Biochem. J. (2011) 439, 333–340 (Printed in Great Britain)
333
doi:10.1042/BJ20110905
Water and urea permeation pathways of the human excitatory amino acid
transporter EAAT1
Robert J. VANDENBERG*1 , Cheryl A. HANDFORD*, Ewan M. CAMPBELL†, Renae M. RYAN* and Andrea J. YOOL†
*Transporter Biology Group, Discipline of Pharmacology, Bosch Institute, School of Medical Sciences, University of Sydney, Sydney, NSW 2006, Australia, and †Discipline of
Physiology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005, Australia
Glutamate transport is coupled to the co-transport of 3 Na + and
1 H + followed by the counter-transport of 1 K + . In addition,
glutamate and Na + binding to glutamate transporters generates
an uncoupled anion conductance. The human glial glutamate
transporter EAAT1 (excitatory amino acid transporter 1) also
allows significant passive and active water transport, which
suggests that water permeation through glutamate transporters
may play an important role in glial cell homoeostasis. Urea
also permeates EAAT1 and has been used to characterize the
permeation properties of the transporter. We have previously
identified a series of mutations that differentially affect either
the glutamate transport process or the substrate-activated channel
function of EAAT1. The water and urea permeation properties of
wild-type EAAT1 and two mutant transporters were measured to
identify which permeation pathway facilitates the movement of
these molecules. We demonstrate that there is a significant rate of
L-glutamate-stimulated passive and active water transport. Both
the passive and active L-glutamate-stimulated water transport is
most closely associated with the glutamate transport process.
In contrast, L-glutamate-stimulated [14 C]urea permeation is
associated with the anion channel of the transporter. However,
there is also likely to be a transporter-specific, but glutamate
independent, flux of water via the anion channel.
INTRODUCTION
for reviews of the roles of water transport by the EAATs in the
central nervous system).
Passive and active water transport by EAAT1 can occur in
the absence of L-glutamate, but are further stimulated in the
presence of L-glutamate. Urea is larger than water, but can be
readily transported by a subset of aquaporins and also by EAAT1,
and has been used to characterize solute transport processes
[23,27–29]. In the present study we have investigated whether
water and urea pass through the same or different pathways
of EAAT1 to gain insight into the molecular determinants of
water and urea permeation. We have used a combination of
pharmacological agents and two mutant transporters, one that has
limited glutamate transport activity and one that has no substrateactivated Cl − conductance, to investigate the water and urea
permeation properties. The mutation of a valine residue in hairpin
loop 2 of EAAT1 to a cysteine (V452C) results in a transporter
with very similar properties to WT (wild-type) EAAT1, but the
application of the positively charged MTS (methanethiosulfonate)
reagent MTSET [sodium (2-sulfonatoethyl) MTS] to V452C
results in a transporter that does not allow L-glutamate transport,
but does permit L-glutamate and Na + binding, which activates
the uncoupled Cl − conductance [30]. To avoid complications
from the addition of MTS reagents, in the present study we
have mutated the valine residue at position 452 to an arginine
residue (V452R) which is similar in size to a cysteine residue
after MTSET treatment. The V452R transporter properties are
indistinguishable from those of V452C after modification with
MTSET. Mutation of Asp112 to alanine (D112A) generates a
transporter that has normal glutamate transport activity, negligible
L-glutamate activation of the uncoupled Cl − conductance and
a considerably larger leak Cl − current [19]. By comparing
the passive and active water transport properties and also urea
Glutamate is the predominant excitatory neurotransmitter in the
mammalian central nervous system [1–3]. Synaptic glutamate
concentrations undergo considerable fluctuations, from approximately 25 nM at the resting state up to 1–2 mM following
synaptic glutamate release [4–6], and it is the role of highly
abundant EAATs (excitatory amino acid transporters) to regulate
these concentrations to maintain a dynamic signalling process
between neurons [7–13].
