Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti
1 Environmental Toxicology Graduate Program
2 Department of Cell Biology and Neuroscience, University of California,
Riverside, Riverside, CA 92521, USA
3 Division of Biology, California Institute of Technology, Pasadena, CA
91125, USA
Author for correspondence (e-mail:
sarjeet.gill{at}ucr.edu)
Accepted 3 April 2003
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Summary |
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Key words: glutamate/aspartate transporter, mosquito, Aedes aegypti, neurotransmitter, amino acid, electrophysiology, localization
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Introduction |
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In mammals, it is believed that Na+- and K+-dependent
high-affinity transport systems are responsible for regulating extracellular
glutamate levels as well as terminating glutamatergic signals in the central
nervous system (for a review, see Danbolt,
2001). Five transporters with the ability to transport
L-glutamate and L-and D-aspartate have been
identified in humans. They have been designated EAAT (Excitatory Amino Acid
Transporter) 15, having 5060% amino acid identity with each
other (Seal and Amara, 1999
).
These proteins drive the uphill transport of glutamate by dissipating the
electrochemical Na+ gradient generated by
Na+/K+-ATPase. It is currently accepted in mammals that
glutamate transport occurs with the cotransport of 3Na+ and
1H+, and a countertransport of 1K+ with every molecule
of glutamate (Levy et al.,
1998
; Zerangue and Kavanaugh,
1996b
). Additionally, glutamate evokes an anion channel activity
intrinsic to the EAATs: a property which, however, is not directly coupled to
substrate translocation (Fairman et al.,
1995
; Wadiche et al.,
1995a
).
In insects, early biochemical studies have shown the existence of
Na+-dependent L-glutamate transport systems in both
muscle and central nervous systems. Examples include Na+-dependent
glutamate transport on isolated abdominal nerve cords from adult
Periplaneta americana, as well as autoradiographic transport studies
in the excitatory neuromuscular junctions of locust localizing
Na+-dependent glutamate transport to nerve endings and surrounding
glia (Evans, 1975;
van Marle et al., 1983
).
The availability of cDNA sequences encoding mammalian EAATs, coupled with
the advent of molecular cloning, has allowed for the cloning and isolation of
EAAT homologues from a number of insect species within the last 5 years. In
particular, insect EAAT homologues have been cloned by independent groups from
the brain and embryo of Drosophila melanogaster (dEAAT1 and dEAAT2)
as well as the brains of caterpillar Trichoplusia ni (TrnEAAT),
cockroach Diploptera punctata (DipEAAT) and honeybee Apis
mellifera (AmEAAT) (Besson et al.,
1999; Donly et al.,
1997
,
2000
;
Kawano et al., 1999
;
Kucharski et al., 2000
;
Seal et al., 1998
). With the
exception of AmEAAT, all insect homologues cloned thus far have been
heterologously expressed and functionally characterized as
Na+-dependent high-affinity glutamate and aspartate transporters
with affinity in the micromolar range (Besson et al.,
1999
,
2000
; Donly et al.,
1997
,
2000
;
Kawano et al., 1999
;
Kucharski et al., 2000
;
Seal et al., 1998
).
Electrophysiological studies on dEAAT1 have illustrated a substrate-elicited
anion conductance that is stoichiometrically uncoupled from substrate
translocation, much like the mammalian EAATs
(Fairman et al., 1995
;
Seal et al., 1998
;
Wadiche et al., 1995b
).
Amongst such similarities, however, certain differences have been noted, such
as a preference for L- and D-aspartate as substrates
over L-glutamate for dEAAT2; a preference for
L-asparatate over D-aspartate by DipEAAT; and abolished
substrate transport by dEAAT1 in the absence of both intracellular and
extracellular chloride (Besson et al.,
2000
; Donly et al.,
2000
; Seal et al.,
1998
).
Attempts at elucidating the spatial localization of the insect EAATs at the
mRNA level for dEAAT1, dEAAT2 and AmEAAT, have demonstrated almost exclusive
localization to parts of the brain insect brain
(Besson et al., 1999;
Seal et al., 1998
;
Soustelle et al., 2002
). A
recent immunohistochemical study on TrnEAAT, however, demonstrated expression
in larval skeletal muscle fibers (Gardiner
et al., 2002
).
In spite of the fact that glutamate performs a plethora of roles in insects, and that a number of insect transporters for glutamate have been characterized, information available for the termination of insect glutamatergic signals is still relatively sparse. In light of this, we present a comprehensive study on a glutamate and aspartate transporter from the mosquito Aedes aegypti (AeaEAAT), which encompasses cloning and characterization from pharmacological, electrophysiological, and spatial localization standpoints. Ae. aegypti is the major vector for the dengue virus; the work presented here therefore represents the first such kind from a blood-feeding insect. Given the recent completion of the Anopheles gambiae genome project, together with the historic interest in the biology of the mosquito as a vector for various human diseases, the work presented here is a timely contribution to the understanding of this disease vector.
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Materials and methods |
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Midgut/Malpighian tubule cDNA library construction
Poly(A)+ RNA was isolated from isolated midgut and Malpighian
tubules of adult female Aedes aegypti. The mRNA was then used for the
construction of cDNA using a cDNA synthesis kit from (GIBCO BRL Life
Technologies, Gaithersburg, MD, USA). The cDNA (approximately 1.5 µg) was
separated by size-exclusion chromatography on a Superose 6 column using a
SMART system (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) as previously
described (Mbungu et al.,
1995). The >2kb cDNA was ligated to
NotI/SalI-cut, dephosphorylated pSport1 vector and
electroporated into DH10B cells. The size of the unamplified library was 130
000 clones, 95% of which were recombinants. Isolation and analysis of
individual cDNA clones demonstrated the presence of large inserts in this
library.
