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The Journal of Neuroscience, October 1, 1999, 19(19):8281-8291
Heteromeric Kainate Receptors Formed by the Coassembly of GluR5,
GluR6, and GluR7
Changhai
Cui and
Mark L.
Mayer
Laboratory of Cellular and Molecular Neurophysiology, National
Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, Maryland 20892
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ABSTRACT |
In the CNS kainate subtype glutamate receptors (GluRs) are likely
to be heteromeric assemblies containing multiple gene products. However, although recombinant kainate receptors from the GluR5-GluR7 gene family have been studied extensively in their homomeric forms, there have been no tests to determine whether these subunits can coassemble with each other. We used the GluR5 selective agonists (RS)-2-amino-3-(3-hydroxy-5-tertbutylisoxazol-4-yl)propanoic
acid (ATPA) and (S)-5-iodowillardiine (I-will) to test
for the coassembly of GluR5 with GluR6 and GluR7 by measuring changes
in rectification that occur for heteromeric receptors containing both
edited and unedited Q/R site subunits. Birectifying ATPA and I-will
responses resulting from polyamine block for homomeric GluR5(Q) became
outwardly rectifying when GluR6(R) was coexpressed with GluR5(Q),
although GluR6 was not activated by ATPA or I-will, indicating the
formation of heteromeric receptors. Similar approaches showed the
coassembly of GluR7 with GluR6 and GluR5. Heteromeric kainate receptors
containing both GluR5 and GluR6 subunits exhibited novel functional
properties, including reduced desensitization and faster recovery from
desensitization than those recorded for homomeric GluR5. Coexpression
of GluR6 with GluR5 also enhanced the magnitude of responses to GluR5
selective agonists. In contrast, the coassembly of GluR7 with GluR6
markedly decreased the amplitude of agonist responses. Our results
indicate that, similar to AMPA receptors, the kainate receptor subunits GluR5-GluR7 exhibit promiscuous coassembly. The formation of
heteromeric kainate receptors may help to explain why the functional
properties of native kainate receptors differ from those that have been
reported for recombinant kainate receptors.
Key words:
kainate receptors; polyamines; glutamate receptors; ATPA; iodowillardiine; coassembly; heteromeric glutamate receptors
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INTRODUCTION |
Kainate receptors are encoded by two
gene families: glutamate receptors (GluRs) GluR5-GluR7, which form
functional homomeric receptors with distinct physiology and
pharmacology (Hollmann and Heinemann, 1994 ; Schiffer et al., 1997;
Dingledine et al., 1999), and KA1 and KA2, which form functional ion
channels only after coassembly with GluR5-GluR7 (Werner et al., 1991;
Herb et al., 1992). The discovery of kainate receptor synaptic currents in diverse areas of the CNS (Castillo et al., 1997; Vignes and Collingridge, 1997; Frerking et al., 1998; Li and Rogawski, 1998; DeVries and Schwartz, 1999; Li et al., 1999), possible modulatory actions of kainate receptors on neurotransmitter release (Clarke et
al., 1997; Rodriguez-Moreno et al., 1997; Vignes et al., 1998), and
studies on GluR6 knock-out mice (Mulle et al., 1998; Bureau et al.,
1999) have stimulated recent interest in kainate receptor biology. The
overlapping expression of mRNAs encoding kainate receptor subunits
(Wisden and Seeburg, 1993; Bahn et al., 1994; Bischoff et al., 1997)
makes it extremely likely that, similar to AMPA receptors, there exist
diverse subtypes of heteromeric kainate receptor assemblies. The
mechanisms regulating the assembly of glutamate receptor (GluR)
subunits are poorly understood but must include signals that allow
coassembly within and between some gene families while restricting
coassembly with other gene families. Thus, AMPA receptor subunits,
which show 68-73% amino acid sequence homology, show promiscuous
coassembly with each other, but not with kainate receptor subunits
(Partin et al., 1993; Brose et al., 1994; Puchalski et al., 1994). In
contrast, although GluR5-GluR7 show similar low homology with both
AMPA receptor subunits (39-41%) and KA1 and KA2 (43-44%), they
coassemble only with the latter (Partin et al., 1993; Brose et al.,
1994; Puchalski et al., 1994; Wenthold et al., 1994).
It seems likely that, similar to AMPA receptors, the coassembly of
GluR5, GluR6, and GluR7 could generate heteromeric receptors with novel
functional properties; however, experimental tests of this have not
been reported. In addition to providing the fundamental knowledge
required for a full understanding of the biology of the
diverse subtypes of GluRs, information on the coassembly of kainate
receptors would be especially useful at the present time, given some
surprising results for kainate receptor-mediated mossy fiber synaptic
responses in CA3 pyramidal neurons (Castillo et al., 1997; Vignes and
Collingridge, 1997). Mossy fiber kainate receptor EPSCs are absent in
GluR6 / mice, suggesting a role for the
GluR6 subunit (Mulle et al., 1998) but also are antagonized by
decahydroisoquinolines that selectively block the activation of
homomeric GluR5, but not GluR6 (Clarke et al., 1997; Vignes et al.,
1997). It seems likely that the mossy fiber EPSC could be mediated by
heteromeric kainate receptors formed by the coassembly of GluR5 and
GluR6, perhaps with additional subunits. To test for the coassembly of
kainate receptors from the GluR5-GluR7 gene family, we took advantage of the recent discovery that two agonists, ATPA, the tert
butyl analog of AMPA (Lauridsen et al., 1985), and
(S)-5-iodowillardiine (I-will), selectively activate
GluR5, but not GluR6 or GluR7 (Clarke et al., 1997; Swanson et al.,
1998). By combining GluR5 subunit selective activation with the
attenuation of polyamine block by the edited (R) forms of GluRs, we
were able to demonstrate the coassembly of GluR5 with both GluR6 and
GluR7. Then analogous approaches were used to demonstrate that
GluR6 and GluR7 can coassemble also. Although further experiments will
be required to determine the subunit composition of native kainate
receptors, and in particular whether there exist heteromeric forms
assembled from more than two subunits (for example, both GluR5 and
GluR6 combined with KA1, KA2, or GluR7), the results of our experiments
are the first step toward this difficult goal.
