For
experiments in Fig. 6, oocytes were injected with a solution containing
monosodium glutamate (MSG, 1 M, 23 nl). For expression
studies, COS7 cells were transiently transfected with the pcDNA3
constructs using the GenePorter transfection kit (Gene Therapy Systems,
San Diego, CA) following the manufacturer's recommendations. The
vector alone was used for mock transfection.
Nt/N0) =
kt, where t is the time, k
is the first-order rate constant, Nt is the radiolabeled substrate released in the medium, and
N0 is the radiolabeled substrate content of the
oocyte at the start of the experiment. Uptake in transfected COS7 cells
was measured for 30 min in the presence of 40 µM
D-aspartate (isotopic dilution, 1:2,500) and in buffer
containing 140 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 1.2 mM CaCl2,
1.2 mM K2HPO4, 10 mM
glucose, and 10 mM HEPES-Na, pH 7.4. Uptake was stopped by
washing the cells three times with ice-cold choline buffer (no sodium).
The uptake values were corrected by the protein content in each Petri
dish. For the experiments with tunicamycin, this compound was added
24 h before measuring the uptake, at a concentration of 5 µg/ml
in 0.1% Me2SO. This concentration and application
time of tunicamycin are compromises between avoiding cellular death and
detachment from the dish and observing a deglycosylation pattern for GLT1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of N206S and wild-type human
GLT1 in transiently transfected COS7 cells.
[3H]D-Aspartate uptake was measured for 30 min in COS7 cells transiently transfected with equal amounts of
cDNA (3 µg/dish). Cells were collected by scraping the dish with
500 µl of 0.1 N NaOH plus 1% SDS. An aliquot was used
for protein determination. The radioactivity content was measured by
liquid scintillation spectroscopy. Data are normalized for the protein
content of each dish and are the mean ± S.E.M. (n = 4) of a representative experiment. Inset, Western blot
performed on lysates prepared from transfected COS7 cells.
hGLT, human GLT; WT, wild type.
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Fig. 2.
Extent of N-linked
glycosylation of human GLT1. A, schematic diagram of
human GLT1 shows the two putative glycosylation sites in the large
extracellular loop between the third and fourth transmembrane domains.
The GLT1 moiety from the sixth transmembrane domain to its end remains
to be fully understood, and it is left undetermined. A novel topology
for GLT1 was recently proposed (24). However, the segment of GLT1
comprising the glycosylation sites is represented as in our diagram.
B, transfected COS7 cells were homogenized and treated with
PNGase F (3 µl; specific activity, 1,800,000 U/mg; New England
Biolabs) for 3 h at 37 °C and resolved on 10%
SDS-polyacrylamide gel electrophoresis. C, COS7 were grown
to near confluence in six-well plates, transfected, and treated with
tunicamycin (5 µg/ml, 0.1% Me2SO, 24 h) before
measuring [3H]D-aspartate uptake (30 min,
room temperature). Cells were collected by scraping the dish with 1 ml
of PBS plus 1% SDS. The lysate was used to determine the protein
content, count of radioactivity, and gel electrophoresis. It should be
noted that tunicamycin caused cell detachment from the wells. The
groups treated with tunicamycin exhibited ~60% less protein content.
Uptake values were normalized by protein content and expressed as
percentage of wild-type GLT1 treated with 0.1% Me2SO.
D, Western blot analysis of transfected COS7 treated with
tunicamycin. The arrow indicates the fully deglycosylated
transporter. hGLT, human GLT; WT, wild
type.
50 mV shown
in Fig. 3C). However, there was a significant reduction in
maximal transport velocity (Fig. 3C). At an external
Na+ concentration of 96 mM, the apparent
affinity for glutamate was 17 ± 4 µM for wild type
and 18 ± 2 µM for N206S. When fitting the
Na+ dependence of the inward currents
(Vm =
50 mV; [Glu] = 100 µM) to
the Hill equation, the apparent affinity for Na+ was
31 ± 4 mM (n = 3;
nHill = 1.6 ± 0.2) for the wild type and 28 ± 5 mM (n = 3;
nHill = 1.5 ± 0.2) for N206S (Fig.