Isolation of clone and construction in expression vector
The gene encoding AeaEAAT, initially named BC10, was identified during an
EST project that randomly sequenced approximately 2000 different genes
expressed in the cDNA library above. The open reading frame (ORF) was
subcloned into the expression vector pBSAMV containing a T7 promoter, through
the NcoI and FseI restriction enzyme sites
(Chiu et al., 2000). These
restriction sites were engineered into the 5' and 3' ends of the
ORF, respectively, by polymerase chain reaction (PCR) using primers containing
the sites. The PCR product generated by Expand (Roche, Palo Alto, CA, USA) was
gel purified, and initially ligated to pCR2.1 (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer's specifications. Clones putatively containing
the desired product were sequenced to confirm the lack of any PCR-generated
errors using ABI Prism cycle sequencing. The clone with the correct sequence
was subsequently subcloned into the pBSAMV vector
(Chiu et al., 2000
), and will
herein be referred to as pBSAMVBC10.
In vitro transcription/translation
The plasmid pBSAMVBC10 containing the AeaEAAT ORF was in vitro
transcribed and translated using the TNT T7-Coupled Reticulocyte Lysate System
(Promega, Madison, WI, USA). A sample (4 µl) of the total reaction was
separated by discontinuous 8% SDS-PAGE as described previously
(Laemmli, 1970). The gel was
stained with Coomassie Blue, destained, treated in Entensify enhancing
solutions (Dupont-NEN, Boston, MA, USA), and dried before exposure to X-ray
film for 3 days.
Transient transfections
HeLa cells were maintained in Minimum Essential Medium (Invitrogen,
Carlsbad, CA, USA) containing 1.5 g NaHCO3, sodium pyruvate
(Invitrogen), non-essential amino acids (Invitrogen), penicillin/streptomycin
(Invitrogen) and 10% fetal bovine serum (Summit Biotechnologies, Greeley, CO,
USA) at 37°C and 5% CO2. Transient transfections utilizing the
T7/vaccinia virus method were generally carried out in 48-well plates using
serum-free medium for all steps (Blakely et
al., 1991). Briefly, 57x104 cells per well
were plated the night before transfection in the above medium lacking
antibiotics, of which 2 wells were counted on the day of the transfection to
obtain an average number of cells per well. A virus multiplicity of infection
of 5, 0.3 µl of lipofectin (Invitrogen), 1 µl PLUS reagent (Invitrogen),
and 100 ng of DNA was used per well in all cases. Lipofectin was diluted in
medium 3035 min before mixing with DNA, which itself was preincubated
with the PLUS reagent for 15 min beforehand. Cells were infected with virus
(30 m, 37°C/5% CO2) with shaking every 10 min, and the
lipofectin/DNA/PLUS mixture was overlayed.
Transport assay in mammalian cells
Transport assays were carried out between 18 and 24 h after
infection/transfection. Cells were washed once with PBS, and incubated for 15
min with uptake buffer (120 mmol l-1 NaCl, 4.7 mmol l-1
KCl, 10 mmol l-1 Hepes, pH 7.5, 5 mmol l-1 Tris-Cl, pH
7.4, 1.2 mmol l-1 KH2PO4, 2 mmol
l-1 CaCl2, 1.2 mmol l-1 MgSO4)
containing glucose before performing the transport assay in duplicate. In
order to determine substrate specificity, cells were incubated with a mixture
of cold substrate (100 µmol l-1) and 3H-labeled
substrate (30 nmol l-1; Amersham, Piscataway, NJ, USA) used as a
tracer, initially for 1020 min. The substrates that were transported
(L-glutamate, L-aspartate and D-aspartate)
were further analysed for ion dependence, time course and doseresponse
studies.
Time course, ion dependence and kinetic analyses
A time course study was performed on all three substrates, where the
duration of transport was varied. Upon determining the time during which
transport followed a linear time course, this time point (5 min) was used in
all subsequent transport assays.
The dependence of substrate transport on the major extracellular ions was determined by substituting equimolar choline chloride for NaCl in the assay buffer, while two types of anions, gluconate and acetate, were used in independent experiments to replace chloride ions.
Doseresponse studies were performed to obtain the affinity and kinetic parameters (Km and Vmax), by varying the cold substrate concentration between 0.1 µmol l-1 and 500 µmol l-1 on a logarithmic scale. Experiments were performed in triplicate to obtain mean Km and Vmax values, calculated by a least-squares fit to the MichaelisMenton equation and EadieHofstee transformation using Origin 6.0 (Microcal, Northampton, MA, USA).
Pharmacology
Inhibition studies were carried out with 1 µmol l-1 cold
substrate and 30 nmol l-1 3H-labeled substrate, so as to render the
concentration of substrate negligible when mathematically determining the
Ki, such that the IC50 approximates the
Ki as described (Cheng
and Prusoff, 1973a). A three-point screen of 3, 100 and 3000
µmol l-1 inhibitor concentration was used initially in order to
eliminate compounds that essentially had no effect on transport. Others were
consequently subjected to a further inhibition assay, where inhibitor
concentrations were varied on a logarithmic scale, typically between 1 nmol
l-1 and 3 mmol l-1.
