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The Journal of Neuroscience, January 1, 2000, 20(1):196-205
GluR5 and GluR6 Kainate Receptor Subunits Coexist in Hippocampal
Neurons and Coassemble to Form Functional Receptors
Ana V.
Paternain1,
María T.
Herrera2,
M. Angela
Nieto2, and
Juan
Lerma1
Departments of 1 Neural Plasticity and
2 Developmental Neurobiology, Instituto Cajal, Consejo
Superior de Investigaciones Científicas, 28002 Madrid, Spain
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ABSTRACT |
We have performed nonradioactive double in situ
hybridization to study the expression of glutamic acid decarboxylase
and GluR6 or GluR5 subunits in hippocampal slices. Our results indicate that although GluR6 is primarily expressed by pyramidal
cells and dentate granule neurons and GluR5 is
prominently expressed in nonpyramidal cells, there is a significant
population of GABAergic interneurons that coexpress the two glutamate
receptor subunits. To assess whether the two subunits could coassemble
to form heteromeric receptors, we studied the electrophysiological
responses when both subunits were coexpressed in HEK293 cells.
Responses evoked by rapid application of either glutamate,
(RS)- -amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionic acid (ATPA) the selective agonist of GluR5 receptors), and AMPA in
cells cotransfected with GluR6(R) and GluR5(Q) presented a similar
degree of outward rectification. This can only be attributed to the
fact that all receptors have at least one GluR6(R) subunit in their
structure, conferring outward rectification, and at least one GluR5(Q)
subunit to confer sensitivity to ATPA and AMPA. More than 80% of the
receptors expressed by a single cell were found to be GluR5/R6
heteromers, presenting different desensitization and gating properties
to homomeric R6 receptors. These results lead us to believe that a
population of interneurons in the hippocampus express receptors made up
of both GluR5 and GluR6 subunits and provide evidence for a greater
diversity of kainate receptors in the brain than previously thought,
that may account for a higher functional complexity.
Key words:
heteromeric kainate receptors; heteromeric glutamate
receptor; ATPA; coassembly; desensitization; hippocampus; interneurons; coexpression
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INTRODUCTION |
Within the glutamate receptor
system, a number of roles have been established for NMDA and AMPA
receptors in brain physiology, whereas the role of kainate receptors
has remained elusive over the years (Lerma, 1997 ). High-affinity
kainate receptors probably account for the kainate-dependent
excitotoxicity and susceptibility to epilepsy of hippocampal circuits
(Nadler, 1981; Coyle, 1983; Ben-Ari, 1985). Moreover, recent
experiments suggest a role for kainate receptors as transducers and
modulators of synaptic transmission (Castillo et al., 1997; Clarke et
al., 1997; Rodriguez-Moreno et al., 1997; Vignes and
Collingridge, 1997; Cossart et al., 1998; Frerking et al., 1998;
Rodriguez-Moreno and Lerma, 1998; Kidd and Isaac, 1999).
Kainate receptors appear to be made up of five different subunits,
namely GluR5, GluR6, GluR7, KA1, and KA2 (for review, see Lerma,
1999). Based on sequence homology, this family has been subdivided into
two branches, GluR5-7 and KA1-KA2 subunits (Egebjerg et al., 1991;
Bettler et al., 1992; Sommer et al., 1992). Both GluR5, GluR6 and GluR7
form homomeric functional channels when expressed in heterologous
systems (for review, see Lerma, 1999). In contrast, KA1 and KA2 do not
yield functional channels in the same systems but generate new
functional receptors when combined with GluR5, GluR6, or GluR7, showing
different properties from the homomeric channels (Herb et al., 1992;
Sakimura et al., 1992). Such a functional analysis of recombinant
subunits has fueled the idea of a heteromeric model for kainate
receptors. The model implies the pairing of members of each branch of
the kainate receptor subunits, i.e., GluRn/KAx, where n and
x are equivalent to 5-7 and 1-2, respectively. The typical
patterns of spatial distribution of the subunits have also supported
this idea. For instance, GluR6, KA1, and K2 are expressed in the CA3
pyramidal cells, whereas GluR6 and KA2 transcripts are more abundant in
the CA1 pyramids. Purkinje cells contain GluR5 and KA1, whereas the
granule cells of the cerebellum express GluR6 and KA2 (Wisden and
Seeburg, 1993; Bahn et al., 1994). However, expression of GluR5 has
also been detected in a small population of hippocampal interneurons
and cerebellar granule cells (Wisden and Seeburg, 1993), and
single-cell RT-PCR from hippocampal cultures demonstrated coexpression
of both GluR5 and GluR6 in a percentage of the cells analyzed (Mackler and Eberwine, 1993; Ruano et al., 1995).
In the present work we have reexamined the distribution of two kainate
receptor subunits, GluR5 and GluR6, in the hippocampus by
nonradioactive in situ hybridization. We have performed
double labeling for GluR5 or GluR6 and glutamic acid decarboxylase
(GAD) as a means to identify the GABA interneurons expressing these subunits. Our results indicate that a population of GABAergic neurons
in the hippocampus may coexpress both GluR5 and GluR6 subunits. We have
also tested the hypothesis that GluR5 and GluR6 could combine to form
heteromeric channels. Our results demonstrate the assembly of both
subunits to form functional receptor channels with pharmacological
properties similar to GluR5 and biophysical properties similar to GluR6
homomeric receptors. Thus, we provide evidence for a greater diversity
of native kainate receptors in the brain.
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MATERIALS AND METHODS |
In situ hybridization in hippocampal slices.
