(Received for publication, March 22, 1995; and in revised form, June 1, 1995)
From the
Application of L-glutamate activates ionic currents in
voltage-clamped Xenopus oocytes expressing cloned human
excitatory amino acid transporters (EAATs). However, even in the
absence of L-glutamate, the membrane conductance of oocytes
expressing EAAT1 was significantly increased relative to oocytes
expressing EAAT2 or control oocytes. Whereas transport mediated by
EAAT2 is blocked by the non-transported competitive glutamate analog
kainate (K Transport of L-glutamate in the central nervous system
and periphery is mediated by a family of membrane proteins postulated
to contain from 6 to 10 transmembrane domains (for review, see (1) ). An important goal in understanding the molecular basis
of transporter function is the identification of the structural domains
involved in substrate recognition and in the formation of the pore or
conduction pathway through which amino acids and other ions pass.
Glutamate uptake is accompanied by an influx of sodium ions and an
efflux of potassium and hydroxyl
ions(2, 3, 4, 5) . Voltage clamp
recording from cells with sufficiently high transporter density can be
used to measure membrane currents associated with glutamate uptake. A
stoichiometry proposed for uptake involves co-transport of
2Na In addition to
differences in ion conduction properties, pharmacological differences
exist between the human excitatory amino acid transporter subtypes.
Many compounds that inhibit radiolabeled glutamate transport (e.g. serine O-sulfate, L-trans-pyrrolidine-2,4-dicarboxylic acid, L-cysteic acid, and D-aspartate) also induce inward
currents, suggesting that they act as competitive substrates for
transport(9) . In contrast, kainate and dihydrokainate, two
conformationally restricted glutamate analogues(10) ,
selectively and competitively block transport of glutamate by the
EAAT2 Studies on various cloned sodium-dependent transporters have
demonstrated the presence of substrate-independent currents (11, 12, 13, 14) , suggesting that
these carriers possess intrinsic pores through which ions pass (for
review see (15) ). Identification of differences in pore
conduction properties between the transporter subtypes will facilitate
identification of residues involved in forming the pore structure. In
this study we demonstrate that EAAT1, but not EAAT2, allows
substrate-independent ion fluxes. We have constructed a chimeric
transporter that mediates a glutamate-independent conductance blocked
by kainate, thereby identifying a domain involved in substrate binding
and allowing analysis of the voltage dependence and ionic selectivity
of this substrate-independent current.
Figure 1:
EAAT1 mediates an
uncoupled ion flux. A, current-voltage plots of oocytes 4 days
following injection of equal amounts of cRNA encoding EAAT1 (opencircles), EAAT2 (opensquares), or
uninjected controls (filledcircles). Data points
represent mean ± S.E. (n = 6). B,
voltage dependence of glutamate-independent current in oocytes
expressing EAAT1 obtained by subtraction of the mean current-voltage
curve of uninjected oocytes from that of oocytes expressing EAAT1. C, the amplitude of the uncoupled current in individual
oocytes between 1 and 5 days following injection of EAAT1 cRNA
(measured as the difference in chord conductance compared with matched
control oocytes) is proportional to the level of expression of the
transporter (measured as the amplitude of the current elicited by 300
µML-glutamate at -100 mV) in the same
oocytes.
Figure 2:
A, schematic diagram of the
chimeric EAAT1/EAAT2 transporter E1-2-1. Filledareas correspond to the EAAT1 cDNA sequence, and the openarea corresponds to the EAAT2-derived domain
inserted into EAAT1 (see ``Experimental Procedures''). Linesabove represent major hydrophobic domains
identified using the Goldman-Engelman-Steitz algorithm (MacVector). B, predicted amino acid sequences of EAAT1 and EAAT2
corresponding to the substituted domain and junction region (numbers correspond to residues in EAAT1). Dashes reflect identical residues between EAAT1 and
EAAT2.