Electrophysiology
Heterologous expression in Xenopus laevis oocytes was utilized to
study the electrophysiological properties of AeaEAAT via
two-electrode voltage clamp (TEV-200, Dagan Instruments, Minneapolis, MN, USA)
interfaced to an IBM-compatible PC using a Digidata 1200 A/D controlled using
the pCLAMP 6.0 program suite (Axon Instruments, Foster City, CA, USA). The
expression construct pBSAMVBC10 was cut with NotI for runoff in
vitro transcription using the Ambion T7 mMessage mMachine kit (Ambion,
Austin TX, USA). Stages V and VI Xenopus oocytes were prepared using
standard protocols, injected with 35 ng of cRNA, and incubated for 36
days in ND96 (96 mmol l-1 NaCl, 2 mmol l-1 KCl, 1 mmol
l-1 CaCl2, 1 mmol l-1 MgCl2, 5
mmol l-1 Hepes, pH 7.5) supplemented with gentamycin at 18°C
before recordings. Current responses were acquired at room temperature and
analyzed with either Clampfit 8.0 (Axon Instruments, Foster City, CA, USA) or
ORIGIN 6.0 (MicroCal Software, Northampton, MA, USA). Signals were filtered
with a 2 kHz low-pass filter and a 3 mol l-1 KCl agar bridge was
used to reduce junction potentials. Microelectrodes were filled with filtered
3 mol l-1 KCl solution, with resistances of less than 1 M
for both current passing and recording electrodes. For the superfusing of
saline solution, a PerkinElmer Model 410 liquid chromatography pump was
used, supplying a continuous flow of ND-96 buffer at a flow rate of 1.0 ml
min-1. A Rheodyne injection valve was used to apply substrates for
doseresponse analysis in 170 µl volumes, providing a 5 s exposure of
substrate to the oocyte. Concentrationresponse relationships to
determine EC50 values were obtained from oocytes clamped at -60 mV
by measuring the peak current amplitude as a function of substrate
concentration. Results were normalized to current evoked by the highest
substrate concentration used, and subsequently fit to the equation
I=ImaxS/(EC50+S),
where I is the peak current amplitude, Imax is
the peak current amplitude evoked by the highest substrate concentration, and
S is the concentration of substrate.
In reversed-transport studies, oocytes clamped at -60 mV were exposed to a high potassium buffer containing 98 mmol l-1 KCl, 1 mmol l-1 MgCl2, 1.8 mmol l-1 CaCl2 and 5 mmol l-1 Hepes (pH 7.5).
Currentvoltage relationships were determined either by measurement
of steady-state currents in response to bath application of substrates or by
off-line subtraction of control current records obtained during 1 ms voltage
pulses to potentials between -60 and +50 mV from corresponding current records
in the presence of substrate. Sodium substitution studies were carried out in
saline containing equimolar choline chloride instead of NaCl. External
chloride was replaced by either gluconate, nitrate or thiocyanate salts as
indicated, at concentrations equivalent to the chloride salts used in ND96.
Intracellular chloride depletion experiments were performed as described in
Wadiche et al. (1995a), by
placing oocytes in chloride-free buffer substituted with gluconate for at
least 20 h prior to use.
Xenopus oocyte transport assay
Substrate-transport assays in Xenopus oocytes were performed by
exposing oocytes to 3H-radiolabeled substrate (30 nmol l-1
L-glutamate, L-aspartate and D-aspartate;
Amersham) for 5 min at room temperature. Oocytes were then rapidly washed
three times in ice-cold buffer in order to prevent any possibility of reversed
transport, upon which they were lysed in 10% SDS and subjected to
scintillation counting.
For cases in which transport assays were performed under two-electrode voltage clamp, oocytes were clamped at -60 mV and exposed to radioactive ligand for 5 s, then washed for 10 s with the respective saline, and subsequently lysed and counted for accumulated radioactivity as described above.
Antibody production and purification
A synthetic peptide of 15 amino acids, corresponding to the C-terminal 14
amino acids of AeaEAAT, plus a cysteine at the N terminus of these 14 residues
(CPSSEINGKTQRNSL) was synthesized (MGIF, University of Georgia). The peptide
was conjugated to a maleimide-activated form of keyhole limpet hemocyanin
(KLH; Pierce, Rockford, IL, USA) according to the manufacturer's instructions,
immunized in rabbits and affinity-purified essentially as described
(Umesh and Gill, 2002).
Preparation of membranes from Ae. aegypti
Membranes were isolated from adult Ae. aegypti using a
differential centrifugation protocol. Head, thorax and abdomen were separated
from male and female Ae. aegypti anesthetized in CO2.
Briefly, tissues were suspended in ice-cold Tris-HCl buffer (50 mmol
l-1, pH 7.4) containing phenylmethylsulfonyl fluoride (PMSF; 1 mmol
l-1) and protease cocktail inhibitor (Roche Diagnostics,
Indianapolis, IN, USA), homogenized with 30 strokes using a Dounce
homogenizer, and centrifuged (3000 g, 4°C, 10 min). The
post-nuclear supernatant was centrifuged (10 000 g, 4°C,
10 min) to remove mitochondria, and the resulting supernatant was further
centrifuged to pellet the membranes (100 000 g, 1 h, 4°C)
using a Beckman Ti 42.2 rotor. Final pellets were resuspended in the
homogenization buffer, upon which protein concentrations were determined with
the BCA Protein Assay Kit (Pierce).