Brains were dissected from anesthetized 14-d-old rats and fixed
overnight in 4% paraformaldehyde at 4°C. After fixing, the brains
were washed three times in PBS and embedded in 7.5% low-melting point
agarose in PBS. The brains were sectioned in a vibratome to obtain 100 µm sagittal slices. The agarose was removed from the slices, which were again fixed overnight, again in 4% paraformaldehyde at 4°C. A
PstI-SalI fragment (649 bp) and a
PstI-XbaI fragment (595 bp) from the
GluR6 and GluR5 plasmids, respectively, were
subcloned into pBluescript-KS. The corresponding fragments were chosen
to contain the most divergent region between two cDNA sequences, with
an average similarity of ~60% at the nucleotide level. At the
stringency used in this protocol, no cross-hybridization is possible
between sequences with <90% similarity. Digoxigenin-labeled antisense
riboprobes were synthesized from these plasmids and from a plasmid
containing the full-length cDNA sequence for
GAD65 (kindly provided by Drs. A. Tobin and
N. Tillakaratne, University of California at Los Angeles, Los Angeles,
CA). In situ hybridization was performed in free-floating
slices as described for whole embryos (Nieto et al., 1996).
Double-labeling experiments were performed by simultaneous
hybridization with two probes. The GluR5 and
GluR6 probes were labeled with digoxigenin-UTP, and the
GAD probe was labeled with fluorescein-UTP from Boehringer
Mannheim (Mannheim, Germany). After hybridization, the slices were
subsequently incubated with alkaline phosphatase-conjugated
anti-digoxigenin and anti-fluorescein antibodies. The alkaline
phosphatase activity was detected by incubation with the following
substrates: nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (BCIP) for GluR5 and GluR6, and
2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyl tetrazolium chloride
(INT)/BCIP for GAD (both substrates from Boehringer
Mannheim) following manufacturer's instructions. Subsequently, sections were cleared in 50% glycerol in PBS and mounted in the same
solution containing 0.02% sodium azide. Sense probes failed to give
any signal. The slices were photographed with a Leica (Nussloch,
Germany) DMR microscope under Nomarski optics.
cDNA transient transfection. Plasmids encoding the VCR or
VCQ versions of GluR6, and the Q- or R-form of
GluR5-2a were a gift of Dr. P. H. Seeburg (Max-Planck
Institute, Heidelberg, Germany). The plasmid encoding the green
fluorescent protein (GFP) was kindly supplied by Dr. T. Hughes (Yale
University). GluR5 and/or GluR6 plasmids were
cotransfected with a GFP expression vector (10 µg of
GluR5, 1 µg of GluR6, and 1 µg
GFP) in HEK 293 cells by electroporation (Gene pulser;
Bio-Rad, Hercules, CA). GluR6 or GluR5 and
KA2 plasmids were cotransfected at 1:1 ratio. Afterwards,
cells were seeded in Petri dishes in DMEM supplemented with 10% fetal
calf serum and antibiotics, and maintained in a humidified incubator at
37°C and 5% CO2. Highly fluorescent cells
(Marshall et al., 1995) were identified and selected for recording.
Electrophysiological recordings and perfusion procedures.
Electrophysiological experiments were performed 1 d after
electroporation. Membrane currents were recorded at a given membrane
potential, using the whole-cell configuration of the patch-clamp
technique using a List EPC-7 amplifier. Series resistance was
compensated by 50-60%. Patch electrodes were fabricated from
borosilicate glass and had a resistance of 5-10 M . Currents were
filtered at 1 kHz (two pole Butterworth filter;  12 dB/octave) and
transferred at a sampling rate of 1-5 kHz into a personal computer for
analysis and display purposes using pClamp software (Axon Instruments, Foster City, CA). All experiments were performed at room temperature (22-25°C). The cells were rapidly perfused using a linear array of
eight glass tubes placed 200-300 µm from the cell body. Ringer's solution with and without agonist flowed from adjacent barrels, and
solution changes were achieved by laterally displacing the whole
perfusion array using a motorized device under the control of a
personal computer (Lerma, 1992). To test the speed of this perfusion
system, the open tip potential of a patch pipette was monitored after
jumping it into a low-strength solution. The current relaxed after a
single exponential with a time constant of <1 msec, and thus, in all
cases total replacement of the solution around the tip of the patch
electrode was achieved in <10 msec (usually 5-6 msec). However, for
receptor activation during whole-cell recordings, diffusion may be
limited by multiple barriers, which may slow down perfusion speed.
Concentration-response relationships were constructed for
glutamate, and sometimes for
(RS)--amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionic acid (ATPA), and fitted with the logistic equation by using
nonlinear regression procedures (SigmaPlot; Jandel Scientific, San
Rafael, CA). For activation curves, peak currents were normalized and pooled for fitting with the equation I = Imax/(1 + (EC50/[agonist])n),
where EC50 is the concentration of agonist
producing half-maximal activation. For steady-state inactivation, the
pooled data were fitted by the equation I = Imax/(1 + ([agonist]/IC50)n),
where IC50 is the concentration of agonist at
which receptors were 50% inactivated. In both equations, n
is the slope factor (i.e., the Hill coefficient). All results are
expressed as the mean ± SEM.
Experimental solutions. The normal external solution was (in
mM) NaCl 160, KCl 2.5, CaCl2 1.8, MgCl2 2, glucose 10, and HEPES 10, pH 7.4. Pipettes were filled with (in mM) cesium methanesulfonate 140, CsCl 5, CaCl2 0.5, MgCl2 5, EGTA 10, and HEPES 10, pH 7.4. Spermine
(100 µM) was routinely included in the pipette solution to prevent washout of inward rectification of unedited receptors (Bowie
and Mayer, 1995; Kamboj et al., 1995; Koh et al., 1995). Osmolarity was
adjusted, when necessary, to 330 and 314 mOsm for extracellular and
intracellular solutions, respectively, by adding sucrose. Agonists were
dissolved in distilled water at 50 mM stock solutions or
directly prepared in normal external solution. Kainate was obtained
from either Sigma (St. Louis, MO) or Tocris Cookson (Bristol, UK), and
AMPA was obtained from Tocris. Disodium glutamate and ConA were from
Sigma, and other salts were obtained from either Sigma or Merck
(Darmstadt, Germany). ATPA was a gift of Dr. J. Drejer (NeuroSearch,
Ballerup, Denmark).