Glutamate transport is coupled to the co-transport of 3 Na + ions
and 1 H + and the counter-transport of 1 K + ion, which allows
the transporters to maintain a 106 -fold gradient across the membrane [14] and ensures that glutamate is efficiently cleared from
the synapse [15]. In addition to the coupled ion fluxes, glutamate
binding to the transporter activates an uncoupled Cl − current,
with the degree of uncoupled current varying between transporter
subtypes [12,16,17]. Furthermore, glutamate transporters allow
Cl − ion leakage in the presence of Na + but in the absence
of glutamate [18–21], and also in the absence of both Na +
and glutamate [22]. The powerful concentrating capacity of the
EAATs, as well as their intrinsic Cl − conductances, means that
the transport process is associated with large fluxes of osmolytes.
MacAulay and co-workers have demonstrated that the human
glutamate transporter EAAT1 also transports a considerable
amount of water [23,24], presumably to counterbalance the flow
of ions. The unitary rate of passive water transport by EAAT1 is
comparable with approximately 10 % of the rate of water transport
by one of the most efficient aquaporins AQP1 [25]. Thus the
additional water permeation function of transporters is likely to
play an important role in maintaining water homoeostasis of glia
and neurons involved in excitatory neurotransmission (see [25,26]
Key words: anion channel, glutamate transport, urea, water
transport.
Abbreviations used: EAAT, excitatory amino acid transporter; MTS, methanethiosulfonate; MTSET, sodium (2-sulfonatoethyl) MTS; TBOA,
benzyloxyaspartate; WT, wild-type.
1
To whom correspondence should be addressed (email [email protected]).
DL-threo-β-
c The Authors Journal compilation c 2011 Biochemical Society
334
R. J. Vandenberg and others
transport rates of WT and mutant transporters, we demonstrate
that the L-glutamate-stimulated water transport is most closely
associated with the transport process. In contrast, urea uptake is
closely associated with the L-glutamate-activated Cl − channel of
EAAT1.
EXPERIMENTAL
Recombinant DNA protocols
EAAT1 cDNA was subcloned into the plasmid oocyte
transcription vector pOTV [16]. Site-directed mutagenesis was
performed using the QuikChange® site-directed mutagenesis kit
(Stratagene), and all mutations were sequenced on both strands by
dye-terminator cycle sequencing (ABI Prism, PerkinElmer Life
Sciences). All EAAT mutants used in the present study have been
characterized previously [19,30]. The WT and mutant transporter
cDNAs were linearized with SpeI and cRNA was transcribed
with T7 RNA polymerase using the mMessage mMachine kit
(Ambion).
Water and solute transport assays
Stage V oocytes were harvested from Xenopus laevis as
described previously, and all surgical procedures were approved
by the University of Sydney and the University of Adelaide
Animal Ethics committees. (The procedures followed protocols
approved under the Australian Code of Practice for the Care
and Use of Animals for Scientific Purposes). Oocytes were
either uninjected (controls) or injected with 6 ng of cRNA
and were maintained at 16–18 ◦ C in ND96 saline [96 mM
NaCl, 2 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 and 5 mM
Hepes (pH 7.55)] supplemented with 100 units/ml penicillin and
100 μg/ml streptomycin, and 10 % (v/v) heat-inactivated horse
serum. All oocytes were transferred into standard isotonic saline
without serum overnight prior to the experimental treatments.
Osmotic swelling was assayed under both isotonic conditions
and in 50 % hypotonic saline containing 0, 100 or 400 μM
L-glutamate or 100 μM TBOA (DL-threo-β-benzyloxyaspartate;
Tocris) applied acutely in the swelling assay without preincubation.
Data collected by videoimaging were captured by ImageJ
software and analysed as described previously [26,27]. Frames
were captured at 20 s intervals for 10 min per assay for 50 %
hypotonic solutions, and at 1 min intervals for 30 min in isotonic
conditions. Data for oocyte volume (V) calculated from the
cross-sectional area were standardized to the initial volume
(V o ), plotted as a function of time, and fit over the linear
component of the swelling response (200 s for hypotonic; 30 min
for isotonic) by linear regression to determine the swelling rate
[%V/V o ·s − 1 (×103 )]. Statistical significance was assessed by twoway ANOVA followed by post-hoc unpaired Student’s t tests.