Preparation of membranes from X. laevis
oocytes
Membranes were prepared from Xenopus oocytes injected with cRNA of
pBSAMVBC10 and that of a D. melanogaster sodium channel, of which the
latter served as a negative control (Buller
and White, 1992). Briefly, oocytes were homogenized in Buffer A
(150 mmol l-1 NaCl, 10 mmol l-1 magnesium acetate, 20
mmol l-1 Tris-Cl, 0.1 mmol l-1 PMSF, pH 7.6) containing
sucrose (10% w/v) at a concentration of 25 oocytes ml-1 of buffer.
Homogenates were overlayed on a discontinuous sucrose gradient consisting of
20% and 50% sucrose, and subsequently centrifuged (15 000 g,
30 min, 4°C). The layer visible at the interface of 20% and 50% sucrose,
representing the membrane portion, was carefully removed, diluted in 5 volumes
of buffer A and recovered by centrifugation (100 000 g, 1.5 h,
4°C).
SDS-PAGE and western analysis
Membranes and cytosol of Ae. aegypti, as well as X.
laevis oocyte membranes, were analyzed by SDS-PAGE and western blotting.
Protein samples were treated with Laemmli sample buffer (37°C, 30 min).
Subsequently, samples were separated by an 8% polyacrylamide gel at a
concentration of 25 and 9 µg per lane for the tissue samples and oocyte
membranes, respectively. For western analysis, samples separated by SDS-PAGE
were electrotransferred onto Immobilon P membranes (0.45 mm; Millipore,
Billerica, MA, USA) using a tank transfer system (80 V, 3 h, 4°C). Upon
blocking (1 h, room temperature) with 3% BSA in PBS containing 0.05% Tween-20
(PBST), membranes were incubated with affinity-purified AeaEAAT antibody (1
µg, overnight, 4°C) diluted in PBST containing 1% BSA. As a negative
control, membranes with identical samples were incubated with primary antibody
that had been preadsorbed with peptide (10 µmol l-1). Upon
washing in PBST, membranes were incubated with horseradish peroxidase
(HRP)-coupled donkey anti-rabbit IgG (Amersham) diluted 1:3000 in 1% BSA/PBST
(1 h, room temperature), washed extensively, and proteins were ultimately
detected using enhanced chemiluminescence (Amersham).
Immunohistochemistry
Immunohistochemical studies were performed on adult thorax. Upon separation
of the thorax and fixation in alcoholic Bouin's solution (overnight, 4°C),
tissues were sectioned after paraffin embedding as described before
(Umesh and Gill, 2002).
Sections were blocked in PBS-Triton X-100 (0.2%) (PBS-Tx, pH 7.4) containing
normal goat serum (NGS, 1%) and BSA (0.1%) for 30 min, and then incubated with
the
-AeaEAAT (1/200 dilution in blocker, 4°C, overnight). After
extensive washes with PBS-Tx, sections were incubated with HRP-labeled goat
anti rabbit IgG (American Qualex, San Clemente, CA, USA) diluted 1/1000 in
blocker, washed thoroughly with PBS-Tx and stained using 3-amino
9-ethylcarbazol as a substrate (Umesh and
Gill, 2002
). Slides were visualized with a Zeiss Axiovert
microscope (Carl Zeiss, Thornwood, NY, USA). All images were ultimately
imported into Adobe Photoshop (version 6.0, Adobe, Sunnyvale, CA, USA) in
which they were assembled and labeled.
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Results |
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The dendrogram analysis (Fig. 1B) includes members of the human EAAT superfamily consisting of hEAAT1-5 and related system ASC transporters, ASCT1 and ASCT2, which transport alanine, serine, threonine and cysteine. Also included are cloned insect EAATs and homologous sequences from the completed genome of Anopheles gambiae. Amongst the five subtypes of mammalian EAATs identified thus far, the above mentioned insect transporters collectively are most identical to hEAAT3, with 47.7% identity to AeaEAAT. The completed genomic sequence of An. gambiae gave three sequences representing putative members of the EAAT family: AAB01008807, AAB01008797 and AAB01008964, with the deduced amino acid sequences yielding proteins with 75.1, 52.0 and 33.5% identity to AeaEAAT, respectively.
Heterologous expression of AeaEAAT
The AeaEAAT open reading frame was subcloned into the expression vector
pBSAMV, whose expression is driven by the T7 promoter. In vitro
transcription and translation of this subclone gave a protein migrating at 42
kDa when separated by SDS-PAGE (Fig.
2, lane 1).
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Heterologous expression of pBSAMVBC10 in both HeLa
(Fig. 3) and CV-1 (data not
shown) cells showed transport of substrates typical for this family of
proteins, namely, L-glutamate, and L- and
D-aspartic acid. Negative controls were initially conducted using
the same vector containing a different transporter, MasIne
(Chiu et al., 2000), but
showed no difference from transfections conducted without DNA (data not
shown).
|
Transport was Na+-dependent, as replacement of Na+ ions in the assay buffer with choline reduced uptake to background levels (Fig. 3A). On the other hand, transport was independent of CL-, since replacement with each of two types of anions, acetate and gluconate, made no significant difference (Fig. 3B).