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RESULTS |
Expression of GluR5 and GluR6 mRNAs in hippocampal neurons
Radioactive in situ hybridization studies have shown
that GluR6 is heavily expressed by hippocampal pyramidal
neurons and dentate granule cells (Wisden and Seeburg, 1993; Bahn et
al., 1994). Similarly, it has been shown that a population of
interneurons in the hippocampus, mainly in the stratum oriens,
expresses the GluR5 subunits (Bahn et al., 1994). We have performed
nonradioactive in situ hybridization studies to allow
single-cell resolution, semiquantitative detection of expression of
GluR6 and GluR5 subunits, and to check whether both might be expressed
by the same type of cells in the hippocampus. As can be seen in Figure
1, GluR6 was abundantly
expressed in pyramidal cells and dentate granule neurons, whereas
GluR5 was prominently expressed by nonpyramidal cells.
However, many neurons laying outside the pyramidal cell layer also
expressed GluR6, and there were some cells within the pyramidal cell layer that contained GluR5 transcripts. This
was observed not only in the CA1 but also in the CA3 field (Fig.
1D-G). Consequently, the possibility exists that
both subunits are expressed by both pyramidal cells and
interneurons.
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Figure 1.
Expression pattern of GluR5
(A), GluR6 (B), and
GAD65 (C) mRNA in the hippocampus.
Whereas GluR5 is predominantly expressed in interneurons
distributed over the whole hippocampus (A),
GluR6 is mainly expressed in the principal cells
(B). The boxes in A
and B indicate the areas shown in D-G.
D, A high-power view of the CA1 layer reveals a high
expression of GluR5 in the stratum oriens
(SO) and stratum radiatum (SR)
interneurons (arrow and white arrowhead,
respectively). Note that, in addition, many cells within the pyramidal
cell layer (Pyr) are also labeled (black
arrowhead). E, A similar zone of the CA1 area
showing high expression of GluR6 in pyramidal neurons
and interneurons both in the stratum oriens and stratum radiatum
(arrow and white arrowhead,
respectively). F, High-power view of the CA3 field
showing expression of GluR5 in several cells both within
and outside the pyramidal cell layer (black and
white arrowheads, respectively). G, A
high-power view of the CA3 field shows GluR6 expression
in numerous interneurons (white arrowheads). For
simplicity, only a few cells have been highlighted in each panel.
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To demonstrate the coexpression of GluR5 and
GluR6, we used double in situ hybridization.
However, the relatively low level of expression of both mRNAs, in
particular that of GluR5, prevented the reliable
determination of their coexpression because the first signal developed
was attenuated during the second development process.
GAD65 mRNA was abundantly expressed by
interneurons throughout the hippocampus (Fig. 1C) and gave a
signal intensity enough to allow double detection. Therefore, we
performed double in situ hybridization with either
GluR5 or GluR6 and
GAD65 as a means to determine whether these
kainate receptor subunits were expressed in pyramidal or GABAergic interneurons.
Figure 2 shows the results obtained from
double in situ hybridization of GluR5 and
GAD mRNA. As can be seen, many neurons expressed both
transcripts throughout the hippocampus (Fig. 2A). To
estimate the percentage of each type of hippocampal neuron expressing
GluR5, we computed in a double-blind test those neurons appearing red (GAD+,
GluR5 ), blue
(GAD,
GluR5+), and brown
(GAD+,
GluR5+). Cells were counted from the
available hippocampal slices (six to eight for each hippocampus;
2500-2900 neurons computed) by at least two different people. The
scores given by the different observers (for each color in a given
slice) were similar (often differing in <1%), and the mean value was
taken. GluR5 was expressed by approximately half of the
interneurons (53.6%; 539 of 1004 neurons) in the stratum oriens of
CA1. This value dropped to one-third (28.4%) in the pyramidal layer,
and only 13.6% of the stratum radiatum interneurons (n = 767) expressed GluR5 mRNA. However, most of the neurons
expressing GluR5 mRNA were GABA interneurons in the CA1
field. Thus, 86% of those neurons expressing GluR5 (1025 neurons) also express GAD (885 neurons) (Fig.
2F), indicating that although in a minority, some
non-GABA neurons express GluR5 subunits. Such a value was estimated to
be slightly larger in the stratum oriens than in the other layers. A
similar pattern was also found in the CA3 field (Fig.
2F).
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Figure 2.
Coexpression of GluR5 subunit and
GAD mRNAs in hippocampal neurons. A,
Composition of low-power pictures showing the hippocampus from the
subiculum (SUB) to the CA3 field. Boxes
in A from left to right
are presented enlarged in B-E, respectively.
GAD expression is labeled red
(white arrowheads), whereas GluR5 mRNA is
labeled blue (black arrowheads).
Coexpression appears as a brown precipitate
(black arrows). F, Cells appearing
blue, red, and brown were counted in the
stratum oriens (SO), pyramidale (SP), and
radiatum of CA1 or lucidum of CA3 (SR/L). The
left histogram represents the fraction of interneurons
(GAD-expressing cells) expressing GluR5 subunits, whereas the
right histogram represents the fraction of
GluR5-expressing cells that are GABA neurons. The histograms are based
on 2500-2900 neurons counted in six to eight slices by at least two
different people.
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GluR6 was abundantly expressed by principal cells in the
hippocampus, and a few interneurons in CA1 stratum oriens (6%) and stratum radiatum (3.3%) contained GluR6 transcripts.
However, most of the GABA interneurons lying in the pyramidal cell
layer also expressed GluR6 (96.2%; Fig.
3B). This was also true in
CA3, where 93.3% of the GABA cells lying in the pyramidal cell layer were labeled (240 of 257 neurons counted; Fig. 3D,F).