For [14 C]urea transport assays, 50 nl of cRNA (20 ng) was
injected into each oocyte and oocytes were incubated in ND96
saline solution (as described above) and incubated with shaking.
The transport of [14 C]urea was measured by placing oocytes in
a dish (five per dish) in ND96 or ND95 diluted 50 % in water
containing 10 μM [14 C]urea (PerkinElmer) with the addition of
100 μM L-glutamate, 100 μM D-aspartate or 100 μM TBOA at
room temperature (22 ◦ C). For uptake under isotonic conditions,
oocytes were incubated for 30 min and then washed three times in
ice-cold ND96, placed in scintillation vials containing 100 mM
NaOH to help dissolve the oocytes and then scintillant added
and radioactivity counted using a MicroBeta TriLux Counter
c The Authors Journal compilation c 2011 Biochemical Society
(PerkinElmer). For uptake under hypotonic conditions (50 %
ND96), the uptake was terminated after 10 min.
Current recordings were made using the two-electrode voltageclamp technique with a Geneclamp 500 amplifier (Axon
Instruments) interfaced with a PowerLab 2/20 chart recorder
(ADI Instruments) using the chart software and a Digidata 1322A
digitizer (Axon Instruments) controlled by an IBM-compatible
computer using the pClamp software (version 10, Molecular
Devices). The current–voltage relationships for substrate-elicited
conductances were determined as described previously [32].
Briefly, steady-state current measurements in the absence of
substrate, obtained during 150 ms voltage pulses from − 30
mV to potentials between − 100 and + 60 in 10 mV steps,
were subtracted from corresponding current measurements in the
presence of substrate. The current–voltage relationship for the
TBOA-blocked leak conductance was determined by subtraction
of the currents measured in the presence of TBOA from currents
measured in the absence of TBOA, using the same voltage
protocol as described above.
Concentration-dependent L-glutamate currents were recorded
by applying L-glutamate between 1 μM and 1 mM at − 100 mV,
and current measurements were fit to the equation:
I / I max = [L-glutamate]/(K m +[L-glutamate])
using least squares non-linear regression, where K m is the
concentration of L-glutamate that generates half-maximal current.
Rates of transport were also measured by applying 100 μM [3 H]Lglutamate to oocytes expressing the various transporters under
voltage clamp at − 100 mV for 1 min followed by washout
of the bath solution with standard buffer for 3 min. Oocytes
were removed from the bath, dissolved in 50 mM NaOH, and
scintillation counting was performed. Background conductances
for oocytes expressing EAAT1, D112A, V452R and uninjected
oocytes were measured over the range − 100 to + 60 mV.
RESULTS
The aim of the present study was to characterize water
and urea movement through WT EAAT1 and two mutant
transporters, and to identify whether this movement is associated
with glutamate transport or the chloride channel function
of EAAT1. Application of L-glutamate to oocytes expressing
EAAT1 generates a current with two components. In standard
frog Ringer’s solution, the transport process generates an
inward current at all membrane potentials up to + 60 mV
[17]. In addition, L-glutamate and Na + binding to the
transporter generates a current that reverses at the Cl − reversal
potential [12,17]. The total current reverses at + 30 mV
(Figure 1A). TBOA is a competitive blocker of L-glutamate
transport [33], but when applied alone to EAAT1, it binds to
the transporter and blocks a small leak current that reverses
direction at the Cl − reversal potential (Figure 1A). Previous
studies have demonstrated that this current is carried by Cl − ions,
and that the ion-permeation properties of the Cl − leak current
differ from those of the L-glutamate-activated Cl − current [19,20].
In uninjected oocytes there is also a background conductance
carried predominantly by Cl − ions (Table 1).