3D). Thus, the strict Na+ dependence of human
GLT1 is not affected by the N206S mutation. To study the possible
effects of N206S on the voltage dependence of the forward uptake
current, we subtracted the currents recorded in the absence from those
measured in the presence of a saturating concentration (200 µM) of L-glutamate and in response to a
series of command voltage pulses. The wild-type- and mutant-mediated glutamate uptake currents exhibited no noticeable difference in the
voltage dependence (Fig. 3E), and the plots could be well superimposed when normalized for the maximal current (data not shown).
The lack of an outward current at depolarized membrane potentials
(
20 mV), despite the presence of Cl
in the external
solution, is consistent with a negligible contribution of a
thermodynamically uncoupled anion conductance to the current-voltage curves generated by both the wild type and N206S mutant. The N206S mutation seemed to have no impact on the affinity for the competitive nontransported inhibitor DHK (38). DHK blocked the glutamate-evoked transport current without inducing a current per se (5 µM to 1 mM DHK; data not shown), and Hanes
analysis revealed no difference in the Ki for DHK in
wild type and N206S (Fig. 3F). Overall, these results
suggest that the N206S mutation in human GLT1 affects maximal forward
uptake capacity but not the apparent affinity of the carrier for
glutamate, Na+, or transport inhibitor.
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Fig. 3.
Properties of N206S expressed in
oocytes. A, wild-type and N206S human GLT1 cRNAs were
injected into Xenopus oocytes. Three days after injection,
oocytes were homogenized in ND96 containing Triton X-100 (1%),
resolved on SDS-polyacrylamide gel electrophoresis, blotted, and probed
with an anti-rat GLT1 antibody (4 µg/ml). B, glutamate
uptake was measured for 30 min in oocytes expressing the glutamate
transporters. Data are normalized to the uptake value of oocytes
expressing wild-type human GLT1 and are the average of at least four
experiments (n = 10 for each group). C,
Michaelis-Menten kinetics for glutamate-evoked uptake current recorded
at 60 mV (n = 4). Data are normalized to the uptake
current evoked by 1 mM glutamate. D, sodium
dependence of Igly. A representative concentration-response
curve for extracellular sodium is shown. The uptake current was
recorded at
50 mV and evoked by application of 100 µM
glutamate. Equimolar choline+ was used to compensate for
the lack of Na+ ions. E, representative
current-voltage relationship of oocytes expressing wild type and N206S
in response to 100 µM glutamate. F, Hanes
analysis for DHK inhibition. The Ki for wild type
and N206S mutant intersected at
19.2 ± 5.5 and
24.1 ± 5.1 µM, respectively (
Ki;
n = 4). Forward uptake current was evoked by
application of 100 µM L-glutamate. S.E.s are
within the size of the symbols. hGLT, human GLT;
WT, wild type.
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Fig. 4.
Reduced surface expression of N206S.
A, representative images from two independent experiments.
Immunocytochemistry was performed on transfected COS7 cells and
cRNA-injected oocytes. The expression of the mutant N206S reduces GLT1
immunoreactivity in the plasma membrane and increases it in the
cytoplasm in comparison with wild-type expression. Serial sections of
0.5 µm were obtained from COS7 cells by confocal microscopy at ×40
magnification. B, biotinylated wild-type and N206S-human
GLT1 expressed in COS7 cells. A representative experiment done in
triplicate is shown. Each lane represents a well containing
the same amount of proteins as assessed by protein measurement and
Western analysis with an anti-GLT1 antibody (not shown).
hGLT, human GLT; WT, wild type.