As could be seen in single in situ hybridizations (Fig.
1B), numerous cells, probably interneurons, in the
stratum lucidum of CA3 contain GluR6 transcripts. This
result was corroborated by double in situ labeling, demonstrating that a large number (85%; Fig. 3E) of
GAD-expressing cells also expressed GluR6 (573 of
671 neurons counted). Although only one-half of the
GluR6-expressing cells in the CA1 stratum oriens were
interneurons (65%), in the stratum oriens of CA3 the majority of the
GluR6-positive cells could be classified as interneurons (93%; 227 of
242 neurons). Similarly, virtually all of the GluR6-containing cells in
the stratum lucidum were interneurons (97%), although in the CA1
stratum radiatum approximately one-half of GluR6-expressing cells also
expressed GAD (48%; Fig. 3F).
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Figure 3.
Coexpression of GluR6 subunit and
GAD mRNAs in hippocampal neurons. A,
Composition of low-power pictures showing the hippocampus from the
subiculum (SUB) to the CA3 field. B-E,
Details of CA1 pyramidal cell layer (B), stratum
radiatum (C), CA3 pyramidal cell layer
(D), and stratum lucidum
(E). F, Cells appearing
blue, red, and brown were counted in the
stratum oriens (SO), pyramidal (SP), and
radiatum of CA1 or lucidum of CA3 (SR/L). GAD expression
is labeled red (white arrowheads),
whereas GluR6 mRNA is labeled blue (black
arrowheads). Coexpression appears as a
brown precipitate (black arrows). The
left histogram represents the fraction of interneurons
(GAD-expressing cells) expressing the GluR6 subunits, whereas the
right histogram represents the fraction of
GluR6-expressing cells that are GABA neurons. The histograms are based
on 1100-1800 neurons counted in six to eight slices by at least two
different people. Calculation of the fraction of GluR6-expressing cells
also expressing GAD was not possible (asterisk) because
all pyramidal cells were labeled blue.
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Taken together, these results indicate that GluR5 and GluR6 subunits
may coexist in a population of hippocampal interneurons, possibly
24-27% of GABA interneurons in the pyramidal cell layer (both CA1 and
CA3) and stratum oriens of CA3 express both subunits. However, it
should be noted that the spread of blue reaction caused by the massive
expression of GluR6 in the pyramidal layer may induce an overestimation
of the number of brown cells in this field. Although coexpression of
GluR5 and GluR6 by interneurons of stratum oriens
in CA1 is less likely, both transcripts appear to be present in at
least 17% of the stratum radiatum (CA1) and stratum lucidum (CA3) interneurons.
GluR5 and GluR6 combine to form functional receptors
A question that arises as a result of the mRNA expression data is
whether or not GluR5 and GluR6 may combine to form heteromeric channels. To address this question, we cotransfected GluR5
and GluR6 in HEK293 cells and studied the agonist
sensitivity of the receptors. Figure
4A shows that homomeric
GluR6 receptors are activated by glutamate but are completely
insensitive to AMPA and ATPA (Lauridsen et al., 1985), the specific
agonist of GluR5 receptors. In our experiments, transfection with the
GluR5 plasmid produced barely detectable currents when the
cells were challenged by glutamate, kainate, or ATPA (data not shown).
However, when GluR6 was cotransfected with GluR5,
large currents were generated by the three agonists (Fig.
4B). It is known that these subunits hold a site in
the second membrane segment that is regulated by mRNA editing (for
review, see Seeburg, 1996). Edited forms (R forms) give rise to
I-V relationships with outward rectification (Fig.
4A), whereas unedited (Q) forms generate receptors
that present strong inward rectification. Because R forms are
phenotypically dominant (Schoepfer et al., 1994), we looked at the
I-V relationship for responses induced by ATPA and AMPA on
heteromeric GluR6(R)/GluR5(Q) receptors. As can be seen in Figure 4,
B and C, the responses evoked by either
glutamate, ATPA, or AMPA in cells cotransfected with both subunits
presented a similar degree of outward rectification. Indeed, the index
of rectification (IR), calculated as the ratio of conductance at 60 and
60 mV, was 1.17 ± 0.05 (n = 13), 1.16 ± 0.04 (n = 13), and 1.5 ± 0.06 (n = 3) for ATPA, glutamate, and AMPA, respectively. This can only be
attributed to the fact that all receptors have at least one GluR6(R)
subunit in their structure, which would confer outward rectification,
and at least one GluR5(Q), which would confer sensitivity to ATPA and
AMPA. We wanted to know whether GluR6(Q) can combine with edited
GluR5(R) subunits. Thus, we cotransfected both plasmids and looked at
the I-V relationships of the expressed receptors. Homomeric
GluR6(Q) generated I-V relationships with a strong inward
rectification (IR < 0.1) and were insensitive to ATPA.
Cotransfection with GluR5(R) not only abolished inward rectification
(IR = 0.84 ± 0.05; n = 6 for
glutamate-induced responses) but also conferred sensitivity to ATPA,
which presented an I-V relationship with a similar degree
of rectification (0.7 ± 0.14; n = 5). These cells
were treated with ConA to remove rapid desensitization (Partin et al.,
1993) and thus facilitate measurements.
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Figure 4.
GluR6 and GluR5 subunits coassemble to generate
heteromeric kainate receptors with new pharmacological properties.
A, Typical responses induced by the rapid perfusion of
glutamate (1 mM) in HEK293 cells voltage-clamped at the
indicated Vm after being transfected with
GluR6(R) plasmid. Cells were treated with ConA to remove
desensitization. Homomeric GluR6 receptors were not activated by ATPA
(100 µM) or S-AMPA (500 µM).
B, In cells cotransfected with GluR6(R) and GluR5(Q),
currents were activated after perfusion of glutamate, AMPA, and ATPA.