AeaEAAT is a high-affinity transporter of its substrates, having Km values of approximately 30 and 10 µmol l-1 for L-glutamate and the two types of aspartate, respectively (Table 1). In all three cases transport was saturable, with the doseresponse relationship following MichaelisMenten kinetics. Similar affinity parameters were observed when AeaEAAT was independently expressed in both HeLa and CV-1 cells (data not shown). The Vmax values were determined to be 6.0, 3.0 and 6.9 fmol cell-1 h-1 for L-glutamate, L-aspartate and D-aspartate, respectively. From these values, the efficacy (Vmax/Km) of transport by AeaEAAT was highest for D-aspartate (0.6), being double that of L-aspartate (0.3), and triple that of L-glutamate (0.2). Substrate transport was not inhibited by D-glutamate (Table 2), indicating that for glutamate, AeaEAAT demonstrates stereoselective transport of the L-congener.
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We tested the ability of the 20 amino acids to compete with transport of D-aspartate (Fig. 4), the non-metabolizable substrate, at a competitor concentration of 5 mmol l-1. As expected, the earlier determined high-affinity substrates (i.e. L-glutamate and L-aspartate) most strongly competed D-aspartate transport. In addition, we found that L-cysteine inhibited D-aspartate uptake to 12% of control levels, and that L-asparagine and L-glutamine also inhibited the substrate transport by 27.5% and 70% of control levels, respectively. At the concentrations used, inhibition by these amino acids was statistically significant at the 99% confidence interval.
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Pharmacology
In an initial screen, various compounds in amounts of 3, 100 and 3000
µmol l-1 were tested for their ability to independently block
the AeaEAAT-mediated transport of L-glutamate and
D-aspartate. 30 nmol l-1 3H-labeled and 100 µmol
l-1 unlabeled substrate were used in the initial screen. Of the
compounds tested, N-methyl-D-aspartate and kainic acid had
no effect on transport, even at 3 mmol l-1
(Table 2). Thereafter, the
amount of substrate used was reduced to a total of 1 µmol l-1,
so as to approximate the IC50 to Ki
(Cheng and Prusoff, 1973b).
The IC50 (equivalent to the calculated Ki)
values of 11 compounds, DL-threo-benzoxyaspartic acid
(DL-TBOA), trans-pyrrolidine-2,4-dicarboxylate
(t-PDC), L-aspartate-ß-hydroxymate,
DL-threo- ß-hydroxyaspartic acid (TBHA),
L-cysteine, L-cysteic acid, serine-O-sulfate,
L-cysteine sulfinic acid,
-amino adipic acid,
ß-glutamate and D-glutamate were determined by using
logarithmically increasing concentrations of the various inhibitors. Overall,
the inhibitors were equally effective at inhibiting the transport of both
D-aspartate (Table
2) and L-glutamate (data not shown). Of the compounds
tested, those involved in the cysteine oxidation pathway such as
L-cysteic acid and L-cysteine sulfinic acid, as well as
TBHA, were the most effective inhibitors, with calculated
Ki values of approximately 6.6±3.3, 7.1±3
and 9.2±2.9 µmol l-1, respectively. The most notable
difference in AeaEAAT pharmacology was inhibition by
serine-O-sulfate, for which the Ki was 29 µmol
l-1. This compound was twice as potent at inhibiting transport by
AeaEAAT than TrnEAAT, and three times as potent than hEAAT3. A universal
inhibitor of all EAAT subtypes, t-PDC, blocked D-aspartate
transport with a Ki of 91±19 µmol
l-1, and DL-TBOA, a non-substrate EAAT inhibitor, gave a
Ki value of 23.7±2.1 µmol l-1.
Expression in Xenopus laevis oocytes
In an attempt to understand the ion transport characteristics of AeaEAAT,
we turned to heterologous expression of AeaEAAT in Xenopus laevis
oocytes where two-electrode voltage clamp analysis was performed.
Xenopus oocytes injected with cRNA in vitro-transcribed from
pBSAMVBC10 displayed an inward current that is characteristic of this family
of transporters upon application of substrate
(Fig. 5A). This is consistent
with a net inward flow of cations during the transport cycle
(Zerangue and Kavanaugh,
1996b). Typically, the current elicited by the application of 100
µmol l-1 substrate was as large as 200400 nA. Similar
currents were produced regardless of the substrate (i.e.
L-glutamate, L-and D-aspartate,
L-cysteine).
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Reversal of extracellular ionic composition by raising K+ ion
concentrations caused outward currents to be elicited by oocytes expressing
AeaEAAT, reflecting the reversed transport of endogenous excitatory amino
acids (Fig. 5B). No outward
current was observed in water-injected oocytes upon exposure to high
extracellular K+ (data not shown). Endogenous excitatory amino acid
concentration in X. laevis oocytes has been reported to be as high as
12 mmol l-1 (Wadiche et al.,
1995a). Reversed transport is a physiologically relevant
phenomenon in mammals that occurs in disease states such as ischemia, when
extracellular concentrations of K+ rise to 60 mmol l-1,
thereby depolarizing cells and allowing the release of glutamate by reversed
functionality of the glutamate transporters
(Attwell et al., 1993
).
Difference IV curves obtained by off-line subtraction of voltage-jump protocols without substrate from those with substrate, revealed a substrate-activated current reversing at 2530 mV for all substrates tested (Fig. 5C). The substrate-activated current was abolished upon substitution of extracellular Na+ with choline (Fig. 5D).