Figure 3:
Substitution of a block of residues from
EAAT2 into EAAT1 confers kainate sensitivity to the chimeric
transporter E1-2-1. Application of kainate alone does not induce
currents in either EAAT1 (top) or EAAT2 (middle),
while co-application of kainate with glutamate blocks the transport
current mediated by EAAT2 but not EAAT1. Application of kainate alone
or co-applied with glutamate resulted in an outward current in cells
expressing E1-2-1 as a result of blockade of a constitutive
uncoupled current in addition to block of the glutamate-induced current
(see text). Oocytes were voltage clamped at -60 mV and superfused
with compounds for duration indicated by the bars.
Figure 4:
A, voltage dependence of L-glutamate-independent (uncoupled) and L-glutamate-dependent currents mediated by the chimeric
E1-2-1 transporter. The glutamate-induced current in a
representative oocyte was obtained by subtraction of the
current-voltage curve in the absence of L-glutamate from that
in the presence of 300 µML-glutamate; the
uncoupled current (in the same cell) was obtained by subtraction of the
current-voltage curve in the presence of 5 mM kainate from
that in the absence of kainate. B, the kainate-sensitive leak
conductance is carried by Na
The nature
of the uncoupled current through the chimeric transporter was examined
by ion substitution experiments. Partial replacement of
Na Substitution of
Cl Comparison of the membrane conductance of control oocytes
with oocytes expressing EAAT1 and EAAT2 suggests that EAAT1, but not
EAAT2, mediates an ionic flux in the absence of glutamate. This
uncoupled flux is likely to be mediated directly by the transporter
since its amplitude is proportional to the level of transporter
expression as measured by the glutamate-induced current. Using cDNAs
encoding the human EAAT1 and EAAT2 subtypes, a chimeric glutamate
transporter was constructed that is sensitive to the EAAT2-selective
blocker kainate. The chimeric transporter is composed predominantly of
EAAT1, with the EAAT2 sequence substituted between the seventh and
ninth hydrophobic domains (residues 366-441; Fig. 2). This
transposed domain encompasses a highly conserved region among members
of the glutamate transporter family. For example, the motif FIAQ
(residues 415-418) is conserved among the rat homologs of EAAT1
(GLAST(18) ) and EAAT2 (GLT-1(19) ), the rabbit homolog
of EAAT3 (EAAC1(20) ), and the Escherichia coli glutamate transporter gltP(21) . Within the chimeric
sequence derived from EAAT2, there are 18 residues that differ from the
corresponding residues in EAAT1. Substitution of this discrete domain
conferred a >50-fold increase in affinity for kainate. However,
because the affinity of kainate is still 7-8-fold lower than that
observed for EAAT2, it is likely that additional amino acid residues
influence kainate sensitivity. The study of further chimeras in
conjunction with site-directed mutagenesis will be required to
determine the precise role of these and other residues in the
interaction of kainate with the transporter. The kainate sensitivity
of the chimeric transporter has allowed identification of the
glutamate-independent current as a flux of monovalent cations through
the transporter. Substrate-independent currents have been observed to
be mediated by cloned sodium-dependent transporters for
glucose(11) , serotonin (12) ,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 14 µM),
EAAT1 is relatively insensitive (K
> 3
mM). Substitution of a block of 76 residues from EAAT2 into
EAAT1, in which 18 residues varied from EAAT1, conferred high affinity
kainate binding to EAAT1, and application of kainate to oocytes
expressing the chimeric transporter blocked a pre-existing monovalent
cation conductance that displayed a permeability sequence K
> Na
> Li
choline
. The results identify a structural
domain of glutamate transporters that influences kainate binding and
demonstrate the presence of a constitutive ion-selective pore in the
transporter.
:1Glu
with countertransport of 1
K
and 1 OH
, resulting in
translocation of one net positive charge(5) . However, recent
studies of a cloned human glutamate transporter have demonstrated that
the quantity of charge translocated with glutamate is
voltage-dependent(6) . This variable charge stoichiometry
occurs as a consequence of an additional current arising from a
thermodynamically uncoupled chloride flux activated by transporter
substrates(7, 8) . Different transporter subtypes
exhibit intrinsic differences in the magnitude of the chloride flux
relative to flux of glutamate(7) .