The electrophysiological properties and transport rates of
EAAT1 and the EAAT1 mutants D112A and V452R have been
characterized previously [19,30] and are summarized below
(Figure 1 and Table 1). The D112A and V452R mutants
have altered relative contributions from the L-glutamate
transport current, the L-glutamate-activated Cl − current and the
Water and urea permeation of glutamate transporters
Figure 1
335
Electrophysiological analysis of WT and mutant EAAT1
(A) Representative current traces measured for membrane potentials between − 100 mV and + 60 mV under conditions as indicated and as described in the Experimental section. Although data
were collected from − 100 mV to + 60 mV in 10 mV increments, we have presented data in 20 mV increments so as to provide greater clarity. (B) L-Glutamate-evoked currents (䊏) were measured
by subtraction of current values in ND96 from currents measured in the presence of 100 μM L-glutamate. TBOA-blocked currents (䉱) were measured by subtraction of the current in the presence
of 100 μM TBOA from the currents in ND96. Background currents in ND96 (䊊) were measured in the absence of substrates and blockers. All currents were normalized to the current measured for
100 μM L-glutamate at − 100 mV. These mutants have been characterized in detail previously [19,30]. Note differences in the reversal potential of the L-glutamate-evoked currents ( + 30 mV for
EAAT1, no reversal for D112A and − 20 mV for V452R), which reflects the different proportions of the currents that are due to the transport and uncoupled components. The L-glutamate-elicited
current amplitudes and the background + leak conductance for EAAT1, D112A, V452R and uninjected oocytes are presented in Table 1.
TBOA-blocked Cl − leak current. L-Glutamate transport by
D112A is similar to WT EAAT1, but L-glutamate does not
activate an uncoupled Cl − conductance in this mutant. In addition,
D112A supports a large TBOA-blocked Cl − leak current, which
is significantly greater than the background current in uninjected
oocytes or in either V452R or EAAT1 [19] (Figure 1B and
Table 1). Although the V452R mutant has compromised L-
glutamate transport, the TBOA-blocked Cl − leak current and the
Cl − current are comparable with those of
EAAT1 [30] (Figure 1 and Table 1). Although all three transporters
have comparable affinities for L-glutamate, and WT EAAT1 and
D112A have comparable rates of [3 H]L-glutamate transport at
− 100 mV, the rate of transport by V452R is substantially reduced
(Table 1).
L-glutamate-activated
c The Authors Journal compilation c 2011 Biochemical Society
336
Table 1
R. J. Vandenberg and others
Kinetic parameters for L-glutamate transport by WT and mutant EAAT1 transporters
Data for EAAT1 and D112A have been published previously [19]. Data for V452R were obtained using the same methods as for the other mutants and are described in the Experimental section.
L-Glutamate did not generate any currents at − 100 mV for uninjected oocytes. Note that the current generated by L-glutamate for D112A is predominately due to coupled transport, the current
generated by V452R is predominantly due to the uncoupled anion current, and for EAAT1 the current generated by L-glutamate is the sum of the coupled transport current and the uncoupled anion
current. Background currents were measured using the standard voltage protocol and the conductance was calculated by dividing the change in current amplitude by the change in membrane
potential. Values are means +
− S.E.M. (n = 5).
Transporter
K m (L-glutamate)
(μM)
[3 H]L-glutamate transport at − 100 mV
(fmol/min per oocyte)
Background conductance
(μS)
100 μM L-glutamate transport
current at − 100 mV (nA)
EAAT1
D112A
V452R
Uninjected
20.0 +
− 3.0
13.0 +
− 2.3
26.6 +
− 3.3
–
414 +
− 26
257 +
− 35
82 +
−5
43 +
−6
1.85 +
− 0.09
2.51 +
− 0.50
1.31 +
− 0.29
1.48 +
− 0.39
888 +
− 16
208 +
−3
409 +
− 34
–
We have compared the rates of swelling of oocytes expressing
WT EAAT1 and each of the mutant transporters in a hypotonic
saline (50 % saline) and a standard isotonic saline to identify
which of the solute conduction pathways are responsible for
water permeation, and also to compare passive and active water
transport. We have used the term passive transport for transport
that occurs where there is a large osmotic gradient (i.e. hypotonic
saline) driving the uptake process, and the term active transport
for transport that occurs in the absence of an osmotic gradient (i.e.
isotonic saline).