It is now well established that excitatory amino acid transporters carry a
substrate-gated anion conductance that is not coupled to transport (reviewed
in Seal and Amara, 1999). The
ability of AeaEAAT to carry this type of current was determined by
investigating the difference IV curves upon replacement of
extracellular CL- with other anions. Outward current was abolished
when external chloride was replaced by gluconate
(Fig. 6A,B), an impermeable
anion, but enhanced when replaced with more permeant anions such as nitrate
and thiocyanate (Fig. 6A,B). We
then compared the substrate-activated currents of oocytes depleted of
intracellular chloride by dialysis, in both the presence and absence of
extracellular chloride (Fig.
6C). Chloride-dialysed oocytes gave identical inward currents
regardless of the presence of extracellular chloride. However, outward
currents were only seen in dialysed oocytes in the presence of extracellular
CL-.
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We addressed the question of whether substrate transport was dependent on the presence of either intracellular or extracellular CL-, by comparing radiolabeled substrate transport of dialysed oocytes to undialysed oocytes while holding the membrane potential at -60 mV (Fig. 6D). Under these conditions, substrate transport was not compromised, in spite of a reduction in the transport associated current.
Western analysis
We addressed the question of AeaEAAT localization by generating a peptide
antibody to a variable region of the protein. Affinity purification produced a
specific antibody with high affinity for AeaEAAT, as characterized by ELISA
(data not shown). When used in immunoblots against adult Ae. aegypti
head membrane fractions (25 µg per lane), as well as membrane preparations
of AeaEAAT expressing Xenopus oocytes (9 µg per lane), the
antibody recognized a band at 52.5 kDa
(Fig. 2, lanes 2 and 4). When
membrane fractions isolated from the adult head, thorax, and abdomen regions
were subjected to western analysis (25 µg per lane), a specific signal of
52.5 kDa was evident in all three regions
(Fig. 7, lanes 1, 3, 5), with
the most intense signal in the thorax (Fig.
7, lane 3). These bands could not be detected when peptide
preadsorbed antibody was used to probe the blots
(Fig. 7, lanes 7, 9, 11).
Immunoreactive bands were absent in the cytosolic fractions
(Fig. 7, lanes 2, 4, 6). The
results shown in Fig. 7 are
from adult males: identical results were obtained from adult females (data not
shown).
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Analogous experiments were conducted with larval and pupal membrane fractions to gather information on the developmental expression profile. Interestingly, only low levels of AeaEAAT immunoreactivity could be detected in larval head, thorax and abdomen membranes fractions even when 40 µg of protein was loaded per lane (data not shown). In the pupal stage, AeaEAAT immunoreactivity was negligible when 40 µg of membranes were probed (data not shown).
Immunohistochemistry
Based on the results from western analysis, we focused on the adult thorax
for immunohistochemical studies. Paraffin sections of the adult thorax probed
with the affinity-purified antibody revealed staining of neuropile regions of
the thoracic ganglia (Fig.
8B,C). Immunoreactivity in this region was abolished when the
antibody had been preadsorbed with the antigenic peptide
(Fig. 8A). Staining of fibers
in the neuropile region, as well as cell bodies peripheral to this region,
could be observed (Fig. 8C, arrowheads). The muscles within the thorax, however, did not show
immunoreactivity to this antibody.
|
![]() |
Discussion |
---|
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---|
At the amino acid level, AeaEAAT has greatest identity to its cloned insect
counterparts, especially to DipEAAT, dEAAT1, and TrnEAAT. When compared to the
five human homologues, AeaEAAT shares highest homology with the neuronal
hEAAT3, whose rabbit homologue was originally isolated from the kidney
(Fig. 1B). For all cloned EAATs
thus far, including AeaEAAT, sequence conservation is greatest at the C
terminus, where 5060% identity is observed. Hydropathy analysis gives
proteins with intracellular N and C termini, having six clear transmembrane
segments in the N terminus. The C-terminal transmembrane topology for this
family of proteins has been a point of much debate, as despite the high degree
of sequence conservation in this region, predictions based on hydrophobicity
gave 04 transmembrane segments for different EAATs
(Slotboom et al., 1999). More
recent experimental studies of some mammalian counterparts suggest that the
C-terminal half comprises a re-entrant loop structure with an outward-facing
hydrophobic linker between the loop and final transmembrane domain
(Grunewald et al., 1998
;
Seal and Amara, 1998
;
Seal et al., 2000
).
In humans, apart from the high-affinity glutamate transporters (EAAT1-5),
there are two Na+-dependent neutral amino acid transporters with
approximately 30% identity to EAAT15, possessing the 6-amino-acid
signature sequence AAI/VFIAQ. The two proteins, ASCT1 and ASCT2, have the
transport properties of system ASC, which transport alanine, serine, threonine
and cysteine (Arriza et al.,
1993; Shafqat et al.,
1993
; Utsunomiya-Tate et al.,
1996
). The five EAAT proteins and two ASCT proteins thus form a
carrier superfamily. As with other EAATs, AeaEAAT shares lower identity
(3035%) with ASCT1 and ASCT2.
Analysis of the two completely sequenced insect genomes, namely D.
melanogaster and Anopheles gambiae, show the presence of two and
three EAAT homologues, respectively. The two in D. melanogaster,
dEAAT1 and dEAAT2, have been cloned and shown to functionally transport
glutamate and aspartate, while the three in An. gambiae (GenBank
accession numbers: AAB01008807, AAB01008797 and AAB01008964) have yet to be
cloned (Besson et al., 1999,
2000
;
Kosakai and Yoshino, 2001
;
Seal et al., 1998
). Based on
this information, it is likely that there are at least two genes in mosquitoes
encoding glutamate/aspartate transporters. However, the lower identity of
AeaEAAT (33.5%) with the third homologous EAAT sequence in An.
gambiae (AAB01008964) resembles the level of identity observed with the
mammalian System ASC transporters (approximately 3035%), suggesting
that it is a functional homologue of System ASC transporters rather than
EAATs.