(
)subtype but are not themselves
transported. EAAT1 and EAAT3 are insensitive to these compounds (K
> 3 mM)(9) .
Schild analysis of the inhibition of EAAT2 by kainate has demonstrated
that the K
is relatively
voltage-independent, which suggests that kainate binds to an external
site on the transporter outside the membrane electric
field(6) . The competitive action of kainate with respect to
glutamate (9) and similar voltage independence of the K
for glutamate activation
(
)suggest that kainate may interact with EAAT2 at the
same site in the outer pore of the transporter where glutamate first
binds.
Construction of a Chimeric Transporter
An in
vivo recombination procedure (17) was used to construct
chimeric glutamate transporters. The EAAT1 and EAAT2 cDNAs were cloned
in tandem into the pCMV plasmid (9) 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 plasmids that contained a
single cDNA and sequenced to determine the junction sites (Sequenase,
U. S. Biochemical Corp.). A number of plasmids were isolated, and all
the chimeras generated using this method had junction sites within a
highly conserved domain from serine 366 to glutamine 415 (EAAT1
numbering). One of these chimeras contained an EAAT1 sequence at the
amino terminus and junctioned with EAAT2 after serine 366. An EcoRI site was incorporated into the EAAT2 segment in this
chimera at a position corresponding to an endogenous EcoRI
site in EAAT1 (residues 442-443), and this construct was used to
generate a three-part chimera using a polymerase chain reaction-based
strategy. Briefly, an antisense oligonucleotide incorporating silent
base changes to introduce the EcoRI site was paired with a
sense oligonucleotide corresponding to the vector polylinker and used
in a polymerase chain reaction with the chimera as template. The
resulting product was subcloned into EAAT1, and the entire coding
region was sequenced to confirm the structure of the construct. The
three-part chimera, termed E1-2-1, was subcloned into pOTV for
oocyte expression(9) .
Electrophysiological Recording
50 ng of capped
cRNA transcribed with T7 polymerase from each of the cDNA constructs
was injected into defolliculated stage V Xenopus oocytes. Two
to seven days later transport was measured by two-electrode voltage
clamp recording, and kinetic analyses were performed as described
previously (16) . In experiments involving ion substitution,
sodium was partially replaced with equimolar choline, lithium, or
potassium. The permeabilities of test ions relative to Na were determined from the shift in reversal potential,
V, of the kainate-sensitive leak current induced by
substitution of one-half the external sodium with ion X from a
modified form of the Goldman-Hodgkin-Katz equation, P
/P
=
2(exp(
VF/RT)) - 1. Mean shifts for each
test ion were calculated by measurement of reversal potentials in
4-6 oocytes following measurement in control (ND96) solution. In
experiments in which the chloride concentration was altered, chloride
was substituted with either gluconate or methane sulfonate. To avoid
offset potentials, a 3 M KCl/agar bridge was used to connect
the bath to ground via a 3 M KCl reservoir containing a
silver/silver chloride electrode.
Substrate-independent Ion Flux
In order to test
for the presence of substrate-independent ionic currents mediated by
the EAAT1 and EAAT2 subtypes, the membrane conductance of oocytes
expressing these transporters was compared with the conductance of
uninjected oocytes in the absence of amino acid (Fig. 1A). The chord conductance (measured between
-40 and +40 mV) in a group of uninjected control oocytes was
1.8 ± 0.4 µS (mean ± S.E., n = 6). In
oocytes from the same group that expressed EAAT2, the chord conductance
was unchanged from control (1.9 ± 0.4 µS; n = 6), while in oocytes expressing comparable levels of
EAAT1, this value was increased significantly (3.4 ± 0.6 µS; n = 6; p < 0.05). Similar results were seen
in different batches of oocytes from three frogs. Subtraction of the
mean current-voltage relationship of uninjected oocytes from that of
oocytes expressing EAAT1 revealed the voltage dependence of the
uncoupled ion flux (Fig. 1B). This current was
approximately linear over the voltage range -100 to +40 mV
and reversed at -16.9 ± 4.9 mV (n = 8). In
order to test whether the glutamate-independent current in oocytes
expressing EAAT1 was mediated by the transporter, the difference in
conductance from the controls was compared with the amplitude of the
current elicited by 300 µML-glutamate at
-100 mV in oocytes expressing different levels of transporter.