Passive water transport
Representative data from one batch of cells (Figure 2A) illustrates
the changes in oocyte surface area as a function of time after
introduction of the EAAT1-expressing or control oocytes into
hypotonic saline, with and without L-glutamate. Oocytes expressing WT EAAT1 showed enhanced rates of swelling in the
presence of L-glutamate that was maximal at 100 μM, without a
further increase at a higher dose (400 μM).
WT EAAT1, D112A and V452R transporters showed elevated
rates of swelling in the untreated condition compared with control
oocytes (Figure 2B), which suggests that the transporters allow
passive water transport above the background rate of oocytes.
Application of 100 μM TBOA (IC50 of 26 μM; [34]) to oocytes
expressing the various transporters reduced the rate of swelling
below that for the untreated oocytes, which was most apparent
for the V452R mutant. In the case of V452R, TBOA reduced the
rate of swelling to rates that were similar to control oocytes. This
suggests that the L-glutamate-independent passive water uptake is
partly due to permeation through the TBOA-blockable Cl − leak
pathway of the transporter.
L-Glutamate was used to stimulate and TBOA was used to
block the different EAAT-dependent permeation pathways [23],
and the water permeation properties were compared between
WT and mutant constructs. Control oocytes have a background
rate of water permeation and neither L-glutamate nor TBOA
significantly altered the rate of water permeation. L-Glutamate
(100 μM) in the hypotonic swelling medium significantly
enhanced the rates of net water influx in oocytes expressing
the WT EAAT1 and D112A, but not the V452R transporters
(Figure 2B). WT EAAT1 and D112A exhibited comparable rates
of [3 H]L-glutamate transport and similar K m values for transport
(Table 1), but L-glutamate did not activate any uncoupled Cl −
conductance in D112A [19] (Figure 1). Although V452R binds
L-glutamate with similar affinity to WT EAAT1 and D112A, Lglutamate transport is significantly reduced compared with the
other transporters. These results demonstrate that the L-glutamate
c The Authors Journal compilation c 2011 Biochemical Society
stimulated passive water transport closely correlates with the
transport process, and suggests that water permeation is activated
by the glutamate transport process.
Active water transport
Water transport rates were also measured under isotonic saline
conditions to estimate the contribution of active water transport by
EAAT1 and the mutant transporters. For all transporters the water
uptake associated with active transport was approximately 10-fold
less than the passive transport in hypotonic conditions. The
magnitudes of the swelling rates in isotonic buffer were very low,
almost negligible in untreated conditions, but were significantly
increased by L-glutamate in the WT and D112A, but not for
the V452R mutant. TBOA also appeared to cause a small (but
not significant) export of water in V452R. Control oocytes did not
show any difference in swelling in the presence of L-glutamate
or TBOA. Although the magnitude of water transport in isotonic
saline is lower than in hypotonic saline for all constructs, the
profiles of responses to L-glutamate for each construct showed
similar trends between the two conditions. In both conditions,
the lack of an enhanced swelling rate response in V452R in the
presence of L-glutamate (as compared with untreated) suggests
that the L-glutamate transporter pathway is required for glutamatestimulated water uptake.
Urea transport
We investigated the [14 C]urea transport rates for EAAT1 and
the mutant constructs to compare the permeability properties
of urea with water. Incubation of oocytes expressing the WT
EAAT1 or the D112A construct in standard isotonic saline with
10 μM [14 C]urea for 30 min led to accumulations greater than
that observed for control oocytes (Figure 3). D112A supported a
large leak current (Figure 1B) and also allowed the largest rate of
background [14 C]urea uptake (open bars, Figure 3A). Inclusion
of 100 μM TBOA caused small reductions in [14 C]urea uptake for
WT EAAT1 and V452R, and a substantial reduction for D112A
compared with the untreated oocytes.
