From the Department of Pharmacology, University of Sydney, New
South Wales 2006, Australia, and the Howard Hughes
Medical Institute, Vollum Institute of Advanced Biomedical Research,
Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S,4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.
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INTRODUCTION |
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L-Glutamate transport in the central nervous system and also in peripheral organs is mediated by a family of structurally related membrane proteins. In the central nervous system, glutamate transporters play an important role by mediating removal of glutamate released at excitatory synapses and also influencing the kinetics of glutamate receptor activation (1, 2). Complementary DNAs encoding five different glutamate transporters have been identified (3-7). The human clones have been named Excitatory Amino Acid Transporters 1-5 (EAAT1-5) (6-8). The recent cloning and expression of recombinant glutamate transporter proteins in heterologous expression systems allow a number of questions concerning the molecular basis for transporter function to be addressed. In particular, an important aspect of such studies is the identification of domains involved in substrate recognition and substrate translocation through the membrane. In this study we have constructed a series of chimeric glutamate transporters, using the EAAT1 and EAAT2 subtypes, and exploited the pharmacological and electrophysiological differences between the two subtypes to map functional domains of these proteins.
To make meaningful predictions about the functional role of various
domains, an understanding of the structure of the transporters is
particularly useful. The predicted amino acid sequences of EAAT1 and
EAAT2 are 65% identical, and if conservative substitutions are allowed
the degree of relatedness is increased to 80% (8). These two proteins
are likely to form very similar structures and therefore are good
candidates for the generation of functional chimeras. Through the use
of hydrophobicity analysis of the amino acid sequences it has been
predicted that there are six -helical transmembrane segments in the
amino-terminal portion of the protein. Tagging amino acid residues
between the various transmembrane segments within this region and
identifying whether the tags are located intracellularly or
extracellularly have confirmed the predictions from the hydrophobicity
analysis (9, 10). Hydrophobicity analysis of the remaining portion of
the proteins has been inconclusive. This region is the most highly
conserved part of the protein and has been suggested to form part of
the pore of the transporter through which glutamate and various ions
pass in the transport process (11). This suggestion has been supported
by evidence from point mutation studies (11-13). Therefore, it is
highly likely that at least some of this part of the protein is closely
associated with the membrane. In two recent studies, molecular tags
have been introduced into the transporters, and the intracellular and extracellular locations of the tags have been used to predict the
topology of this region (10, 14). Two very different models for the
transmembrane topology of the transporters have been predicted from the
two studies. In both models, four transmembrane domains have been
predicted, but in the model suggested by Slotboom and co-workers (14),
the four transmembrane segments are
-helical, whereas in the model
suggested by Wahle and Stoffel (10), the four transmembrane segments
are
-sheets. In most membrane proteins the most highly conserved
domains are located within the transmembrane regions. Although other
topological models are also
possible,1 for the purposes
of this study, we tentatively favor the
first model, consisting of 10
-helical transmembrane segments,
because this model places a larger proportion of the highly conserved domain within the membrane. However, further work is clearly required to resolve this issue.
Despite the high degree of amino acid sequence conservation between EAAT1 and EAAT2, the transporter subtypes show quite distinct pharmacological differences. The most striking pharmacological difference between the transporter subtypes is the selective sensitivity of the EAAT2 subtype to competitive inhibition or blockade by dihydrokainate, kainate, and threo-3-methylglutamate (8, 15). These compounds block glutamate transport by EAAT2 in a voltage-independent manner, suggesting that they block transport by competing with glutamate at an extracellular recognition site. Another compound, (2S,4R)-4-methylglutamate, shows some very distinctive differences in its mechanism of action on the transporters. (2S,4R)-4-Methylglutamate is a voltage-independent, competitive blocker of glutamate transport by EAAT2, but it is also a potent substrate of the EAAT1 transporter subtype. As such these compounds provide a set of selective molecular probes for the extracellular glutamate recognition site of the transporters.
In contrast to the competitive blockers of glutamate transporters, a number of other pharmacological agents have been shown to inhibit glutamate transport by competing with glutamate as a substrate for the transporters (8). One pharmacological substrate that shows some clear differences in transport parameters between EAAT1 and EAAT2 is L-serine-O-sulfate. L-Serine-O-sulfate is a substrate for both EAAT1 and EAAT2, but it is a less potent substrate on EAAT2 than EAAT1. In addition, the EC502 values for L-serine-O-sulfate transport by EAAT2 are steeply voltage-dependent but relatively constant for transport by EAAT1 (16). This difference between EAAT1 and EAAT2 in L-serine-O-sulfate transport is likely to reflect a difference in the structure of the pore of the two transporters. Thus, L-serine-O-sulfate may be used as a molecular probe for the pore of the transporters.