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MATERIALS AND METHODS |
Mutagenesis cell culture and expression of recombinant
receptors. The GluR5 cDNA used in our experiments was the GluR5-2a splice variant described by Sommer et al. (1992); DNA sequencing confirmed that the construct used in our experiments had only 16 amino
acids in the C terminus that follows the last membrane-spanning domain
and no insert in N-terminal domain. The GluR6 construct we used (I567V
and Y571C) was fully edited in the first membrane domain (Köhler
et al., 1993). The GluR7 construct that was used was the GluR7a splice
variant (Schiffer et al., 1997). To create GluR7(R), we
performed site-directed mutagenesis by amplification in
vitro of pBSGluR7a(Q), using complimentary mutagenic
oligonucleotides (Life Technologies, Rockville, MD) and
Pfu polymerase (Stratagene, La Jolla, CA). Selection of
mutants was achieved by the subsequent digestion of parental plasmid
with DpnI. A 563 bp BglII fragment that included
the Q to R mutation was sequenced completely and subcloned into a
GluR7a eukaryotic expression vector construct in which a
cytomegalovirus promoter controls the transcription of cDNAs
(Keinänen et al., 1990). HEK 293 cells (CRL 1573, American Type
Culture Collection, Manassas, VA) were maintained at 70-80% confluence in Life Technologies' minimal essential medium with Earle's salts, 2 mM glutamine, and 10% fetal
bovine serum. Then 24 hr after being plated at low density (2 × 104 cells per ml) onto 35 mm Petri dishes,
the cells were transfected via the calcium phosphate technique (Chen
and Okayama, 1987). Kainate receptor cDNAs, 2.4 µg per 35 mm dish,
were cotransfected with 0.6 µg of the cDNA for green fluorescent
protein (S65T mutation) to aid in the identification of transfected
cells. For the coexpression of different receptor subunit combinations,
cDNAs were transfected at the desired ratios with the total cDNA
maintained at 3 µg per dish. The cells were washed with PBS 12-18 hr
after transfection and used for electrophysiological recordings after
another 24-48 hr.
Recording conditions. Electrophysiological recordings were
performed by using whole-cell patch clamp. The external solution contained (in mM) 145 NaCl, 5.4 KCl, 5 HEPES, 1 MgCl2, and 1.8 CaCl2. The
internal solution contained (in mM) 105 CsMeSO3, 10 CsF, 15 CsCl, 5 Cs4-BAPTA, 10 HEPES, 1 MgCl2, and 0.5 CaCl2 to which 60 µM spermine was added. The pH of both the
external and internal solutions was adjusted to 7.3; their osmolarity
was adjusted to 295 mOsm with sucrose. Before recording, we applied 0.3 mg/ml concanavalin A to individual cells for 4 min to attenuate
desensitization. ATPA and (S)-5-iodowillardiine were
gifts from Drs. P. Krogsgaard-Larsen (Royal Danish School of Pharmacy,
Copenhagen, Denmark), J. C. Watkins, and D. E. Jane
(University of Bristol, UK); additional samples of these drugs were
purchased from Tocris (Ballwin, MO). BAPTA was purchased from Molecular
Probes (Eugene, OR); all other reagents were purchased from Sigma (St.
Louis, MO) or Aldrich (Milwaukee, WI). Recordings were made with an
Axopatch-200B amplifier (Axon Instruments, Foster City, CA), using
fire-polished thin-walled borosilicate glass pipettes (2-5
M ). Series resistance (3-10 M) was compensated routinely by 90%. Records
were stored on a Power Macintosh G3 computer with a 16 bit
analog-to-digital converter (ITC-16; Instrutech, Elmont, NY) under the
control of the data acquisition program Synapse (Synergy Research,
Monrovia, MD). Agonists were applied via a stepper motor-based fast
perfusion system, as described previously (Vyklicky et al., 1990).
Data analysis. Current-voltage (I-V)
plots were generated with voltage ramps from  105 to +105 mV (0.42 V/sec). Procedures in the Igor program (WaveMetrics, Lake Oswego, OR)
were used to generate and analyze conductance-voltage
(G-V) plots. First, the reversal potential for
I-V plots was estimated by using a fifth-order polynomial
fit to the average of four leak-subtracted responses. Then
G-V plots were generated and fit with appropriate
equations. For GluR5(Q) and GluR6(Q) homomeric receptors the
G-V plots were fit with the Woodhull equation over the
range from 100 to +20 mV:
![G=G<SUB><UP>max</UP></SUB><FENCE><FENCE>1+<FR><NU>[<UP>Spm</UP>]</NU><DE>K<SUB><UP>D</UP>(<UP>0</UP>)</SUB><UP> exp</UP><FENCE><FR><NU>−V<SUB><UP>m</UP></SUB> z &thgr; F</NU><DE>RT</DE></FR></FENCE></DE></FR></FENCE></FENCE> ,](/content/vol19/issue19/fulltext/8281/img001.gif)
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(1)
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where Gmax is the conductance
at a sufficiently hyperpolarized potential to produce full relief from
block by polyamines; [Spm] is the cytoplasmic polyamine concentration
(60 µM spermine was added to the internal
solution); KD(0) is the dissociation constant for spermine at 0 mV membrane potential;
Vm is the membrane potential;
z is the valence and electrical distance for polyamine block. F, R, and T have their standard
values. For GluR6(R) responses and for heteromeric receptors with
outwardly rectifying G-V plots, the ratios of conductance
values at +80 to 80 mV were used to describe the extent of rectification.
For responses with intermediate rectification recorded on the
coexpression of the edited and unedited forms of GluRs, the G-V plots were fit with the sum of two Boltzmann functions
over the range of 100 to +50 mV:
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(2)
|
where Gmax and
Vm have the same meaning as defined
above; Vb is the membrane potential
for a half-block of each component by polyamines;
kb describes the voltage dependence of
the block for each component; and C is a constant. Then the
KD(0) values for polyamine block were
calculated from the relationship:
![K<SUB><UP>D</UP>(0)</SUB>=[<UP>Spm</UP>]<UP> exp </UP><FENCE><FR><NU>V<SUB><UP>b</UP></SUB></NU><DE>k<SUB><UP>b</UP></SUB></DE></FR></FENCE>.](/content/vol19/issue19/fulltext/8281/img003.gif)
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(3)
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Values in the text are mean ± SEM unless noted
differently. Statistical tests of differences between data sets were
performed with Student's t tests.