Under isotonic conditions, 100 μM L-glutamate caused
significant increases in the rates of transport of [14 C]urea for
WT EAAT1 and V452R, but not D112A-expressing and control
oocytes (Figure 3A). Previous work has shown that the D112A
mutant transports L-glutamate, but L-glutamate does not activate
an additional uncoupled Cl − conductance [19]. Thus, for the
D112A mutant, there does not appear to be an appreciable
amount of transport of [14 C]urea that is associated with the Lglutamate-stimulated transport pathway. L-Glutamate binds to the
Water and urea permeation of glutamate transporters
Figure 2
337
Effects of L-glutamate and TBOA on the osmotic water permeabilities of WT EAAT1 and mutant constructs
(A) Osmotic swelling responses of control and WT EAAT-expressing oocytes measured as the relative surface area as a function of time after introduction into 50 % hypotonic ND96 saline, with 0, 100
or 400 μM L-glutamate. Values are mean +
− S.D. for nine oocytes per group. Compiled data from four batches of oocytes for swelling rates from all osmotic swelling assays under hypotonic (50 %
ND96) (B) and isotonic (100 % ND96) (D) conditions, standardized to the mean swelling rate of the untreated WT EAAT response within the same batch of oocytes. The L-glutamate concentration
used was 100 μM and the TBOA concentration was 100 μM. A comparison of the rate of swelling of oocytes in hypotonic saline and in isotonic saline from untreated oocytes expressing WT EAAT1,
the two mutants and control oocytes from the same batch are included (C). Statistical analyses were as described in the Experimental section (*P < 0.05; **P < 0.01).
V452R transporter and activates the Cl − conductance, but Lglutamate transport is significantly reduced [30] (Figure 1). Although this mutant has impaired transport, L-glutamate stimulates
the rate of [14 C]urea transport by 6.6-fold above untreated levels.
Thus, for the V452R mutant, the levels of [14 C]urea transport
correlate with the activation of the L-glutamate-stimulated Cl −
conductance and not the transport process.
Passive water transport by EAAT1 and the mutants
(hypotonic saline) is considerably higher than active transport
(with isotonic saline). We also investigated whether the osmotic
gradient influenced the rate of [14 C]urea uptake. The rates of
[14 C]urea uptake in hypotonic saline were similar to those
measured in isotonic saline (Figure 3B). This suggests that the
rate of [14 C]urea uptake is independent of the osmotic gradient.
Furthermore, the rate of [14 C]urea transport was not significantly
reduced by a lower extracellular Na + concentration, as imposed
in the hypotonic saline condition, as compared with the standard
concentration of Na + in the isotonic saline.
D-Aspartate is transported by EAAT1 with a transport constant
similar to L-glutamate (K 0.5 = 23 +
− 2 μM for D-aspartate and
K 0.5 = 20 +
− 3 μM for L-glutamate), but the maximal rate of
transport of D-aspartate is approximately 50 % of that measured
for L-glutamate [19,35,36]; nonetheless, the amplitudes of the Cl −
currents that are activated by the two substrates are similar [30].
The rates of [14 C]urea transport by EAAT1 when stimulated by Daspartate as compared with L-glutamate are also similar (Figure 4),
which suggests that the rate of substrate transport itself was not
the major determinant for the rate of [14 C]urea transport.
We also investigated whether the nature of the external
anion influenced the untreated and L-glutamate-stimulated rates
of [14 C]urea uptake by EAAT1. Although the predominant
physiological anion is Cl − , other anions show differential
levels of permeability through the anion channel of L-glutamate
transporters. For example, nitrate (NO3 − ) is 17-fold more
permeant than Cl − , whereas gluconate does not permeate the
channel [17,19]. Despite differences in permeability of the anions,
no significant differences were observed for either the untreated
or L-glutamate-stimulated rates of [14 C]urea uptake (Figure 4).
Although urea uptake appears to be most closely associated with
the channel activity of EAAT1, the nature of the permeant anion
does not appear to influence the rate of [14 C]urea uptake.