Responses activated by either agonist presented a similar degree of
outward rectification, indicating the dominance of the R forms
(outward-rectifying) over the Q forms (inward-rectifying).
C, Current-voltage relationship for these three
agonists from cells transfected with GluR5(Q) and GluR6(R). Points are
mean ± SEM from eight (ATPA), seven (glutamate), and three (AMPA)
cells.
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In cells not treated with ConA, 100 µM ATPA induced
responses that largely but not totally desensitized (11.6 ± 1%
of the peak current remained; n = 21). To determine the
population of heteromeric receptors formed after cotransfection of both
plasmids, we looked at the efficiency of ATPA to cross-desensitize
glutamate-induced response. Figure
5A shows that the response to
glutamate was largely reduced by a previous pulse of ATPA. In the 17 cells in which GluR5(Q) and GluR6(R) were coexpressed, a prepulse of
ATPA depressed the subsequent glutamate-induced peak current by 81.7%
(Fig. 5B). In contrast, ATPA did not induce a depression of
the glutamate-induced response in cells expressing GluR6 only. The
results were similar when GluR5(R) and GluR6(Q) were cotransfected
(84.5 ± 2.7%; n = 5 of the glutamate-induced
response was cross-desensitized). This result indicates that a large
fraction of receptors expressed in these experiments included both
GluR5 and GluR6 subunits and that the editing site has no influence on
subunit assemblage.
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Figure 5.
In cells cotransfected with
GluR5 and GluR6, most of the receptors
formed are heteromeric receptors. A, ATPA
cross-desensitized most of the response activated by glutamate (1 mM; arrows) in cells cotransfected with both
subunits and not treated with ConA, indicating the existence of a
largely homogeneous population of heteromeric receptors.
B, Average data for the glutamate-induced current
remaining after a prepulse of ATPA (100 µM) in cells
transfected with GluR6(Q) (black bar), with GluR5(Q) and
GluR6(R) (light gray bar), or with GluR5(R) and GluR6(Q)
(dark gray bar). The white bar represents
the remaining steady-state response in ATPA-induced responses
(St. St.; i.e., nondesensitizing).
Numbers in parentheses correspond to the
number of cells studied.
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Activation-inactivation properties of kainate receptors
Because most of the receptors susceptible to glutamate activation
in cells cotransfected with GluR5 and GluR6 were
heteromeric, we wanted to further characterize this new type of kainate
receptor. Therefore, the glutamate dose-response relationship for the
peak current was calculated and compared with homomeric GluR6 receptors (Fig. 6). Data from different cells were
normalized and pooled for computer fitting with the logistic equation.
GluR5/R6 receptors expressed in HEK293 cells did not differ from
homomeric GluR6 receptors in their activation or steady-state
desensitization in response to glutamate (Fig. 6). The curve that best
fitted the dose-response data from GluR5/R6 receptors presented an
EC50 value of 1.34 mM for
glutamate, with a slope factor of 0.6. The inactivation curve was
constructed as previously described (Paternain et al., 1998; Fig.
6A). Briefly, after a period of incubation of at
least 10 sec, enough time to attain desensitization in a steady-state
situation, this solution was rapidly replaced by another one containing
a saturating concentration of kainate (Fig. 6A). The
peak response was measured and compared to that obtained when no
glutamate was included in the conditioning pulse. The fitted curve
revealed a half-maximal inactivation at a submicromolar concentration
of glutamate (0.33 µM) with a slope factor of
1. These values were close to those observed for GluR6 homomeric receptors in these (Fig. 6) and previous experiments (Heckmann et al.,
1996; Paternain et al., 1998).
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Figure 6.
Activation-inactivation properties of heteromeric
GluR5/R6 kainate receptors. A, To calculate the
steady-state inactivation curve, cells were exposed to different
concentrations of glutamate for 10 sec before jumping to a solution
containing kainate. The effect of 1 µM of glutamate on a
test response evoked by kainate (300 µM; thin
trace) is shown (thick trace). B,
Activation and inactivation curves of homomeric (dotted)
and heteromeric (solid) kainate receptors. The lines
through the points represent the best least-squares fit of the Hill
equation to pooled data and presented a half-saturated concentration
(EC50) of 898 and 1338 µM for GluR6
and GluR5/R6 receptor activation, respectively, and 0.34 and 0.33 µM for inactivation (IC50). Points are
the mean ± SEM of data collected from three to seven cells.
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The onset and recovery of desensitization was also determined for
heteromeric GluR5/R6 receptors. In the continuous presence of agonist,
the current desensitization followed a single exponential time course
(Fig. 7A). It was observed
that the onset of desensitization was slightly faster in cells
transfected with GluR5 and GluR6 than with
GluR6 alone. As for GluR6 receptors, the time constant of
desensitization was concentration-dependent, decreasing as the agonist
concentration increased. To calculate the actual rate of the onset of
receptor desensitization, we measured the time constant of current
decay at several concentrations of agonist. The concentration versus
desensitization rate (i.e., 1/ onset) plot
reveals a saturating process, which was well described by a rectangular
hyperbolic function. The asymptotic value of this function reflects the
rate of desensitization when the binding rate of glutamate is no longer
a limiting step (Paternain et al., 1998). This value was significantly
different in heteromeric and homomeric channels (126 and 92 sec1, respectively; p < 0.05; normal distribution comparison), indicating that heteromeric
GluR5/R6 receptors desensitize slightly but significantly faster than
homomeric GluR6 formations.
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Figure 7.
Desensitization kinetics in GluR6 and
GluR5/R6 kainate receptors. A, Onset of desensitization
was faster in heteromeric than in homomeric receptors. The superimposed
lines represent single exponential fits to the desensitization process
with the indicated time constants (Des).