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RESULTS |
Selective activation of GluR5 by ATPA and
(S)-5-iodowillardiine
The design of our experiments required that we be able to
distinguish responses mediated by heteromeric kainate receptors containing more than one type of subunit from those mediated by homomeric receptors. To do this, we used kainate receptor subtype selective agonists and the change in rectification that occurs on
coassembly of the edited (R) and unedited (Q) forms of GluR subunits.
To facilitate the analysis of rectification that used ramp changes in
membrane potential, we attenuated the strong desensitization of kainate
receptors by a treatment of HEK cells with 0.3 mg/ml concanavalin A for
4 min (Partin et al., 1993; Everts et al., 1999). We began our study by
testing for the coassembly of GluR5 and GluR6. These experiments
revealed that, although both GluR5(Q) and GluR6(R) were activated by
100 µM kainate as expected, the amplitude of responses
for GluR5(Q), 354 ± 88 pA (n = 12) at 60 mV,
was approximately one-fifth of that for GluR6(R), 2024 ± 540 pA
(n = 7). Desensitization of kainate responses was
attenuated to a similar extent for both subtypes, such that the ratio
of peak responses to those measured at 200 msec was 1.08 ± 0.02 for GluR5(Q) and 1.02 ± 0.01 for GluR6(R). In contrast to the
nonselective agonist action of kainate, 10 µM
ATPA and 100 µM I-will produced responses only
for cells transfected with GluR5(Q), whereas for GluR6(R) these
agonists were completely inactive (Fig.
1A,B). On average, the
amplitude of GluR5(Q) responses to 10 µM ATPA was 0.83 ± 0.05 (n = 6) of those to kainate
recorded from the same cell, whereas for I-will the ratio was 1.29 ± 0.06 (n = 7). The attenuation of desensitization by
concanavalin A was less for responses to ATPA and I-will than to
kainate, but it was sufficient to generate large-amplitude equilibrium
responses (Fig. 1A).
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Figure 1.
Selective activation of GluR5 by ATPA and I-will.
A, Responses from a HEK cell transfected with GluR5(Q)
to 100 µM kainate, 10 µM ATPA, and 100 µM I-will at 60 mV. B, When the same
agonists were applied to a cell transfected with GluR6(R), only kainate
produced inward current responses. C, Ramp
I-V plots for responses to 100 µM I-will
recorded from HEK cells transfected with GluR5(Q) or both GluR5(Q) and
GluR6(R) at a cDNA ratio of 1:1. D, Ramp
I-V plot for responses to 100 µM kainate
recorded from a HEK cell transfected with GluR6(R). In all experiments
the cells were treated with concanavalin A to attenuate
desensitization.
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Analysis of rectification was performed by using ramp changes in
membrane potential from 105 to 105 mV (0.42 mV/msec) during equilibrium responses to selected agonists. As expected, the
I-V plots for GluR5(Q) responses to I-will were
birectifying because of a voltage-dependent polyamine block (Fig.
1C), whereas I-V plots for GluR6(R) responses to
kainate were outwardly rectifying (Fig. 1D). In
contrast, when GluR5(Q) and GluR6(R) were coexpressed at a cDNA ratio
of 1:1, the I-V plots for responses to 100 µM I-will were outwardly rectifying (Fig.
1C) even though, as shown above, I-will is inactive at
homomeric GluR6(R). This indicates that GluR5 and GluR6 can coassemble
to form heteromeric kainate receptors.
Overexpression of GluR6 dominates heteromer formation
with GluR5
To facilitate the analysis of the properties of heteromeric
kainate receptors generated by the coassembly of GluR5 with GluR6, we
used conductance-voltage plots (see Materials and Methods). When
GluR5(Q) and GluR6(R) were cotransfected at a cDNA ratio of 1:1, this
analysis revealed weak outward rectification for responses to both 10 µM ATPA (Fig.
2A) and 100 µM I-will (Fig. 2B). In
contrast, for homomeric GluR5(Q) the responses to these agonists showed
strong biphasic rectification similar to that reported previously for
the activation of unedited AMPA and kainate receptors (Bowie and Mayer,
1995). Surprisingly, when the cDNA ratios for GluR5(Q) to GluR6(R) were
changed over the range of 1:1, 2:1, and 5:1, the responses remained
outwardly rectifying (Fig. 2B), such that the ratio
of the conductance at +80 to 80 mV was 1:1 = 1.53 ± 0.03 (n = 8), 2:1 = 1.73 ± 0.06 (n = 13), and 5:1 = 1.68 ± 0.04 (n = 5). Only when the cDNA ratio for GluR5(Q) to
GluR6(R) was increased to 20:1 did we record responses with biphasic
rectification intermediate between that for homomeric GluR5(Q) and
GluR6(R) (Fig. 2C). The rectification of responses for the
20:1 ratio of cDNAs varied markedly from cell to cell (Fig.
2C); such extreme variation was not observed for the GluR5 to GluR6 cDNA ratios of 1:1, 2:1, or 5:1.
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Figure 2.
Functional overexpression of GluR6 dominates
heteromer formation with GluR5. Shown are G-V plots for
responses to 10 µM ATPA (A) and 100 µM I-will (B, C) recorded from HEK cells
transfected with homomeric GluR5(Q) or GluR5(Q) plus GluR6(R) at cDNA
ratios of 1:1, 2:1, 5:1, and 20:1, as indicated. In A
and B, the data points show the mean ± SEM for
4-12 cells per experiment; at cDNA ratios for GluR5(Q) to GluR6(R) of
1:1, 2:1, and 5:1 the responses to GluR5 selective agonists show a
similar weak outward rectification. C,
G-V plots for individual cells normalized to the
conductance at 100 mV; at cDNA ratios of 20:1 the rectification
varied from cell to cell.