DISCUSSION
L-Glutamate transporters catalyse ion-coupled transport, but also
have an intrinsic anion channel. We and others have demonstrated
that L-glutamate transport can be modulated without affecting
anion channel activity, suggesting that discrete molecular domains
or conformational states govern the two functions [16,19,30,37–
39]. In the present study we used constructs of EAAT1 with
mutations that differentially target the glutamate transport and
channel functions. The results suggest that the L-glutamateinducible active and passive water permeation pathways become
available concurrently with the transport of L-glutamate. The
mutant transporter D112A and WT EAAT1 support similar
rates of L-glutamate transport, and have similar levels of Lglutamate-stimulated water permeation. In contrast, the transportimpaired mutant V452R does not display significant L-glutamate
stimulation of water transport. The results of the present study also
provide evidence for a L-glutamate-independent passive water flux
pathway [24]. However, the nature of this permeation pathway
remains obscure. EAAT1 and D112A display higher levels
of passive water transport than control oocytes in the absence of
c The Authors Journal compilation c 2011 Biochemical Society
338
Figure 3
R. J. Vandenberg and others
[14 C]Urea uptake by WT and mutant EAAT1
[14 C]Urea uptake was measured in untreated oocytes, in the presence of 100 μM L-glutamate
and in the presence of 100 μM TBOA. (A) Isotonic conditions, ND96 buffer; (B) hypotonic
conditions, ND96 diluted 50 % in water. Levels of significance are as marked (***P < 0.001).
Rates of uptake for untreated control oocytes were significantly different to oocytes expressing
the EAAT1 mutant D112A (all at P < 0.001), but not the mutant V452R. glut, glutamate; untr,
untreated.
Figure 4 The rate of substrate transport or the external anion does not
affect the rate of [14 C]urea uptake by WT and mutant EAAT1
The background rates of [14 C]urea uptake by oocytes expressing EAAT1 were not significantly
different between the Cl − -based buffer, the gluconate − -based buffer or the NO3 − -based
buffer. In the Cl − -based buffer, the rate of L-glutamate-stimulated [14 C]urea uptake (solid
bars) was significantly greater than that of untreated oocytes (open bars) (P < 0.001), but not
significantly different to the rate of D-aspartate-stimulated urea uptake (grey bar). asp, aspartate;
glut, glutamate; untr, untreated.
the substrate L-glutamate. The EAAT inhibitor TBOA does not
appear to reduce these background levels of uptake to control
levels, which suggests that an alternative conformational state
that is not associated with the glutamate transport function may
be responsible for this additional water permeation. In contrast,
the V452R mutant displays an unusual effect in that TBOA
does reduce the background rate of passive water transport to
control levels, suggesting that the nature of the L-glutamate
c The Authors Journal compilation c 2011 Biochemical Society
independent passive water flux is different in V452R compared
with WT EAAT1 or D112A. The crystal structure of a prokaryotic
homologue of EAAT1, GltPh , has been determined in the presence
of the substrate L-aspartate and the blocker TBOA [40,41]. The
key difference between the substrate and blocker-bound forms
of GltPh is that the hairpin loop structure HP2 is held open
in the TBOA-bound form, which has two consequences. First,
it prevents closure of the external gate of the transporter and,
secondly, it prevents the formation of a Na + -binding site that is
required for transport. The V452R mutation is within HP2, and
a possible reason for the impaired transport by this mutant is the
inability of the external gate to close down on the substrate and/or
the inability to form the Na + -binding site. TBOA bound to V452R
may also lead to stabilization of an alternative conformational
state, which is not formed by EAAT1 or D112A, and prevents
the background water permeation observed in EAAT1 and
D112A.