In addition to the pharmacological differences, the different transporter subtypes allow a varying degree of a thermodynamically uncoupled chloride flux through the transporter (6, 7, 17-19). Different transporter substrates also appear to be coupled differentially to the activation of chloride fluxes (6, 17, 19). In particular, the chloride conductance activated by D-aspartate transport by EAAT1 is significantly greater than the chloride conductance activated by D-aspartate transport by EAAT2 (17). These differences in substrate-activated chloride conductance between transporter subtypes provide another independent molecular probe for identifying the domain responsible for forming the pore of the transporters.
We have constructed a series of chimeric glutamate transporters with the aim of investigating the importance of transmembrane domains 7-10 in determining functional differences between EAAT1 and EAAT2. The chimeric glutamate transporters were characterized in terms of electrogenic glutamate transport; sensitivity to the transport blockers kainate, threo-3-methylglutamate, and (2S,4R)-4-methylglutamate; L-serine- O-sulfate transport; and chloride permeability.
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EXPERIMENTAL PROCEDURES |
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Chemicals-- L-Glutamate (Na+ salt), D-aspartic acid, L-serine-O-sulfate (K+ salt), kainic acid, and HEPES were obtained from Sigma, Sydney, Australia. (2S,4R)-4-Methylglutamate and threo-3-methylglutamate were obtained from Tocris Cookson, Bristol, U. K. All other chemicals used were of analytical grade.
Construction of Chimeric Transporters--
The chimeric
glutamate transporters were constructed using cDNAs encoding the
EAAT1 and EAAT2 transporter subtypes. Two procedures were used to
construct the various chimeras. First, an in vivo recombination procedure was used to construct the chimeras based on the
method described by Buck and Amara (20). EAAT1 and EAAT2 cDNAs were
cloned in tandem into the pCMV plasmid (21) with a unique
XbaI site between the cDNAs. The tandem construct was linearized with XbaI, and 100 ng of DNA was used to
transform competent DH5 cells. Colonies were screened for
recombinant plasmids that contained only a single cDNA and
sequenced to determine the junction sites (Sequenase, U. S.
Biochemical Corp.). A number of chimeras were isolated, and all of them
had junction sites within the most highly conserved domain from serine
366 to glutamine 415 (EAAT1 numbering). One of these chimeras, termed
EAAT1-EAAT2-Chimera-7 (E12C7), contained the EAAT1 sequence at the amino terminus and junctions with EAAT2 at leucine 404. The chimera was subcloned into
pOTV for synthesis of cRNA and expression in Xenopus
oocytes. In all of the remaining chimeras unique restriction sites were engineered into the cDNAs, using the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and then the cDNA fragments of one of the transporters were cut out of the plasmid and
ligated into corresponding sites of the other transporter cDNA.
More than 20 different chimeric glutamate transporters were constructed
in this way, but only the chimeras in Fig. 1 were functional in terms
of supporting electrogenic glutamate transport. All of the functional
chimeras were sequenced using a Perkin-Elmer 377 sequencer with the
Catalyst 800 robotic work station at the Sydney University and Prince
Alfred Macromolecular Analysis Center. Selected nonfunctional chimeras
were also sequenced to check that polymerase chain reaction-generated
errors were not introduced into the chimeric cDNAs. The functional
chimeras are named according to the orientation of cDNA fragments,
with the numbering referring to the transmembrane closest to the
junction site. For example, E12C7 contains EAAT1 sequence from the
amino terminus to transmembrane domain 7 and then the EAAT2 sequence to
the carboxyl terminus. E121C7-8 contains the EAAT1 sequence from the
amino terminus to transmembrane domain 7 followed by the EAAT2 sequence
to the end of transmembrane 8 and then EAAT1 sequence to the
carboxyl-terminal end.