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Previous studies have shown that the single-channel conductance of
GluR5(Q) is at least 50 to 100 times larger than that for GluR6(R)
(Swanson et al., 1996). The fivefold larger amplitude of kainate
responses for GluR6(R) versus GluR5(Q) described above suggests that in
HEK cells the functional expression of GluR6 is considerably more
efficient than that of GluR5 (see Fig. 1). We also observed a large
(>40-fold) difference in response amplitudes for GluR6(Q) versus
GluR5(Q) in Xenopus oocytes injected with equal amounts of
cRNAs for these subunits, suggesting similar behavior in multiple
heterologous expression systems. Replacing either the 5'-untranslated
region (UTR) or both the 5'-UTR and signal peptide of GluR5 with that
from GluR6 failed to increase the amplitude of GluR5 responses,
suggesting either that the translation, assembly, cell surface
expression, or stability of GluR5 is much less than that for GluR6 or
that GluR5 opens with low probability as compared with GluR6. However,
the latter possibility seems unlikely given similar maximum open
probabilities for native kainate receptors in dorsal root ganglion
(DRG) neurons (Huettner, 1990) and recombinant GluR6 (Traynelis and
Wahl, 1997) and given results suggesting that kainate receptors in DRG
neurons are generated by homomeric GluR5 (Partin et al., 1993; Swanson
et al., 1998).
Recent experiments on polyamine block of heteromeric AMPA receptors
suggest that the affinity for spermine varies with Q/R site
stoichiometry (Washburn et al., 1997). It thus seemed likely that the
strong attenuation of polyamine-mediated rectification observed for
heteromeric kainate receptors generated by the coexpression of GluR5(Q)
and GluR6(R) at cDNA ratios of 1:1, 2:1, and 5:1 (Fig. 2A,B) reflects a constrained stoichiometry of
assembly under these conditions rather than any functional dominance in
heteromeric kainate receptors of edited versus unedited subunits.
Evidence in support of this was obtained by examining changes in
rectification produced by the cotransfection of GluR5(Q) with GluR5(R)
or GluR6(Q) with GluR6(R). In single HEK cells we were unable to record
responses for homomeric GluR5(R) because of the low amplitude of
responses for this subunit (Sommer et al., 1992; Swanson et al., 1996); when expressed in Xenopus oocytes, the responses for
GluR5(R) were large enough to permit reliable G-V plot
analysis and revealed weak outward rectification indistinguishable from
that recorded for homomeric GluR6(R). Subsequent experiments on HEK
cells transfected with cDNAs for the Q and R forms of GluR5 at ratios
of 1:1 and 5:1 revealed rectification intermediate between that for
homomeric assemblies of edited or unedited GluR5 subunits (Fig.
3A). Similar results were obtained
on coexpression of GluR6(Q) and GluR6(R) (Fig. 3B).
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Figure 3.
Coassembly of edited and unedited GluR5 or
GluR6 generates multiple families of kainate receptors.
A, G-V plots of the responses to 100 µM kainate for homomeric GluR5(Q) or GluR5(Q) plus
GluR5(R) transfected at cDNA ratios of 1:1 and 5:1. B,
G-V plots for homomeric GluR6(Q), homomeric GluR6(R),
or GluR6(Q) plus GluR6(R) transfected at cDNA ratios of 1:1 and 5:1.
The symbols show the mean ± SEM of responses to
100 µM kainate for 5-11 cells per experiment; the
dotted line in A shows the responses for
homomeric GluR6(R). C, Responses to 50 µM
domoate for three cells transfected with GluR6(Q) plus GluR6(R) at a
cDNA ratio of 5:1; open circles indicate the fits of the
sum of two Boltzmann functions over the range of 100 to +50 mV.
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Although these results established that Q/R site stoichiometry in
kainate receptors regulates polyamine affinity in a manner similar to
that observed in previous studies on AMPA receptors (Washburn et al.,
1997), we found that, when the ratio of cDNAs for the Q and R forms was
increased from 1:1 to 5:1, there was considerable heterogeneity in the
extent of rectification from cell to cell. Such variability resembled
that described previously when GluR5 and GluR6 were coexpressed at a
cDNA ratio of 20:1 (see Fig. 2). In many cases the G-V
plots for individual cells transfected with both the Q and R forms of
either GluR5 or GluR6 clearly showed multiple components of polyamine
block, visible as inflections on the descending limb of the
G-V plot; this was particularly true for GluR6 (Fig.
3C), although similar results were obtained in some cells
for GluR5. As a result, in these cells the G-V plots were
fit well by the sum of two Boltzmann functions, but not by a single
Boltzmann (Fig. 3C). The simplest explanation for this would
be the existence in single cells of multiple populations of kainate
receptors with different KD values for
polyamine block because of the incorporation of Q and R forms at
different stoichiometry. Indeed, if the assembly of GluRs within a
single gene family is dictated mainly by the relative concentrations of
subunits within the endoplasmic reticulum, one would expect to observe
multiple receptor populations with different sensitivity to polyamine
block rather than the apparently homogeneous receptor population
described in previous studies on heteromeric AMPA receptors expressed
at different ratios of Q to R forms (Washburn et al., 1997). Thus, our
results are explained best by the existence in individual cells of
multiple receptor populations with different affinities for polyamines.
For the coexpression of GluR6(Q) and GluR6(R) at a 5:1 ratio, the
KD values for spermine block,
calculated from fits of the sum of two Boltzmann functions (Eqs. 2, 3),
were 4.5 ± 1.1 µM for the high-affinity
component of the block, similar to previous estimates for homomeric
GluR6(Q) (Bowie and Mayer, 1995; Bähring et al., 1997), and
238 ± 99 µM (mean ± SEM) for the
low-affinity component of the block for the three cells shown in Figure
3C, with the proportion of receptors with high and low affinity for spermine varying from cell to cell.
Responses to GluR5 selective agonists upregulated by
coexpression with GluR6
The amplitude of responses to I-will appeared to be larger when
GluR5 was coexpressed with GluR6 as compared with the responses that
were recorded when GluR5 was expressed alone, suggesting that
heteromeric kainate receptors assembled from GluR5 and GluR6 may have
novel functional properties. This was true both for the coexpression of
GluR5(R) with GluR6(Q) and for the coexpression of GluR5(Q) with GluR6(R).
In individual HEK cells we were unable to record responses for
homomeric GluR5(R) larger than a few picoamps, in agreement with the
results of previous studies (Sommer et al., 1992; Swanson et al.,
1996). In contrast, when GluR5(R) was coexpressed with GluR6(Q) at a
1:1 ratio of cDNAs, outwardly rectifying responses were evoked reliably
by the GluR5 selective agonist 100 µM I-will, with a mean
amplitude of 165 ± 38 pA at 100 mV (Fig.