Urea can permeate some aquaporins [27,28] and has been
shown to permeate EAAT1 (the present study and [23]). Our
experiments with [14 C]urea highlight some interesting differences
between water and urea permeation of EAAT1. In contrast
with water permeation, the uptake of [14 C]urea correlates with
the L-glutamate-activated Cl − channel and the L-glutamateindependent Cl − leak channel activities of the transporters rather
than the transport process. WT EAAT1 and V452R have large Lglutamate-activated chloride currents, whereas the L-glutamateactivated chloride current is essentially absent in the D112A
mutant. L-Glutamate-stimulated urea uptake rates show similar
trends, being largest in WT EAAT1 and the V452R mutant, and
absent in the D112A mutant. Furthermore, the D112A mutant has
a large leak current that is blocked by TBOA, and the background
rate of urea uptake is significantly greater than WT and can also
be blocked by TBOA. These differences in urea uptake compared
with water transport may reflect the different sizes of water
and urea (molecular masses of 18 Da and 60 Da respectively).
Urea is larger than water and may still pass through the
Cl − channel, which can accommodate anions up to 5 Å
(1 Å = 0.1 nm) [42], but urea may be too large to pass through the
L-glutamate translocation pathway.
One conundrum that arises from the present study is that
urea passes through the anion channel in a glutamate-dependent
manner, but it appears that the smaller water molecule does not.
The behaviour of the V452R mutant may provide some clues. A
possible explanation is that water does in fact pass through the
channel, but as it is smaller than urea it can do so in a glutamateindependent manner. The V452R mutant has a background water
uptake that is greater than the untreated control oocytes. This
additional water flux could be via the channel, and glutamate may
not stimulate this any more than the background level. In GltPh ,
with TBOA bound, the transport domain is locked into a state that
may prevent movement of the transport domain relative to the
trimerization domain [44]. Thus, in the condition where TBOA is
bound to V452R, the transporter may be locked into a position that
prevents water passage through the background state and thereby
reduces the water flux compared with the untreated condition. It
may not be possible to measure this state in the WT and D112A
mutant because TBOA bound to these transporters may still allow
the required conformational changes for the passage of water
through the channel.
Another surprising result from the present study was that
urea transport by EAAT1 is not affected by the nature of the
extracellular anion. It is possible that although urea (and probably
water) passes through the anion channel, the anions and urea may
have different selectivity filters within the channel, which would
allow their independent movement through the channel.
Water and urea permeation of glutamate transporters
Water molecules are likely to be highly promiscuous in their
interactions with transporters, yet the permeation pathways in the
glutamate transporter family of proteins appear to be discrete. It
appears that glutamate binding to the transporter induces a series
of conformational steps that also create a permeation pathway
for water molecules. Previous studies on transporters from the
SLC6 family, which included the γ -aminobutyric acid transporter
GAT1, the insect neutral amino acid transporter KAAT1 and
the bacterial leucine transporter LeuTAa , demonstrate that this
family of transporters also allows a significant amount of passive
water flux [43,44] but, in contrast with the EAAT family, water
permeation was not substrate-activated. Two mutant transporters
of KAAT1 and GAT1, one that is devoid of transport function
and another with enhanced transport function, retained the same
rates of water uptake as their respective WT transporters, which
suggests that active transport is not required for water uptake by
these transporters. Although there is a growing list of transporter
proteins that allow water movement, it appears that these proteins
utilize a range of mechanisms to support water permeation.
Understanding these mechanisms will help us to delineate the
role transporters play, in combination with the aquaporins, in
water homoeostasis in the human body.
AUTHOR CONTRIBUTION
Robert Vandenberg designed experiments, conducted experiments, analysed data and
wrote the paper; Cheryl Handford conducted experiments and analysed data; Ewan
Campbell conducted experiments and analysed data; Renae Ryan designed experiments,
analysed data and wrote the paper; and Andrea Yool designed experiments, analysed data
and wrote the paper.
ACKNOWLEDGEMENTS
We thank L. O’Carroll for skilled technical assistance.
FUNDING
This work was supported by the National Health and Medical Research Council [Senior
Research Fellowship to R.V.; Career Development Award to R.R.; Australian Research
Council project grant number DP1092729]; and the Channel 7 Children’s Research
Foundation [grant number 257 (to A.Y.)].
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