Based on the initial amino acid sequence analysis, as well as the fact that
AeaEAAT was originally isolated from a midgut/Malpighian tubule cDNA library,
we heterologously expressed AeaEAAT in mammalian cells and Xenopus
oocytes to test the hypothesis that it is the functional homologue of hEAAT3.
Heterologously expressed AeaEAAT indeed transports glutamate and aspartate
with high affinity (Table 1),
sharing many of the properties seen with the mammalian EAATs, such as a strict
dependence on extracellular Na+ for function, and ability to
perform reversed transport in the presence of high concentrations of
extracellular K+. A few lines of evidence prove our hypothesis that
AeaEAAT is the functional homologue of hEAAT3. For example, analysis of
AeaEAAT pharmacology excludes it from being the functional homologue of
hEAAT2, as AeaEAAT is insensitive to DHK and KA, a property distinct to hEAAT2
(Table 2). With the exception
of a unique sensitivity to serine-o-sulfate, pharmacological analysis of
AeaEAAT using known blockers of glutamate transport shows its similarity to
hEAAT1 and hEAAT3. Examination of substrate-elicited currents shows AeaEAAT to
be responsive to L-cysteine
(Fig. 5C), an amino acid which
causes neuronal damage in mammals (Olney
and Ho, 1970). Application of L-cysteine produces
transporter-mediated currents specific for mammalian EAAT3, and thus
additionally classifies AeaEAAT as an hEAAT3 orthologue
(Zerangue and Kavanaugh,
1996c
).
Profiles of difference IV curves representing the
substrate-activated transporter currents demonstrate AeaEAAT to be most
similar to hEAAT3 as both reverse between +35 and +40 mV
(Fig. 5C)
(Wadiche et al., 1995a).
Difference IV curves for mammalian EAATs show clear reversal
potentials ranging from +9 to +40 mV for hEAAT13, to -20 mV in the case
of hEAAT4 and 5 (Arriza et al.,
1997
; Fairman et al.,
1995
; Wadiche et al.,
1995a
). Sodium-dependent solute transporters acting as secondary
active carriers are not expected to reverse in polarity when exposed to
extracellular substrate, regardless of the membrane potential, as seen for the
rat GABA transporter (Mager et al.,
1993
). While reversals are not expected of classical secondary
active carriers, they have been observed with transporters mediating
substrate-evoked conductances that are not coupled to transport
(Chaudhry et al., 2001
;
Fairman et al., 1995
;
Wadiche et al., 1995a
). In
particular, for mammalian EAATs the reversal has been attributed to a
stoichiometrically uncoupled substrate-activated anion conductance, which is a
property intrinsic to EAATs comprising (1) an anion leak in the absence of
substrate, and (2) an anion channel-like activity evoked by the substrates of
glutamate transport, but is not required for substrate transport
(Arriza et al., 1997
;
Bergles and Jahr, 1997
;
Fairman et al., 1995
;
Otis and Jahr, 1998
;
Seal et al., 1998
;
Wadiche et al., 1995a
;
Wadiche and Kavanaugh, 1998
).
Although termed `stoichiometrically uncoupled', this channel activity requires
the presence of Na+ and substrate, and is thought to be activated
early in the transport cycle (Otis and
Kavanaugh, 2000
). The necessity for Na+ and substrate
is also apparent in our studies, since absence of both Na+ and
substrate evokes no transporter-mediated current
(Fig. 5D).
While all five human EAATs exhibit a substrate-activated anion conductance,
its contribution to the overall substrate-activated current differs. For
example, more than 95% of the substrate-induced current is carried by
CL- ions for hEAAT4 and hEAAT5 and 5073% for hEAAT1-3
(Arriza et al., 1997). dEAAT1,
the only other insect glutamate transporter studied by two-electrode voltage
clamp, possesses a substrate-activated anion conductance that is responsible
for most of the substrate-induced current. Furthermore, in the absence of
intracellular and extracellular CL-, both substrate transport and
substrate-induced currents by dEAAT1 are completely abolished
(Seal et al., 1998
).
Difference IV curves upon anion substitution show
AeaEAAT to elicit an analogous substrate-activated anion conductance, although
the effect of CL- on AeaEAAT is not as dramatic as for dEAAT1 or
hEAAT4 and 5. Replacement of extracellular CL- with gluconate, a
more bulky anion, reduces the amplitude of both inward and outward currents,
as for dEAAT1 (Fig. 6A,B),
whereas substitution with more permeant anions such as
NO3- and SCN- increases the outward currents
and shifts the reversal potential to values more negative than
ECl, as seen for mammalian EAATs
(Wadiche et al., 1995a;
Zerangue and Kavanaugh,
1996a
). The gluconate-mediated effects can be explained by
employing explanations similar to those used for dEAAT1, where (1) the reduced
outward current results from a reduction in inward anion permeation, and (2)
the reduced inward current results from a reduced outward flow of
CL- due to a change in the chemical gradient
(Seal et al., 1998
). On the
other hand, effects of NO3- and SCN- are due
to increased permeation of these anions through AeaEAAT. Removal of both
extracellular and intracellular CL- by dialysis with gluconate
abolishes the reversal of the substrate-elicited current, while retaining an
inward current (Fig. 6C),
reflecting the coupled transport of substrate, Na+, H+
and countertransport of K+. We conclude that the anion channel
activity is not stoichiometrically coupled to substrate transport, since
radiolabeled substrate transport under voltage clamp is not compromised
regardless of the presence of CL-
(Fig. 6D).