There was a linear correlation in individual oocytes (Fig. 1C), demonstrating that the amplitude of the
glutamate-independent current was related to the transporter expression
level (r = 0.89). These results suggest that EAAT1,
but not EAAT2, mediates a substrate-independent ion flux.
Expression and Kinetic Parameters of a Chimeric
Transporter
Kainate competitively antagonizes the EAAT2-mediated
current induced by L-glutamate with a K of 14 µM but has no effect on the steady state
current in the absence of glutamate at membrane potentials between
-160 mV and +80 mV(6) . In contrast to EAAT2,
glutamate transport by EAAT1 is relatively insensitive to
kainate(9) . Application of up to 3 mM kainate alone
to oocytes expressing EAAT1 does not induce any steady-state current
over this voltage range (results not shown). In order to further
investigate the properties of the glutamate-independent current
mediated by EAAT1 and to identify domains involved in the interaction
of the transporter with kainate, chimeric transporters were constructed
and assessed for their kainate sensitivity. One such chimera, termed
E1-2-1, is comprised of the EAAT1 sequence from the amino
terminus through Ser-366 followed by the EAAT2 sequence through
Ala-441, followed by the EAAT1 sequence through the carboxyl terminus (Fig. 2). E1-2-1 contains 18 residues from EAAT2 between
Ser-366 and Ala-441 that differ from the corresponding EAAT1 residues.
Injection of RNA transcribed from the E1-2-1 chimeric cDNA into
oocytes resulted in a >10-fold increase in
H-labeled L-glutamate uptake (data not shown). Application of glutamate
to voltage-clamped oocytes expressing E1-2-1 resulted in inward
currents similar to those observed in oocytes expressing the parent
transporters (Fig. 3). The K
for L-glutamate exhibited by the chimeric transporter was 9
± 3 µM (n = 6), slightly lower than
that for EAAT1 (19 ± 3 µM) and EAAT2 (17 ± 2
µM). Because the substrate serine O-sulfate
displays marked differences in kinetic parameters between EAAT1 and
EAAT2(9) , chimera-mediated currents induced by this compound
were examined. EAAT1 displayed a K
of 35
± 2 µM for serine O-sulfate and an I
(relative to glutamate) of 1.02 ± 0.05,
whereas the corresponding values for EAAT2 were 237 ± 14
µM and 0.54 ± 0.02. The K
and I
values for serine O-sulfate transport by E1-2-1 were 17 ± 3
µM and 1.0 ± 0.1 (n = 5),
suggesting that the structural domain of the chimera involved in
determining the kinetic parameters for substrate translocation may be
comprised of EAAT1-derived sequences.
Kainate Actions on the Chimeric Transporter
In
contrast to EAAT1 and EAAT2, application of kainate to oocytes
expressing the E1-2-1 chimeric transporter generated a small
outward current at -60 mV (Fig. 3C). This outward
current was increased in a dose-dependent and saturable fashion by
kainate (EC = 120 ± 16 µM at
-60 mV; n = 4). In addition, co-application of 10
µM glutamate with 3 mM kainate to the
E1-2-1 chimera similarly resulted in an outward current rather
than a simple block of the inward transport current as seen with EAAT2.