B, The time constant value for onset of desensitization
was measured in several cells at different concentrations of agonist,
normalized and pooled for fitting. The following rectangular hyperbolic
function was fitted to the data points: y = a · x/(b + x), where y is the onset rate, measured as
1/Des at any concentration of glutamate
(x), a is the asymptote of the
function, and b is the agonist concentration at which
rate is 50% of the maximum. The asymptotic values obtained for each
receptor type are indicated and were significantly different
(p < 0.05). Data points are the mean ± SEM of five or six cells. C, The desensitization
recovery of heteromeric receptors was slower than that of homomeric
assemblies. Receptors were completely desensitized by applying a 0.6 sec pulse of glutamate (1 mM). Conditioning pulses were
followed by test pulses of the same duration delivered at different
intervals. The peak amplitude was measured and normalized with respect
to the average of all conditioning pulses. This value (recovery) was
plotted against the interval between applications
(D). The data collected from two
(asterisks) to four cells were pooled and fitted with
single exponential functions (dotted and solid
lines for GluR6 and GluR5/R6 receptors, respectively) with the
illustrated time constants of recovery (rec)
which significantly differed (p < 0.05)
when normal distributions were statistically compared. Data points are
the mean ± SEM.
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We then investigated whether the rate of recovery from the desensitized
state was different in heteromeric and homomeric receptors. Thus, we
applied test pulses of glutamate at different intervals after the
receptors were desensitized by a conditioning pulse of agonist (Fig.
7C). Peak responses were measured and normalized to permit
the comparison of several cells. Homomeric GluR6 receptors recovered
from desensitization after an exponential time course with a time
constant of 2.2 ± 0.1 sec (cf. Paternain et al., 1998). In cells
expressing heteromeric GluR5/R6 receptors, the time course of recovery
was also well fitted by a single exponential function but showed a
slower time constant (3.2 ± 0.4 sec; Fig. 7D). These two values differed statistically (p < 0.05;
normal distribution comparison)
These results indicate that coassembly of GluR5 and GluR6 subunits
alters the kinetics of both onset and recovery of desensitization such
that heteromeric GluR5/R6 receptors enter more rapidly into the
desensitized state and leave it more slowly.
We wanted to further analyze how the addition of new subunits into
receptors made of GluR5 (plus KA2 or GluR6) or GluR6 subunits (plus KA2
or GluR5) modifies the activation of the functional receptor. In Figure
8, the response patterns induced by rapid application of the three different agonists are compared in receptors expressed after the cotransfection of the three different subunits. Glutamate induced similar responses when activating receptors of either
configuration. In contrast, AMPA activated the three heteromeric
receptors with remarkably different patterns. Although this agonist
desensitized rapidly and completely GluR5/KA2 receptors, it produced
currents with GluR6/KA2 that inactivated partially and slowly.
Something similar happened when the AMPA derivative ATPA was applied.
This agonist did not desensitize heteromeric GluR6/KA2 formations,
whereas it produced a rapid and strong desensitization in GluR5/KA2
receptors. Interestingly, as described above, ATPA rapidly desensitized
heteromeric GluR5/R6, but left some steady-state current. This
distinctive behavior in terms of desensitization makes it possible to
differentiate this type of receptor from the other formations. In
addition, to generate responses with different properties, ATPA
presented a higher affinity for heteromeric GluR5/R6
(EC50 = 21 ± 2.8 µM) than for
GluR6/KA2 (EC50 = 84 ± 9.7 µM). As for glutamate and kainate, the treatment of
kainate receptors with ConA to remove desensitization drastically
increased the apparent affinity (Paternain et al., 1998) because
the EC50 for ATPA was decreased by 19-fold
(EC50 = 1.1 ± 0.08 µM; Fig. 8C).
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Figure 8.
Activation patterns of heteromeric
kainate receptors by different agonists. A, HEK cells
were cotransfected with either GluR6 and KA2, GluR5 and KA2, or GluR6
and GluR5, and the current response to rapid applications of glutamate
(Glu, 300 µM), AMPA (500 µM), and ATPA (100 µM, except where otherwise indicated) were studied. Note
that glutamate evoked responses with similar shapes in the three
receptor types. However, GluR6/KA2 responded to AMPA and ATPA
differently than the other receptors. ATPA differentiated GluR5/KA2
from GluR5/GluR6 in that it did not completely desensitize
GluR6-containing GluR5 receptor. Except for the responses induced by
ATPA in GluR5/GluR6 receptors, the illustrated recordings were
collected from the same cell in each case. B,
Dose-response curve for ATPA activation of heteromeric GluR6/KA2
receptors. The current amplitudes were expressed as a percentage of the
current induced by 500 µM AMPA. The solid
line is the result of fitting the Hill equation with an
EC50 of 84 µM and a Hill coefficient of 1.7. Points are the mean ± SEM of data collected from 2 (asterisk) to 26 cells. C, Dose-response
curve for ATPA in heteromeric GluR5/GluR6 receptors. Solid
lines are the result of fitting the Hill equation with
EC50 of 21 and 1.1 µM in cells untreated or
treated with ConA, respectively, to remove desensitization. Points are
the mean ± SEM of data collected from three to seven cells.
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It is noteworthy that ATPA indeed behaves as a partial agonist of
GluR6-containing receptors when these also contain the KA2 or GluR5
subunits. In heteromeric GluR6/KA2 receptors, and unlike glutamate or
AMPA, ATPA induced nondesensitizing responses (<5% of current
attenuation in a 20 sec pulse at 100 µM), and
ATPA-induced currents always were smaller than the peak response
induced by glutamate (1 mM) or S-AMPA (500 µM). On average, the current induced by 100 µM ATPA was ~30% of the peak current induced
by 500 µM S-AMPA. Similarly, in
GluR6/R5 receptors, the peak response to 100 µM
ATPA was 25 ± 1.5% (n = 51) of that induced by
glutamate (1 mM), of which only a fraction desensitized.