4A,C). Although we could
not quantify the effect, this suggests that functional upregulation
must occur for GluR5(R) on coexpression with GluR6(Q). Strikingly,
responses in the same cells to 100 µM kainate,
a nonselective agonist that activates both GluR5 and GluR6, on average
were 50 times larger than those to 100 µM
I-will and displayed strong biphasic rectification, albeit somewhat
weaker than that for homomeric GluR6(Q), suggesting that many receptors
had failed to incorporate GluR5(R).
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Figure 4.
Coassembly of GluR5 and GluR6 upregulates the
responses to GluR5 selective agonists. A,
G-V plots for responses to 100 µM I-will
and 100 µM kainate scaled to have the same amplitude at
100 mV for cells transfected with both GluR5(R) with GluR6(Q) at a
cDNA ratio of 1:1; the symbols show the mean ± SEM
of responses for six cells; the unscaled response to I-will is plotted
as a dotted line. B, G-V
plots for responses to 100 µM I-will and 100 µM kainate normalized to have the same amplitude at 100
mV for cells transfected with both GluR5(Q) and GluR6(R) at a cDNA
ratio of 1:1. The symbols show the mean ± SEM of
responses for nine cells; the unscaled response to I-will is plotted as
a dotted line. C, Box plots of the mean
amplitude of I-will-evoked currents at 100 mV for the edited and
unedited versions of GluR5 expressed alone or with GluR6 at a cDNA
ratio of 1:1, as indicated; the cells were treated with concanavalin A
to attenuate desensitization. The top and bottom
boundaries of the boxes indicate 25 and 75% of
the data, and the whiskers indicate 10 and 90% of the
data. The median is indicated by a bold bar; the
shaded areas indicate the mean ± SD. Note that the
coexpression of GluR6 upregulates the amplitude of responses to
I-will.
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The amplitude of responses to the GluR5 selective agonist I-will also
were upregulated when GluR5(Q) was coexpressed with GluR6(R) (Fig.
4C). On average, after attenuation of desensitization by
concanavalin A the responses to 100 µM I-will
at 100 mV were 250 ± 46 pA (n = 9) for
homomeric GluR5(Q), 933 ± 203 (n = 8) when
GluR5(Q) was coexpressed with GluR6(R) at a 5:1 ratio, 1321 ± 620 pA (n = 10) at a 2:1 ratio, and 1609 ± 259 pA
(n = 9) at a 1:1 ratio. When GluR5(Q) and GluR6(R) were
coexpressed at a 1:1 cDNA ratio, the responses to both kainate and
I-will showed weak outward rectification (Fig. 4B).
The amplitude of responses to kainate was, on average, 2.2-fold larger
than those to I-will recorded in the same cells. This suggests
functional overexpression of GluR6(R) versus GluR5(Q), because after
concanavalin A treatment the responses of homomeric GluR5(Q) to kainate
and to I-will had similar steady-state amplitudes, whereas while
GluR6(R) has a much smaller single-channel conductance than GluR5(Q)
(Swanson et al., 1996). Alternatively, as discussed later, a larger
single-channel conductance of GluR5(Q) plus GluR6(R) heteromers when
all four subunits are activated by the nonselective agonist kainate, as compared with the response when only GluR5 subunits in the same GluR5
plus GluR6 heteromers are activated by I-will, also would be consistent
with the results that were obtained.
Coexpression of GluR5 and GluR6 regulates kainate
receptor desensitization
Previous studies have revealed that for homomeric GluR6 the
responses to kainate desensitize rapidly and almost completely for Q/R
site-edited and unedited forms (Partin et al., 1993; Swanson et al.,
1997; Traynelis and Wahl, 1997). For homomeric GluR5(Q) and for native
kainate receptors in DRG neurons, the responses to I-will also show
strong desensitization with fast kinetics (Wong et al., 1994; Swanson
et al., 1998). The present study revealed that, when GluR5 and GluR6
are coexpressed, the resulting heteromeric kainate receptors show
reduced desensitization with an enhanced steady-state current in
response to either I-will or kainate (Fig. 5). For responses to 100 µM
I-will the steady-state currents were 1.4 ± 0.4% of peak
(n = 12) for homomeric GluR5(Q) but 10.7 ± 0.9%
(n = 13) for GluR5(Q) plus GluR6(R) expressed at a cDNA
ratio of 1:1. In these experiments there is no doubt that the change in
desensitization results from the formation of heteromeric receptors, because GluR6(R) is unresponsive to I-will. On average, the
steady-state amplitude of responses to 100 µM
kainate was only 1.3 ± 0.2% of peak (n = 7) for
homomeric GluR6(R), whereas for GluR5(Q) plus GluR6(R) coexpressed at a
cDNA ratio of 1:1, 10 of 19 of cells showed much larger steady-state
currents, on average 6.0 ± 1.0% (n = 10) of the
peak response. Given the evidence for the functional overexpression of
GluR6 (see Fig. 2), it is likely that in the remaining nine cells the
responses to kainate were dominated by homomeric GluR6(R). Compared
with responses for homomeric assemblies of GluR6(Q) or GluR6(R), the
reduced desensitization observed on the coexpression of GluR5 and GluR6
brings to mind the enhanced steady-state response reported in some
experiments on native kainate receptors in hippocampal neurons (Ruano
et al., 1995; Wilding and Huettner, 1997; Paternain et al., 1998),
suggesting that, at least in some cells, native kainate receptors may
be heteromers containing GluR5 and GluR6, perhaps in combination with
other subunits.
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Figure 5.
Coassembly of GluR5 and GluR6 reduces kainate
receptor desensitization. A, Responses to 100 µM kainate recorded from HEK cells expressing homomeric
GluR6(R) or GluR5(Q) plus GluR6(R) at a cDNA ratio of 1:1.
B, Responses to 100 µM I-will recorded
from HEK cells expressing homomeric GluR5(Q) or GluR5(Q) plus GluR6(R)
at a cDNA ratio of 1:1. C, Desensitization, expressed as
a steady-state/peak current, for homomeric GluR6(R), homomeric
GluR5(Q), and GluR5(Q) plus GluR6(R) heteromers. The steady-state
current amplitude was measured at 1 sec after the start of the
application of the agonists. The box plots were generated by the method
described in Figure 4C.