The outward current induced by kainate at -60 mV resulted from a
conductance decrease due to the block of a constitutive inward current,
as revealed by subtraction of the current-voltage plot in the presence
of kainate from that in its absence (Fig. 4A). The
kainate-sensitive current reversed direction at -17.9 ±
1.0 mV (n = 16) in ND96 bathing solution, similar to
the reversal potential of the EAAT1-associated uncoupled flux
(-16.9 ± 4.9 mV, Fig. 1B). Thus, the small
segment of EAAT2 sequence in the E1-2-1 chimera confers
sensitivity of the uncoupled current to block by kainate.
ions. The reversal
potential of the kainate-sensitive uncoupled currents was measured as
in A using external bath solution (ND96) with
[K
] = 0 and in which
[Na
] was varied by equimolar substitution
with choline
. The line shows least squares
fit to data points (mean ± S.E.; n = 5) with a
slope of 58.2 mV.
by choline (reducing
[Na
]
from 100.5 to 22.5
mM) caused a 38.2 ± 4.2 mV (n = 6)
shift in the reversal potential, indicating that sodium ions contribute
to the substrate-independent leak current. A plot of the reversal
potential of the current blocked by kainate versus the
logarithm of the external sodium concentration revealed that the
reversal shifted 58.2 mV/decade change in
[Na
]
, in accord with the
prediction of the Nernst equation for a Na
-selective
electrode (Fig. 4B). The monovalent cation selectivity
of the uncoupled conductance was further studied by equimolar
replacement of one-half the external Na
with
K
, Li
, or choline. From the reversal
potential shifts, the permeability sequence was determined to be
K
(1.64) > Na
(1) >
Li
(0.83) choline (<0.04).
with gluconate or methanesulfonate
had no effect on the reversal potential of the uncoupled current (n = 5). However, after depletion of intracellular chloride by
16-h incubation in gluconate-substituted medium, removal of
extracellular chloride reversibly abolished the kainate-sensitive
uncoupled current (n = 5). These results indicate that,
although chloride ions are not permeant, they are required for
expression of the uncoupled monovalent cation current. When measured in
chloride-free conditions to abolish the leak current, kainate blocked
the inward current induced by 30 µML-glutamate
with a K
of 97 ± 18
µM (n = 3), similar to the potency
observed for the inhibition of the substrate-independent leak current
in the normal ND96 bath solution (126 ± 6 µM). This
result is consistent with the hypothesis that block of the leak
conductance and glutamate transport are both mediated by kainate
binding at a single site on the transporter.
-aminobutyric
acid(13) , dopamine(14) , and phosphate.
(
)Similar to results in the present study, a
sodium-dependent leakage current attributed to the glutamate
transporter(s) endogenous to salamander retinal glial cells has also
been reported(22) . The presence of uncoupled currents in
various sodium co-transporters may provide clues about the structural
requirements for the permeation path involved in translocation of large
substrate molecules in addition to small inorganic ions. The similarity
of the inhibition constants for kainate block of the
glutamate-dependent and -independent currents suggests that the ions
that carry the uncoupled current are likely to permeate the same pore
region of the transporter as glutamate. While transporter-mediated
substrate translocation is commonly modeled by an alternating access
scheme(23) , the permeation and possible gating mechanisms
underlying substrate-independent ion fluxes are not well understood.
The steady-state uncoupled cation conductance of the glutamate
transporter is relatively insensitive to membrane potential.
Nevertheless, the requirement of the cation flux for chloride suggests
a possibility of gating, perhaps via an allosteric effect of chloride
binding. Alternatively, interactions between cations and chloride may
occur in the transporter pore as have been observed in a neuronal
chloride channel(24) . Although both EAAT1 and EAAT2 mediate a
chloride flux, this flux requires amino acid for activation (7) and is thus distinguished from the uncoupled cation current
that occurs in the absence of amino acid. Further work to elucidate the
molecular mechanisms underlying these fluxes, including localization of
the precise residues that comprise the permeation pathway, will allow
the construction of increasingly more detailed models of transport.
We thank Jacques Wadiche for discussion and Weibin
Zhang for preparation of Xenopus oocytes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.