Electrophysiological Recording--
Xenopus laevis
were obtained from the African Xenopus Facility, Republic of
South Africa. A lobe of the ovary was surgically removed while the frog
was anesthetized with 3-aminobenzoic acid ethyl ester methanesulfonate
salt (Sigma), and stage V-VI oocytes were isolated after collagenase
treatment. 10-50 ng of cRNAs encoding the various glutamate
transporters were injected into oocytes. 2-7 days later transport was
measured by two-electrode voltage clamp recording using a Geneclamp 500 amplifier (Axon Instruments) and a MacLab 2e recorder (ADInstruments).
In transport experiments the membrane potential of the oocytes
expressing the given transporter was clamped at 60 mV and superfused
continuously with frog ringer buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.55).
When measuring dose responses for various substrates, this buffer was
changed to one containing the indicated concentration of substrate.
Voltage-dependent L-glutamate or
D-aspartate transport currents were measured by clamping
the membrane at various potentials between
60 and +60 mV and applying
the substrate to the bath solution.
Analysis of Kinetic Data--
Current (I) as a function of
substrate concentration ([S]) was fitted by least squares to I = Imax·[S]/(EC50 + [S]), where Imax is the maximal current, and EC50 is the
concentration of substrate which generates half-maximal current. The
Imax values for L-serine- O-sulfate
and, where appropriate,
(2S,4R)-4-methylglutamate, are expressed relative
to a maximal dose of L-glutamate in the same cell. A
selectivity ratio, EC50R, represents the EC50
value for each substrate divided by the EC50 for glutamate
for EAAT1, EAAT2, and each chimeric transporter. Imax and
EC50 values are expressed as mean ± S.E. and were
determined by fitting data from individual oocytes. Current (I) as a
function of blocker concentration ([B]) was fitted by least squares
to I = Imax (Imax·[B]/(IC50 + [B])), where
Imax is the current generated by 30 µM
glutamate, and IC50 is the concentration of the blocker
that causes a 50% reduction in the glutamate transport current. The
transport blockers, (2S,4R)-4-methylglutamate,
threo-3-methylglutamate, and kainate, have all been shown to
be competitive blockers of glutamate transport by EAAT2 using Schild
analysis (8, 15, 22), allowing the calculation of inhibition constants
(Ki) from IC50 values using the equation
Ki = IC50/(1 + [Glu]/EC50), where [Glu] = 30 µM, and
EC50 for glutamate was calculated for each transporter as
described above. Ki values are expressed as
mean ± S.E. and were determined by fitting data from individual oocytes.
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RESULTS |
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Generation of Functional Chimeric Transporters-- A number of chimeric glutamate transporters (>20) were constructed using the EAAT1 and EAAT2 subtypes, but only the five chimeric transporters depicted in Fig. 1 showed high affinity electrogenic glutamate transport in oocytes expressing the corresponding cDNAs. The EC50 values for glutamate transport by EAAT1 and EAAT2 are 14.7 ± 2.9 µM and 23.2 ± 1.2 µM, respectively, and the corresponding values for the five functional chimeric transporters fall within the range 3.2-37.2 µM (Fig. 2 and Table I).
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Characterization of Chimeric Transporters-- The wild-type and chimeric glutamate transporters have been characterized in terms of potency of L-serine-O-sulfate as a substrate, chloride permeability, sensitivity to the transport blockers kainate and threo-3-methylglutamate, and whether (2S,4R)-4-methylglutamate is a substrate or a blocker of transport. In each of these functional characteristics there are clear differences between EAAT1 and EAAT2 (Figs. 2-4 and Tables I-III).
Functional Domain Conferring Substrate Selectivity-- The EC50 values for glutamate did not differ more than by a factor of 2 for the chimeras, with the exception of E121C7-8, EC50 3.2 µM. To allow meaningful interpretation of EC50 data a selectivity ratio has been included for each chimera. This ratio represents the EC50 value for each substrate divided by the EC50 for glutamate for each chimera, allowing the comparison with wild-type transporters with standardization for the EC50 differences observed.