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Although desensitization is reduced on the coexpression of GluR5(Q) and
GluR6(R), the rate of onset of desensitization was not greatly
affected. Previous studies found that homomeric GluR6 displays
fast-desensitizing responses to kainate (Heckmann et al., 1996),
whereas homomeric GluR5 shows profound heterogeneity in the rate of
onset of desensitization (Swanson and Heinemann, 1998). When GluR5(Q)
was coexpressed with GluR6(R), we found that responses to 100 µM kainate also exhibited inter-cell variability. We
observed two types of desensitization: a "fast" type that can be
well fit by a single exponential,  =12.9 ± 0.8 msec
(n = 9), and a "slow" type best fit by the sum of
two exponentials, 1 = 8.7 ± 0.8 msec
(A1 = 74 ± 7.1%) and
2 = 49.2 ± 10.4 msec (n = 10). The "fast" type of desensitization appeared not to differ from that for the responses of homomeric GluR6(R) to 100 µM kainate, = 12.5 ± 0.8 msec
(n = 6). However, both the "fast" and "slow" types of desensitization developed with smaller time constants than
those for responses of homomeric GluR5(Q) to 100 µM kainate: "fast"
1 = 17.7 ± 2.7 msec (A1
41 ± 9.6%) and 2 = 432 ± 85 msec (n = 7); "slow" 1 = 117 ± 20 msec (A1 33.2 ± 8.0%) and
2 = 452 ± 56 msec (n = 3). These results suggest that GluR6(R) is dominant over GluR5(Q) in
controlling the rate of onset of desensitization. Similar to the
results obtained for kainate, the responses to 100 µM I-will for homomeric GluR5(Q) also exhibited
cell to cell heterogeneity in the rate of onset of desensitization. For
3 of 12 cells the responses were well fit by a single exponential, = 8.8 ± 1.3 msec; for 9 of 12 cells the onset of
desensitization was fit by the sum of two exponentials,
1 = 8.0 ± 1.1 msec
(A1 = 78 ± 3.3%) and 2
= 132 ± 19 msec. When GluR5(Q) was coexpressed with
GluR6(R), the responses of individual cells to I-will also displayed
"fast" and "slow" types of desensitization, with time constants
similar to those for homomeric GluR5(Q).
For homomeric GluR5 expressed in HEK cells and for native kainate
receptors expressed in DRG neurons, the recovery from desensitization evoked by I-will proceeds extremely slowly, with time constants of 2.5 and 4 min, respectively (Wong et al., 1994; Swanson et al., 1998).
Rundown proceeds on the same time scale, making an accurate measurement
of the kinetics of recovery from desensitization for I-will extremely
difficult. To avoid this, we compared the extent of recovery from
desensitization at a single 30 sec time point for homomeric GluR5(Q)
and for GluR5(Q) plus GluR6(R) coexpressed at a cDNA ratio of 1:1 (Fig.
6). With twin-pulse applications of 500 µM I-will separated by 30 sec, we observed 10.9 ± 1.7% (n = 5) recovery for homomeric GluR5(Q) but
48.2 ± 2.6% (n = 8) recovery for the 1:1
cotransfection of GluR5(Q) and GluR6(R). If we assume that recovery
from desensitization follows single-exponential kinetics, the time
constants given by these values would be 4.9 ± 1.0 min for
homomeric GluR5(Q), in good agreement with previous estimates, and
0.78 ± 0.06 min for the 1:1 cotransfection of GluR5(Q) to
GluR6(R). These experiments clearly reveal that the coexpression of
GluR5(Q) with GluR6(R) speeds up recovery from desensitization, which
might help to explain why hippocampal mossy fiber kainate receptor
EPSCs do not inactivate during high-frequency presynaptic stimulation
(Castillo et al., 1997; Vignes and Collingridge, 1997). We performed
these experiments with the GluR5 selective agonist I-Will because,
although knowledge of the changes in the kinetics of recovery from
desensitization for glutamate is the physiologically relevant parameter
that we need to measure to understand the behavior of EPSCs, the large
population of homomeric GluR6 present when GluR5 and GluR6 are
coexpressed (see Fig. 2) would interfere with the required
observations.
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Figure 6.
Coassembly of GluR5 and GluR6 speeds recovery from
desensitization. A, Responses to paired applications of
500 µM I-will separated by 30 sec intervals reveal
persistent desensitization for homomeric GluR5(Q). B,
Recovery from desensitization is much faster when GluR5(Q) is
coexpressed with GluR6(R) at a 1:1 ratio of cDNAs. C,
Box plots for recovery from desensitization measured at 30 sec
intervals between paired applications of I-will. The box plots were
generated by the method described in Figure 4C.
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Coassembly of GluR7 with GluR5 and GluR6
Because I-will does not activate homomeric kainate receptors
assembled from GluR7 (Swanson et al., 1998), we were able to use the
same strategy to test for the coassembly of GluR5 with GluR7 as was
used in experiments with GluR6. Because GluR7 does not undergo Q/R site
RNA editing (Lomeli et al., 1992), it was necessary first to generate
the required construct by site-directed mutagenesis. When GluR5(Q) was
coexpressed with GluR7(R) at cDNA ratios of 1:1 and 5:1, the responses
to I-will recorded after concanavalin A treatment showed reduced
biphasic rectification as compared with those for homomeric GluR5(Q)
(Fig. 7A). This result indicates
that GluR5 and GluR7 can coassemble to form heteromeric receptors. Two
notable differences were observed in these experiments when compared
with the results obtained for the coexpression of GluR5 with GluR6.