For the mammalian EAATs, the anion channel activity was recently separated
from transport activity on a molecular level, where sulfhydryl modification of
mutated residues V449C or V452C in hEAAT1 abolished substrate transport but
not the anion conductance (Ryan and
Vandenberg, 2002; Seal et al.,
2001
). These amino acids are conserved in AeaEAAT (V398 and V402)
and could thus provide the basis for its ability to also exhibit an anion
conductance. In summary, studies on the anion channel properties of AeaEAAT
demonstrate its similarity to hEAAT1, 2 and 3, rather than hEAAT4 or hEAAT5.
Coupled with the sequence comparisons and pharmacological studies discussed
previously, these data strengthen the hypothesis that this protein is a
functional homologue of hEAAT3.
Competition studies for D-aspartate transport by the 20 amino acids revealed statistically significant inhibition by L-glutamine and L-asparagine, amidated forms of the excitatory amino acid substrates (Fig. 4). While preliminary studies in our laboratory implicate the ability of AeaEAAT to transport these amidated amino acids (data not shown), these results, as well as the L-glutamine and L-asparagine inhibition noted here, may be an artifact of trace contamination of L-glutamate and L-aspartate in the commercially available amino acids.
The final question addressed in this study is the localization of AeaEAAT,
as it is still unclear whether insect EAATs participate in signal termination
at invertebrate neuromuscular junctions. Only two recent reports have
addressed this issue, where Soustelle et al.
(2002) examined dEAAT1 and
dEAAT2 expression via in situ hybridization, and Gardiner et al.
(2002
) studied TrnEAAT
localization via immunohistochemistry. Results from both groups
demonstrate the respective EAATs to be glial rather than neuronal. Gardiner et
al. (2002
) provided additional
evidence for TrnEAAT localization in glia at both neuromuscular junctions and
in neuropile regions of larval T. ni. The glial localization of these
insect transporters illuminates the importance of glia in regulation of
extracellular glutamate concentrations. Indeed, in humans it is believed that
the glial transporters (hEAAT1 and hEAAT2) principally contribute to glutamate
clearance in the brain, thereby preventing glutamate induced excitotoxicity.
This has been demonstrated by EAAT knockout mice, in which mice lacking glial
EAATs develop neurodegeneration and paralysis that are symptomatic of
glutamate excitotoxicity (Rothstein et
al., 1996
; Tanaka et al.,
1997
).
Initial experiments to localize AeaEAAT by western analysis showed the
greatest immunoreactivity in membrane fractions of the adult thorax
(Fig. 8). Given the intensity
of the immunoreactive band in thorax membranes compared with the head and
abdomen membrane fractions, we initially hypothesized AeaEAAT to be present in
adult thorax muscles, since the adult thorax is rich in muscles involved in
flight and leg movement. However, immunohistochemistry results from the adult
thorax showed specific staining in the neuropile regions of the thoracic
ganglia but not in muscles (Fig.
8). The present data do not discriminate between the neuronal or
glial localization of AeaEAAT, an issue which is currently being pursued.
Nonetheless the results do suggest that AeaEAAT is not involved in termination
of glutamatergic signals at the adult mosquito neuromuscular junction. Yet,
based on the findings of Gardiner et al.
(2002) that insect EAATs are
indeed localized in muscles, as well as our hypothesis that a second
functional EAAT exists in the mosquito genome (as discussed earlier), we
postulate that the second mosquito EAAT would be responsible for termination
of glutamatergic signals at the mosquito neuromuscular junction.
It is interesting to note that western analysis of analogous membrane
fractions from fourth-instar larvae and day-2 pupae gave virtually negligible
immunoreactivity, implying an increase of the AeaEAAT signal with development.
Indeed, this is the case for dEAAT1, where the authors have noted that the
onset of expression occurs in larval stages rather than in embryonic stages
(Soustelle et al., 2002).
Tissue-specific RTPCR performed to localize AeaEAAT gave inconclusive
results, as PCR products from genomic and cDNA gave identical sizes,
indicating a lack of intronic sequences (data not shown).
In spite of the fact that this clone was originally isolated from an EST library of the adult female midgut/Malpighian tubule library, whole-mount immunolocalization studies using these tissues did not yield any positive results. We believe that this can be attributed to one of the following: (1) the present antibody is not capable of detecting AeaEAAT in the whole-mount preparations used, (2) the transcript, and therefore the protein is expressed below the limits of detection of immunocytochemical procedures, or (3) the clone was isolated from contaminating nerve or muscle tissue surrounding the midgut and Malpighian tubule tissues used to create the cDNA library.
The results presented in this study provide an insight to the role played
by excitatory amino acid transporters in the mosquito, Aedes aegypti.
Mosquitoes have historically been and still are the cause of many human
diseases worldwide, including malaria, dengue, lymphtic filariasis, yellow
fever, Japanese encephalitis and West Nile virus
(Roberts, 2002). Together,
there are over 500 million cases a year of mosquito-borne illnesses. Amidst
the heightened interest in mosquito biology, the work presented is a timely
contribution to the further understanding of this disease vector.
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Acknowledgments |
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Footnotes |
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