Wild-type transporters EAAT1 and EAAT2 display a 9-fold difference in L-serine- O-sulfate transport parameters (EAAT1; EC50 18.4 ± 3.4, EC50R 1.3; EAAT2, EC50 174 ± 20, EC50R 7.6) (Fig. 2 and Table I). Differences in maximal current are also displayed between EAAT1, which attains the same Imax as glutamate, and EAAT2, which attains 70% of the Imax observed for glutamate. The five chimeras transport L-serine-O-sulfate within the EC50 range 8.4-213 µM. Chimeric transporters that displayed transport parameters more aligned with EAAT1 were E21C8 (EC50 18.7 ± 2.2, Imax 0.94 ± 0.01, EC50R 1.4), E12C10 (EC50 27.9 ± 7.1, Imax 1.07 ± 0.03, EC50R 1.0), and E121C7-8 (8.4 ± 1.5, Imax 1.3 ± 0.03, EC50R 2.6) (Fig. 2 and Table I). EAAT2 transport parameters were represented in chimeric transporters E21C10 (EC50 178 ± 26, Imax 0.74 ± 0.03, EC50R 10.5) and E12C7 (EC50 213 ± 8, Imax 0.62 ± 0.01, EC50R 5.7) (Fig. 2 and Table I). The chimeric transporters define a functional domain (region 3) that influences the substrate selectivity of L-serine-O-sulfate observed with EAAT1 and EAAT2. Chimera E21C8, which contains EAAT1 sequence in region 3 and 4, has EAAT1-like L-serine-O-sulfate selectivity. Chimera E12C10 showed EAAT1-like L-serine-O-sulfate selectivity, and the reverse chimera E21C10 had EAAT2-like L-serine-O-sulfate selectivity, implying that the intracellular region 4 does not confer L-serine-O-sulfate selectivity. L-Serine-O-sulfate transport by chimera E12C7, which contains EAAT2 sequence in regions 2, 3, and 4, displayed EAAT2 selectivity, whereas E121C7-8, which contains EAAT1 sequence in regions 3 and 4, displayed EAAT1 selectivity, confirming the importance of region 3. In summary, these results suggest that region 3, which includes the 9th and 10th transmembrane domains, is a major determinant of differences in substrate selectivity between EAAT1 and EAAT2.Functional Domain Determining Differences in Chloride Permeability-- Chloride permeation of both wild-type and chimeric transporters was estimated by measuring voltage-dependent transport currents of D-aspartate and L-glutamate. Differences in chloride permeation between EAAT1 and EAAT2 are most obvious for D-aspartate transport (17) (Fig. 3 and Table II), and the membrane potential at which D-aspartate transport currents reverse direction may be used as an indicator of relative chloride permeability. The chimeric transporters for the most part clearly resembled either EAAT1 or EAAT2. Chimeric transporters E21C8 (Erev D-aspartate +7.1 ± 1.5; Erev L-glutamate > +50 mV) and E12C10 (Erev D-aspartate +2.3 ± 1.2; Erev L-glutamate +37.0 ± 3.8 mV) both have reversal potentials that are similar to EAAT1 (Fig. 3 and Table II). Chimera E12C7 (Erev D-aspartate > +50 mV; Erev L-glutamate > +50 mV) did not show a reversal of current in the membrane potential range up to > +50 mV, indicative of EAAT2-like chloride permeability (Fig. 3 and Table II). Chimera E21C10 (Erev D-aspartate +23.9 ± 2.5 mV; Erev L-glutamate > +50 mV) did not obviously correspond to either EAAT1 or EAAT2 classification (Fig. 3 and Table II).
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Domain Conferring Blocker Sensitivity-- A defining feature of the differences between EAAT1 and EAAT2 transporters is the selective sensitivity of EAAT2 to the transport blockers kainate and threo-3-methylglutamate. EAAT2 glutamate transport currents are inhibited selectively by kainate (Ki 17.9 ± 2.2 µM) and threo-3-methylglutamate (Ki 23.2 ± 2.7 µM), whereas EAAT1 is relatively insensitive (Fig. 4 and Table III). The chimeric transporter that clearly displayed blocker sensitivity was E21C10 (kainate Ki 15.5 ± 0.6 µM; threo-3-methylglutamate Ki 20.5 ± 1.8 µM).
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Functional Characteristics Defined by (2S,4R)-4-Methylglutamate-- (2S,4R)-4-Methylglutamate has proven to be a useful tool in discerning differences between EAAT1 and EAAT2 transporter subtypes. The chimeric transporters E21C8, E12C10, and E12C7, which are insensitive to kainate and threo-3-methylglutamate, transport (2S,4R)-4-methylglutamate (Fig. 4 and Table III). Chimera E21C10, which displayed sensitivity to kainate/threo-3-methylglutamate blockade, also displayed (2S,4R)-4-methylglutamate blocker sensitivity (Fig. 4 and Table III). Thus, the molecular determinants of whether (2S,4R)-4-methylglutamate is a blocker or substrate for the most part coincide with the determinants for kainate and threo-3-methylglutamate sensitivity.