First, biphasic rectification because of polyamine block was reduced to
a much greater extent for the 1:1 cDNA ratio of GluR5(Q) to GluR7(R)
than for the 5:1 cDNA ratio. This is similar to results obtained when
the edited and unedited forms of GluR5 or GluR6 were coexpressed (see
Fig. 3) but different from results obtained on coexpression of GluR5(Q)
and GluR6(R) (see Fig. 2). Second, the amplitude of responses to I-will
did not show clear evidence for upregulation on the coexpression of
GluR5(Q) with GluR7(R). This was in marked contrast to results obtained
for the coexpression of GluR5(Q) with GluR6(R). The mean amplitude of
responses to 100 µM I-will was 250 ± 46 pA (n = 9) for homomeric GluR5(Q), 312 ± 76 pA
(n = 8) for the 5:1 ratio of GluR5(Q) and GluR7(R), and
355 ± 100 pA (n = 4) for the 1:1 ratio; these
values were not statistically different (p > 0.05). In the same cells the amplitude of responses to 100 µM kainate decreased slightly as the ratio of
cDNAs for GluR5(Q) to GluR7(R) was decreased (Fig. 7B); the
mean amplitudes were 254 ± 38 pA (n = 9) for
homomeric GluR5(Q), 167 ± 29 pA (n = 8) for the
5:1 ratio of GluR5(Q) to GluR7(R), and 126 ± 31 pA
(n = 4) for the 1:1 cDNA ratio. Although these values
were also not statistically different (p > 0.05), the decline in the ratio of the amplitude of responses to
kainate and I-will in individual cells was highly significant; the
ratios were 1.09 ± 0.09 for homomeric GluR5(Q), 0.69 ± 0.10 (p < 0.01) for the 5:1 ratio of GluR5(Q) to
GluR7(R), and 0.36 ± 0.02 (p < 0.01) for
the 1:1 cDNA ratio (Fig. 7B).
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Figure 7.
Coassembly of GluR5 and GluR6 with GluR7.
A, G-V plots of responses to 100 µM I-will for homomeric GluR5(Q) or GluR5(Q) and GluR7(R)
transfected at cDNA ratios of 1:1 and 5:1; the symbols
indicate the mean ± SEM for three to five cells per experiment.
B, Mean amplitude of responses to 100 µM
kainate and 100 µM I-will recorded from cells transfected
with homomeric GluR5(Q) or GluR5(Q) plus GluR7(R) at cDNA ratios of 5:1
and 1:1; the error bars indicate the mean ± SEM.
C, G-V plots of responses to 500 µM kainate for homomeric GluR6(Q590E) or GluR7(R) and
GluR6(Q590E) transfected at a cDNA ratio of 1:1; the
symbols indicate the mean ± SEM for five to six
cells per experiment. D, Mean amplitude of responses to
500 µM kainate recorded from cells transfected with
homomeric GluR6(Q590E) or GluR7(R) plus GluR6(Q590E); the error bars
indicate the mean ± SEM. In all experiments the cells were
treated with concanavalin A to attenuate desensitization.
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The low affinity of GluR7(Q) for glutamate and kainate, rapid and
complete desensitization to these agonists, and the lack of effect of
concanavalin A on desensitization for GluR7(Q) (Schiffer et al., 1997)
made it possible to test for the coassembly of GluR6 with GluR7 by
recording responses to concentrations of kainate <1 mM,
which are ineffective in activating GluR7 (Schiffer et al., 1997). In
contrast to the results obtained for coexpression with GluR5(Q), when
GluR7(R) was coexpressed with GluR6(Q) at cDNA ratios of 1:1, 2:1, and
5:1, extremely variable results were obtained. For some cells biphasic
rectification was strongly attenuated, which indicates the formation of
heteromers between GluR6(Q) and GluR7(R), whereas other cells showed
little change in biphasic rectification as compared with the responses
for wild-type GluR6(Q). The later case is probably attributable to a
much larger macroscopic conductance for homomeric GluR6(Q) that in many
cells most likely obscures the response of heteromers formed by the
coassembly of GluR6(Q) and GluR7(R). At high (10:1) ratios of cDNAs for
GluR7(R) to GluR6(Q), in many cells the responses to 500 µM kainate were too small to analyze accurately. These
results suggest that coassembly with GluR7(R) downregulates the high
levels of functional expression typical for homomeric GluR6(Q). When we
repeated these experiments by using a mutant, GluR6(Q590E), which gives
functional responses ~100 times smaller than for wild-type GluR6(Q)
but which has a threefold lower affinity for spermine than wild-type
GluR6(Q) (Panchenko et al., 1999), more consistent results were
obtained on coexpression with GluR7(R) at cDNA ratios of 1:1. For all
of the cells that were tested, coexpression with GluR7(R) strongly attenuated the biphasic rectification characteristic of polyamine block
for homomeric GluR6(Q590E) (Fig. 7C). In addition, these experiments revealed marked attenuation of the amplitude of responses to kainate on coexpression with GluR7(R) (Fig. 7D). The mean
amplitudes of responses to 500 µM kainate were
2160 ± 740 pA (n = 6) for homomeric GluR6(Q590E)
but only 110 ± 41 pA (n = 5) for the 1:1 ratio of
GluR6(Q590E) to GluR7(R).
| |
DISCUSSION |
Our experiments were designed to address the issue of whether
kainate receptor subunits from the GluR5-GluR7 gene family can coassemble to form heteromeric receptors with novel functional properties. Our results show clearly that they do. However, our results
also raise a number of general issues related to the properties of
heteromeric GluRs, ranging from the biophysical details of the
mechanism of receptor gating to the signals mediating subtype-specific assembly of selected GluR subtypes and the likely subunit composition and role of kainate receptors in vivo.
Functional properties of heteromeric kainate receptors
It would be predicted that, at saturating concentrations of GluR5
selective agonists, the maximal occupancy of heteromeric receptors
generated by the assembly of GluR5 with GluR6 or GluR7 will be less
than for homomeric GluR5 and will vary with subunit composition. The
recent study of Rosenmund et al. (1998), which relates AMPA receptor
single-channel conductance to the number of subunits occupied by an
agonist, argued that GluRs were tetramers and that occupancy of a
minimum of two agonist binding sites was required to allow the Q form
of GluR3 to open to a subconductance state of 5 pS, with the binding of
three and four agonist molecules producing openings to states of 15 and
23 pS. This scheme has the consequence that some of the subunit
combinations likely to be generated under the conditions of our
experiments will not generate currents in response to GluR5 selective
agonists, even though an agonist has bound to the receptor, whereas
other subunit combinations will produce much less than the maximum
response possible when all agonist binding sites are occupied (Fig.