These results indicate that ATPA can no longer be considered as a
specific agonist of GluR5-containing receptors. Although in heteromeric
GluR6/KA2 receptors, the ATPA-induced response would be a fraction of
the amplitude of glutamate-induced rapidly desensitizing response, it
is worth noting that ATPA induces nondesensitizing currents, and in
terms of steady-state activation, it should be much more effective than
kainate and the endogenous ligand.
| |
DISCUSSION |
In the present work, we have studied the coexpression and
coassembly of two of the most abundantly expressed kainate receptor subunits, GluR5 and GluR6, in the hippocampus. Our results indicate that both subunits are able to form heteromeric receptors and that both
colocalize in a population of GABA neurons in the hippocampus, implicating these heteromeric receptors in the transduction machinery for glutamate signaling in inhibitory interneurons.
In contrast to other authors' observations (Sommer et al., 1992;
Swanson and Heinemann, 1998), under our experimental situation, we were
unable to obtain expression of homomeric GluR5 receptors after
transfection in HEK293 cells. This was not attributable to unsuccessful
plasmid transfection or a failure in cDNA transcription or RNA
translation because when the same construct was contransfected with
KA2 (a subunit unable to form functional channels by
itself), responses were induced in 100% of the cases. We have no
obvious explanation for this phenomenon, but it is possible that the
functional state of the cell line used (e.g., passage number, time in
culture, etc.) might influence the assembly of GluR5 subunits into
functional receptors, giving different results in different
laboratories. In addition, it is well known that only the shortest
splice variant of GluR5 (i.e., GluR5-2a) is able
to form functional homomeric channels (Sommer et al., 1992), yet with
low efficiency when compared to that of GluR6.
Interestingly, most of the receptors assembled after expression of
GluR5 and GluR6 subunits were heteromeric. Cross-desensitization
experiments allowed us to estimate that >80% of the functional
receptors expressed after cotransfection of both cDNAs contained enough
GluR5 subunits to confer ATPA sensitivity. This analysis was based on
the rationale that whereas glutamate could activate the whole
population of receptors (homomeric and heteromeric), ATPA would only be
able to activate and desensitize those receptors containing GluR5
subunits because it was unable to activate homomeric GluR6. Together,
these results indicate that cotransfection of GluR5 and GluR6 induces a
large proportion of heteromers present in the membrane, allowing us to
further characterize them.
Responses induced by the endogenous agonist in heteromeric and
homomeric GluR6 receptors did not differ in that the current was
rapidly activated, and receptors rapidly and almost completely desensitized. However, R5/R6 heteromeric constructions desensitized 30% faster and recovered from desensitization slower (~40%) than homomeric GluR6. It is worth noting that desensitization rate constants
were estimated from the asymptote of fitted curves (Fig. 7A), i.e., when the rate becomes independent of agonist
concentration and therefore independent of the perfusion speed
(Paternain et al., 1998). The desensitization rate measured in this way
should be fairly accurate, making the observed differences reliable. Indeed, such a difference in the onset rate of desensitization would in
principle account for the difference in the EC50
for glutamate from dose-response curves (Fig. 6), because rapid
desensitization may curtail activation at even high concentrations of
agonist. Therefore, we conclude that heteromeric R5/R6 and homomeric R6 have similar apparent affinities for glutamate. Previous work has
reported lower EC50 values for homomeric GluR6 as
well as faster desensitization rates (Heckmann et al., 1996; Swanson et al., 1997; Traynelis and Wahl, 1997). Although the slower
perfusion speed attained by us could account for larger
EC50 values, in the mentioned works fast
perfusion was achieved by excising patches or by lifting the cells from
the bottom of the dish. It is presently unclear how these manipulations
may affect desensitization kinetics and receptor affinity. Indeed, with
our perfusion system we have been able to resolve GluR6-mediated
responses in excised patches with desensitization time constants of  2
msec. Whatever the explanation, it is unlikely that the small changes
detected by us in desensitization kinetics and affinity would establish
relevant differences in physiological responses mediated by this kind
of heteromeric kainate receptor, as compared to homomeric GluR6.
Conversely, the gating properties of heteromeric receptors for agonists
other than glutamate presented remarkable differences. GluR6/KA2 and
GluR5/KA2 were both sensitive to ATPA and AMPA. However, although
responses were poorly or nondesensitizing in the former, currents
rapidly faded in the latter (Fig. 8). These same agonists activated
heteromeric GluR5/GluR6 with a different pattern. In particular, ATPA
desensitized these receptors rapidly but only partially, because a
clear steady current remained at all concentrations. In addition, the
affinity of heteromeric GluR5/GluR6 receptors for ATPA was greater than
heteromeric GluR6/KA2 formations (21 vs 84 µM in
EC50, respectively) and as such, more similar to
that of native kainate receptors expressed by DRG cells and human
homomeric GluR5 receptors (Clarke et al., 1997). However, many other
receptor types would predictably be activated by ATPA, because the
presence of additional receptor subunits in the hippocampus, such as
KA1 and KA2, leaves open the possibility that GluR5/KAx, GluR6/KAx, and
GluR5/GluR6/KAx receptors could also exist.