Chimera E121C7-8, which displays a kainate/threo-3-methylglutamate-sensitive leak current, allows (2S,4R)-4-methylglutamate to be transported (EC50 1.5 ± 0.02 µM, Imax 0.49 ± 0.02, EC50R 0.5) (Fig. 4 and Table III). The EC50 for transport of (2S,4R)-4-methylglutamate for E121C7-8 is lower than that of EAAT1 EC50 16.8 ± 1.9 µM, but this may be a reflection of the lower EC50 for glutamate for E121C7-8 (EC50 3.2 ± 0.4 µM) (Fig. 4 and Table III). The importance of region 3 playing a role in determining substrate versus blocker functionality of (2S,4R)-4-methylglutamate is highlighted by chimeras E21C8, E12C10, and E21C10. Chimera E12C7, on the other hand, supports the suggestion that other regions are also required for the blockade of glutamate transport functionality. ![]() |
DISCUSSION |
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Various methods for identifying functional domains of glutamate transporters are possible. We have used the chimeric protein approach because it has a number of advantages over other methods such as site-directed point mutations. The principal advantage is that only fully functional proteins that show characteristics of one or both the parental transporters are used in the analysis. As such the conclusions are based on positive results rather than alteration of function or in some instances loss of function associated with the generation of site-directed mutations. One limitation of this approach is that only information concerning domains that confer functional differences will be obtained. These domains may not necessarily coincide with a region that forms the glutamate recognition site.
Although more than 20 different chimeric glutamate transporters were constructed, electrogenic glutamate transport was detected only in the five chimeras depicted in Fig. 1. This suggests that the formation of functional transporters requires a number of specific interactions among amino acid residues from multiple regions. Subtle differences in these specific interactions between EAAT1 and EAAT2 may explain the functional differences between these two glutamate transporters. In the formation of the chimeric transporters, some of these interactions may be altered, leading to loss of function.
The utility of the chimera approach is based on both the degree of the structural relatedness and the functional differences between the two proteins being studied. This study presents structure-function analyses of chimeric constructs of two subtypes of glutamate transporters, EAAT1 and EAAT2. These two glutamate transporters were chosen because they display a high degree of homology, 65%, and the greatest degree of difference in substrate selectivity, blocker sensitivity, and chloride permeability among transporter subtypes while transporting glutamate with a similar EC50 (8, 15). The pharmacological and electrophysiological differences were exploited to define domains of the transporters which confer particular characteristics.
Three different molecular probes were employed to characterize EAAT1, EAAT2, and chimeric transporters. The first molecular probe, L-serine-O-sulfate, is a substrate for transporters which displays a difference in affinity (8, 16, 23). Chloride permeability, the second molecular probe, is markedly different between EAAT1 transport of D-aspartate and EAAT2 transport of D-aspartate (17) and was employed to delineate further the differences in EAAT1 and EAAT2 related to the pore of the transporter. Kainate (8), threo-3-methylglutamate, and (2S,4R)-4-methylglutamate (15) provide probes for an extracellular glutamate recognition site on EAAT2. (2S,4R)-4-Methylglutamate provided added information in that it is transported by EAAT1 (15).
The chimeric transporters define a domain including transmembrane regions 9 and 10, which is of primary importance in determining functional differences between EAAT1 and EAAT2. This region consists of 57 amino acids of which 17 are not conserved between EAAT1 and EAAT2. Three of the 17 amino acids have been allocated to extracellular domains, 10 are located in the two transmembrane domains, and 4 are positioned intracellularly according to the model by Slotboom and colleagues (14). This particular region is highly conserved among the five different human glutamate transporters. In this region EAAT1 has 68% identity with EAAT2, 78% identity with EAAT3, 92% identity with EAAT4, and 81% with EAAT5. It is generally accepted that highly conserved amino acid sequences form functionally important structures, and it may be predicted that critical changes in amino acid sequence in such a region will alter the functional properties of the protein. One would predict that one or more of the nonconserved 17 amino acids in transmembrane domains 9 and 10 contribute to differences in L-serine-O-sulfate substrate selectivity, differences in chloride permeability, and differences in blocker sensitivity between EAAT1 and EAAT2.