20 mV) and measured as DHK-sensitive currents in the presence of high
extracellular potassium and low extracellular sodium and the absence of
external glutamate. Xenopus oocytes have a free pool of
glutamate in the cytoplasm (~10 mM), making this amino
acid available as an intracellular substrate of the transporter (42).
Under the above-mentioned conditions, wild-type human GLT1 generated an
outwardly directed current, which could be blocked by extracellular
application of DHK (Fig. 5, A
and B, insets;
20 mV; Ref. 36). This
DHK-sensitive reverse transport current at +60 mV was 34 ± 4%
(n = 12) of the forward uptake current recorded at
60
mV in Na+ buffer (ND96). In contrast, for N206S, the
DHK-sensitive reverse transport current was 69 ± 8% of the
forward uptake current (Fig. 5, compare A and B).
Moreover, in N206S, the reverse transport current at 0 mV represented
~80% of the total transporter-mediated currents (forward and reverse
transport currents), whereas in wild-type hGLT1, the reverse transport
current was ~ 35% of the total transport currents (Fig.
5C). This suggests a marked propensity of the N206S mutant
to extrude glutamate out of the cell. The same ratio was observed at
20 mV, whereas at +20 mV the outward current tended to be the
predominant current both for wild type and N206S.
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Fig. 5.
Reverse transport mediated by wild type and
N206S. A and B, forward uptake current was
evoked by bath application of 25 µM glutamate in ND96
(insets, black-filled bars; 20 mV). Reverse transport
current was evoked in the absence of extracellular glutamate by bath
application of ND80 (open bars), and the extent was
determined by application of DHK (1 mM; insets,
gray-filled bars;
20 mV). A series of voltage pulses of 100 msec
duration from a holding potential of
50 mV was applied to generate
current-voltage curves of forward (filled symbols) and
reverse (open symbols) transport current. For forward uptake
current determination, recordings in the presence of glutamate (25 µM) were subtracted off-line from recordings in ND96 in
the absence of glutamate. For reverse uptake currents, recordings in
the presence of DHK (1 mM) were subtracted from currents in
ND80. C, percentage of glutamate transporter-mediated efflux
current compared with the total transporter mediated currents (forward
plus reverse) at the indicated membrane potentials. Data are mean ± S.E.M. from 12 cells in each group. *, p < 0.05;
**, p < 0.01. D, oocytes expressing
N206S (open symbols) and wild-type human GLT1 (filled
symbols) were injected with 23 nl of radiolabeled plus cold
D-aspartate. Radioactivity injected into each oocyte was
~10,000 cpm. A nonspecific leak of D-aspartate was
measured in oocytes that did not express the transporter. Data are from
a representative experiment (n = 10 in each group) and
represent the value obtained by subtracting the nonspecific leak of
D-aspartate and the DHK-insensitive component (measured in
the presence of 1 mM DHK). The nonspecific leak
represents ~ 60% of the total release of
D-aspartate, and the DHK-insensitive component of the
remaining release was not statistically different in wild type (42 ± 2.3%) and N206S (39 ± 4%). hGLT, human GLT.
3/min for N206S and 0.34 × 10
3/min for wild-type human GLT1. On average,
the ratio of the N206S and wild-type efflux rate constants from three
independent experiments was 1.81 ± 0.26. The difference in the
efflux rate constant was even more pronounced if the expression of the
wild type was adjusted down to the approximate level of expression of
N206S, assessed as the extent of glutamate-evoked uptake current. At 30 min, the efflux rate constant of D-aspartate for N206S
was 2.24-fold that of wild type (N206S-cRNA, 25 ng/50 nl,
versus wild-type cRNA, 10 ng/50 nl; data not shown).
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Fig. 6.
Cytoplasmic injection of MSG evoked reverse
transport currents in wild-type and N206S-human GLT1 oocytes.
A and B, recordings from representative oocytes
show activation of outward currents after intracellular injection of
MSG, as indicated by the arrows (23 nl, 1 M).