The pharmacological distinction between heteromeric kainate receptors
is not limited to the combination of one of the GluR5-7 subunits with
one of the KA subunits, an assumption that surprisingly has been the
overall accepted scheme for the assembly of kainate receptors
(for review, see Lerma, 1999). This assumption may be, at least in
part, responsible for a number of discrepancies observed between
recombinant and native kainate receptors. For instance, by using
single-cell RT-PCR of electrophysiologically characterized neurons, we
found that ~20% of the hippocampal embryonic cells, maintained in
culture for a few days, expressed GluR5 and GluR6 mRNAs (Ruano et al.,
1995). Interestingly, in those cells we could never observe responses
such as those obtained in recombinant systems or DRG neurons,
indicating the presence of homomeric GluR5 receptors. This observation,
surprising at that time, can be now explained if heteromeric GluR5/R6
receptors were indeed present in the membrane of those cells: the
response induced by kainate would not have differed from that obtained
in neurons expressing only GluR6. Furthermore, Vignes et al. (1997)
observed that two new compounds, LY293558 and LY294486, that antagonize
human GluR5 receptors, also antagonize the depolarization induced by
kainate in the CA3 region as well as the synaptic response induced by high-frequency stimulation of mossy fibers, which has been
unequivocally identified as GluR6-mediated (Mulle et al., 1998). Our
demonstration of the assembly of GluR5 and GluR6 subunits reconciles
both observations and indicates that the presence of a population of
receptors including both subunits should be taken into account. It is
worth noting, however, that the presence of heteromeric GluR5/GluR6
receptors may not be restricted to the hippocampus (Mackler and
Eberwine, 1993) but may also occur in other parts of the brain (e.g.,
striatal cells; Ghasemzadeh et al., 1996; Porter et al., 1998).
Finally, what is the role of heteromeric R5/R6 receptors? Is there any
advantage in forming heteromeric R5/R6 over homomeric receptors? At the
present time, when the functional role of kainate receptors is still
largely ignored, it is difficult to define a role for a heteromeric
kainate receptor. However, it is likely that the different subunits may
confer different targeting capabilities to the corresponding receptors.
For instance, it has been reported that GluR6 subunits interact with
PSD95/SAP90 proteins through binding to their PDZ1 domain. In contrast,
the KA2 C-terminal domain interacts with SH3 and GK domains of the same
protein (Garcia et al., 1998). Obviously, the clustering capabilities
of heteromeric GluR6/KA2 receptors performed by this kind of structural
proteins are wider than those of homomeric GluR6, as would be the case for their coupling to signaling proteins. It is not yet known which
proteins are able to bind and cluster GluR5 subunits, but the existence
of three different C-terminal variants indicates multiple possibilities
for specific receptor targeting. A given binding motif may serve to
associate the receptor to a particular signal transduction cascade
and/or to localize it to particular regions of the cell (axon terminal
vs dendritic spine), implying different functional roles. Although all
these possibilities should be studied in detail, the unique
pharmacological and kinetic properties described here for heteromeric
receptors should prove useful to identify native kainate receptors. The
possibility of coassembly of subfamily members provides evidence to
indicate that the molecular diversity of kainate receptors in the brain
is larger than previously thought and adds new excitement into the
study of this receptor type, a receptor that is just beginning to be understood.
During the review process of this paper, a report by Cui and Mayer
(1999) appeared demonstrating coassembly of GluR5, GluR6, and GluR7 in
HEK 293 cells. The conclusion from this study is essentially the same
as ours in that, similar to AMPA receptors, kainate receptor subunits
exhibit promiscuous coassembly. This study also revealed that
heteromeric GluR5/R6 receptors showed reduced desensitization with
enhanced steady-state current in response to kainate. In keeping with
this, we also observed some enhanced steady-state current in
kainate-induced responses after coexpression of both subunits (steady
state of 8 ± 2%; n = 8 of peak amplitude; data
not shown) as compared to homomeric GluR6 receptors (<2% of the peak
current remained). However, any additional alteration of
activation-inactivation kinetic properties of heteromeric receptors is
not comparable in both studies because, unlike Cui and Mayer (1999), we
evaluated heteromeric receptor properties for the endogenous ligand glutamate.
| |
FOOTNOTES |
Received Aug. 30, 1999; revised Oct. 11, 1999; accepted Oct. 13, 1999.
This work was supported in part by grants to J.L. from the
Dirección General de Enseñanza Superior e
Investigación Científica (P.M.96/0008) and to J.L. and
M.A.N. from the Community of Madrid (08.5/0042/1998). M.T.H. held a
fellowship from the Instituto de Cooperación Iberoamericana. We
thank Dr. P. H. Seeburg for the cytomegalovirus expression
plasmids encoding GluR6, GluR5, and KA2, Drs. A. Tobin and N. Tillakaratne for the GAD65 plasmid, Dr. T. E. Hughes
for the plasmid encoding green fluorescent protein (GFP), Dr. J. Drejer
for the gift of ATPA, Dr. M. Sefton for proofreading, and D. Guinea for technical assistance.
Correspondence should be addressed to Dr. J. Lerma, Instituto Cajal,
Consejo Superior de Investigaciones Científicas, Avenue Doctor
Arce 37, 28002 Madrid, Spain. E-mail: lerma{at}cajal.csic.es.
| |
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Copyright © 2000 Society for Neuroscience 0270-6474/0/201196-10$05.00/0
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Z. Ren, N. J. Riley, L. A. Needleman, J. M. Sanders, G. T. Swanson, and J. Marshall
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Z. Ren, N. J. Riley, E. P. Garcia, J. M. Sanders, G. T. Swanson, and J. Marshall
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M. F. M. Braga, V. Aroniadou-Anderjaska, J. Xie, and H. Li
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G. A. Kerchner, T. J. Wilding, J. E. Huettner, and M. Zhuo
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I. Khalilov, J. Hirsch, R. Cossart, and Y. Ben-Ari
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J. Lerma, A. V. Paternain, A. Rodriguez-Moreno, and J. C. Lopez-Garcia
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A. B. Ali, J. Rossier, J. F. Staiger, and E. Audinat
Kainate Receptors Regulate Unitary IPSCs Elicited in Pyramidal Cells by Fast-Spiking Interneurons in the Neocortex
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T. J Wilding and J. E Huettner
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P. J. Brockie, D. M. Madsen, Y. Zheng, J. Mellem, and A. V. Maricq
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G. A. Kerchner, T. J. Wilding, P. Li, M. Zhuo, and J. E. Huettner
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A. Contractor, G. T. Swanson, A. Sailer, S. O'Gorman, and S. F. Heinemann
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A. Rodriguez-Moreno, J. C. Lopez-Garcia, and J. Lerma
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TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