Although this region is clearly involved in determining functional differences, other regions must also be involved. A recent report, using chimeras generated from GLT-1 (mouse equivalent of EAAT2) and EAAC1 (mouse equivalent of EAAT3) has suggested that an intracellular region between transmembrane domain 6 and transmembrane domain 7, from amino acid residues 301 to 338 of EAAC1, contributes to the formation of a dihydrokainate recognition site (25). The results of Kanai and co-workers (25) are consistent with the observation in this report, that other regions in addition to region 3 contribute to the formation of the kainate recognition site. These results pose an interesting conundrum as to how an intracellular site and two transmembrane domains form an extracellular recognition site for kainate or dihydrokainate. Two possibilities may be considered. First, the topological model is incorrect, and the 38-amino acid residue domain identified by Kanai et al. (25) is not intracellular but within the membrane or extracellular. The second possibility is that the intracellular segment may associate with transmembrane domains 9 and 10 to alter the conformation of an extracellular site to that kainate or dihydrokainate binds. Clearly, finer resolution of the topological model is required before these alternate possibilities may be resolved.
The intracellular carboxyl-terminal domain may also contribute to differences between EAAT1 and EAAT2, defined by region 4. The intracellular carboxyl terminus has very low sequence homology between the two transporter subtypes, and predictions of which amino acid residues are conferring these minor effects is difficult. The intracellular carboxyl terminus appears to confer functional differences to EAAT1 and EAAT2 in conjunction with other regions. The effects on chloride permeability may result from an association of the intracellular carboxyl terminus with other intracellular domains, which in turn influence the conformation of the pore of the transporter.
It has been suggested that the highly conserved region from amino acid residues 383-415 (EAAT1 numbering) forms part of the pore of the transporter, based on point mutations (11, 13) and cysteine scanning mutagenesis.1 Mutation of glutamate 404 in transmembrane domain 7 of GLT-1 (rat equivalent to EAAT2) to aspartate disrupts glutamate transport without altering glutamate binding (13). The mutation of glutamate 404 to aspartate changes substrate selectivity and prevents both forward and reverse transport of potassium ions, whereas sodium transport and chloride ion fluxes are unaffected (11, 13). These results suggest that glutamate 404 does not disrupt substrate recognition but alters the permeation pathway and ion coupling of the transporter (11, 13). Thus, Kavanaugh and co-workers (11) concluded that this residue is closely associated with the pore of the transporter.
The importance of transmembrane domains 9 and 10 in determining differences in L-serine-O-sulfate substrate selectivity and chloride permeability has been identified in this study. This result implies either that transmembrane domains 9 and 10 form part of the pore region of the transporter or that they have an interactive role with the pore region, influencing how substrates and chloride permeate the pore of the transporter. Hence, an interaction between transmembrane domains 9 and 10 and the highly conserved region which includes transmembrane domain 7 may form the basis of the differences observed in transport parameters between EAAT1 and EAAT2.
In this study we have identified an important region, transmembrane domains 9 and 10, which plays a role in defining functional differences in substrate selectivity, chloride permeability, and blocker sensitivity between EAAT1 and EAAT2. Other regions are also involved in defining the blocker sensitivity of EAAT2. A minor role is identified for the intracellular carboxyl-terminal region conferring an effect on chloride permeability. Further point mutations in these regions will assist in defining better the molecular basis for substrate recognition, substrate and ion translocation through the transporter, and release of substrates at the intracellular surface. As the topology of the excitatory amino acid transporters becomes more conclusively defined, the regions identified in this study will allow more directed predictions of the amino acid residues responsible for the differences in the transport process of EAAT1 and EAAT2.
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ACKNOWLEDGEMENTS |
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We thank Suzanne Habjan and Kong Li for expert assistance and Michael Kavanaugh and Jill Maddison for helpful comments.
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FOOTNOTES |
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* This work was supported by the Australian National Health and Medical Research Council, the Ramaciotti Foundation, and the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 61-2-9351-6734; Fax: 61-2-9351-3868; E-mail:robv{at}pharmacol.usyd.edu.au.
1 B. Seal and S. G. Amara, unpublished observations.
2 The abbreviations used are: EC50, concentration of substrate which generates half-maximal current; I, current; S, substrate; EC50R, selectivity ratio; B, blocker; IC50, concentration of blocker which causes a 50% reduction in glutamate transport current; Erev, reversal potential.
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REFERENCES |
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