Oocytes were held at 20 mV, and 50 µM glutamate was
applied to evoke the forward uptake current (filled bars).
A, glutamate was applied in ND96 either before or after
cytoplasmic injection on the steady level of the outward current. A
decrease in the forward uptake current was noticed when glutamate was
applied on top of the outward current because of the reduction of ionic
gradients caused by the intracellular injection of MSG. The outward
current was partly abolished by application of DHK (1 mM),
and this component of the current was regarded as specific for human
GLT1. B, the DHK-sensitive component of the outward current
was compared in oocytes expressing similar levels of functional
glutamate transporters. Oocytes were injected with 10 ng/50 nl of cRNA
for wild type and 25 ng/50 nl of cRNA for N206S and clamped at
20 mV.
C, Averaged forward and reverse transport currents recorded
from wild-type (n = 8) and N206S-human GLT1
(n = 6) at
20 mV as shown in B. hGLT, human GLT; WT, wild type.
Our analysis on mutations in the human GLT1 gene in patients with ALS indicated that N206S is present in one allele of
chromosome 11 (37). Because both normal and mutated genes coexist in vivo, we investigated whether N206S had any influence on
the activity of wild-type human GLT1. A subsaturating amount of cRNAs (10 ng each) encoding wild-type or N206S-human GLT1 was injected in
combination or alone into oocytes, and the resulting glutamate uptake-evoked current was measured at different membrane potentials. The neuronal glutamate transporter human EAAC1 was coinjected together
with N206S or wild-type as a control to evaluate the selectivity of the
effect of the N206S mutant. Although wild-type human GLT1 manifested
greater activity then N206S, when coexpressed with N206S, the latter
dominantly down-regulated the wild-type to the level of activity
determined by the mutant alone (Fig. 7).
In contrast, coexpression of N206S with wild-type EAAC1 had no
statistically significant effect on the uptake current mediated by the
neuronal subtype.
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Fig. 7.
The N206S mutant exerts a dominant negative
effect on wild-type human GLT1 activity. 10 ng of cRNA was
injected for each transporter. Current-voltage curves were obtained by
bath application of 100 µM glutamate. Data are normalized
either to the uptake-evoked current generated by wild-type human GLT1
(WT; A) or by human EAAC1 (B) at 100
mV and are averaged from three different experiments (n = 12).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. S. G. Amara and J. L. Arriza for providing the EAAT2 cDNA.
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FOOTNOTES |
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* 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.
§ Supported by the Amyotrophic Lateral Sclerosis Association. To whom correspondence should be addressed: Brigham and Women's Hospital, Harvard Medical School, Harvard Institutes of Medicine Bldg., Rm. 570, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5827; Fax: 617-525-5830; E-mail: dtrotti@rics.bwh.harvard.edu.
Supported by the Norwegian Research Council.
§§ Supported by the Amyotrophic Lateral Sclerosis Association, the Muscular Dystrophy Association, the Pierre L.de Bourgknecht Amyotrophic Lateral Sclerosis Research Foundation, National Institutes of Health NINDS Grants 1PO1NS31248-05 and 5F32HS10064, and National Institutes of Health NIA Grant 1PO1Ag12992-04.
¶¶ Supported by the Amyotrophic Lateral Sclerosis Association and the Muscular Dystrophy Association. To whom correspondence should be addressed: Brigham and Women's Hospital, Harvard Medical School, Harvard Institutes of Medicine Bldg., Rm. 570, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5827; Fax: 617-525-5830; E-mail: mhediger@rics.bwh.harvard.edu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M003779200
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ABBREVIATIONS |
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The abbreviations used are: GLT, glutamate transporter; ALS, amyotrophic lateral sclerosis; DHK, dihydrokainate; MSG, monosodium glutamate; PBS, phosphate-buffered saline. GLAST, glutamate-aspartate transporter; EAAC, excitatory aminoacid carrier; EAAT, excitatory aminoacid transporter.
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