8). When GluR5 is coexpressed with GluR6,
according to the scheme of Rosenmund et al. (1998), we should not
detect any response to GluR5 selective agonists for receptors of
subunit composition 1GluR5:3GluR6, leaving only 2GluR5:2GluR6,
3GluR5:1GluR6, and homomeric GluR5 as possible functional combinations
(Fig. 8). However, because of the functional overexpression of GluR6
when GluR5 and GluR6 are coexpressed at cDNA ratios of 1:1, 2:1, and even 5:1, the receptors of composition 2GluR5:2GluR6 are likely to be
the major population that responds to GluR5 selective agonists rather than 3GluR5:1GluR6 or homomeric GluR5. If GluRs are pentamers and not tetramers, the quantitative details will be different but the
result remains that single-channel conductance, and thus macroscopic
current amplitude, will increase with the number of subunits occupied
by an agonist. Such a scheme helps to explain the similar rectification
for I-will responses when GluR5(Q) is coexpressed with GluR6(R) or when
GluR5(R) is coexpressed with GluR6(Q) (see Figs. 2, 4), although we
would expect functional overexpression of GluR6(R) in the first case
and of GluR6(Q) in the second case. We suggest that, for coexpression
of GluR5(Q) and GluR6(R), the dominant functional heteromer activated
by GluR5 selective agonists will be 2GluR5(Q):2GluR6(R), whereas for
coexpression of GluR5(R) and GluR6(Q) the dominant functional heteromer
most likely will be 2GluR5(R):2GluR6(Q). Our results suggest that, no
matter which form (Q or R) is activated by GluR5 selective agonists,
heteromers of GluR5 and GluR6 containing 2R and 2Q subunits will
exhibit similar outward rectification.
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Figure 8.
Molecular mechanisms for kainate receptor
diversity. The scheme illustrates possible receptor combinations that
could be formed by coassembly of the top row, GluR5(Q)
with GluR6(R), and the bottom row, GluR5(R) with
GluR6(Q), assuming a tetrameric stoichiometry.
Imax indicates the maximum response that
could be generated by a GluR5 selective agonist according to the values
published by Rosenmund et al. (1998) for the activation of homomeric
AMPA receptors at the appropriate receptor occupancies by GluR5
selective agonists; NR indicates that these forms would
not be expected to respond to I-will or ATPA. Mean  indicates the maximum values for single-channel conductance recorded by
Swanson et al. (1996) for the Q and R forms of homomeric GluR5 and
GluR6.
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The results of experiments in which GluR5(Q) and GluR5(R) or GluR6(Q)
and GluR6(R) were coexpressed (see Fig. 3) help to address the issue of
the identity of the subunit composition of receptors with intermediate
affinity for polyamine block and provide information for an
interpretation of responses from heteromers containing more than one
kainate receptor gene product. Although outward rectification that is
independent of polyamine block (Bähring et al., 1997) limits the
resolution of these experiments, in cells transfected at a 5:1 ratio of
GluR6(Q) to GluR6(R) the G-V plots were well fit, assuming
only two polyamine-sensitive receptor populations, expressed at
different relative amounts (see Fig. 3C). One of these, with
an affinity for spermine similar to that for homomeric GluR6(Q), most
likely is a tetramer of unedited subunits. The second population, with
50-fold lower affinity, most likely represents the form 3Q:1R. If this
is true, it seems reasonable that the forms 2Q:2R and 1Q:3R would have
even lower affinity for polyamines and not be detectable as inflections
on G-V plots under the conditions of our experiments.
Returning to the case of heteromers formed by coassembly of GluR5 and
GluR6 (see Figs. 2, 4), our results support the proposal that 2Q:2R heteromers, which we believe are not blocked by cytoplasmic
concentrations of polyamines under the conditions of our experiments,
underlie the weak outward rectification observed for I-will responses
for both GluR5(Q):GluR6(R) and GluR5(R):GluR6(Q) expressed at 1:1 cDNA ratios.
The coexpression of GluR6 or GluR7 with GluR5 had different effects on
the amplitude of equilibrium responses to agonists expected to activate
GluR5 subunits selectively (see Figs. 4, 7). In the case of GluR6,
which generates functional receptors with much higher efficiency than
other AMPA or kainate receptor subunit combinations, it is possible
that assembly or cell surface expression is facilitated, as compared
with other GluR subunits, and that GluR6 acts as a chaperone when
combined with GluR5. It is also possible that the upregulation of the
amplitude of I-will responses is attributable to an effect of GluR6 on
the gating of GluR5. Evidence consistent with such an effect is the
observation that the coassembly of GluR5 with GluR6 reduces the extent
of and speeds recovery from desensitization for GluR5 selective
agonists. When GluR7 was coexpressed with GluR5, there was a reduction
in the relative amplitude of responses to kainate versus I-will (see Fig. 7B). This indicates that GluR7 and GluR6 have different
effects on GluR5. When GluR7 was coexpressed with GluR6, the amplitude of equilibrium responses to kainate was reduced dramatically (see Fig.
7C). Although high concentrations of kainate are required to
activate GluR7 channel gating (Schiffer et al., 1997), lower concentrations most likely produce desensitization; it is possible that, when GluR7 combines with GluR6, strong desensitization mediated by GluR7 interferes with the activation of GluR6 subunits even after
treatment with concanavalin A. At the present time it is unknown
whether the desensitization of GluRs occurs independently in individual
subunits or whether this is a cooperative process in which individual
subunits are coupled allosterically to their neighbors.
Subunit composition of native kainate receptors
Kainate receptor mRNAs are expressed widely throughout the CNS
with levels that are higher at early developmental stages (Bettler et
al., 1990; Wisden and Seeburg, 1993; Bahn et al., 1994; Bischoff et
al., 1997). Although the brain region-specific expression patterns for
GluR5, GluR6, and GluR7 are unique, they overlap in multiple regions.
However, relatively few studies directly address whether single cells
express more than one member of this gene family. Acutely isolated,
purified cerebellar granule cells were shown by PCR to express RNA for
both GluR5 and GluR6 (Belcher and Howe, 1997), with similar results
obtained by single-cell PCR after short-term culture (Pemberton et al.,
1998). The extent of RNA editing was different for the two kainate
receptor subunits and increased during development but to different
extents such that, by postnatal day 15, the most common forms were
GluR5(Q) and GluR6(R). Although pharmacological approaches convincingly
showed functional kainate receptors to be present in cerebellar granule
cells (Pemberton et al., 1998), their physiological role and subunit
composition remain unknown, but it is noteworthy that kainate receptors
are expressed in the external germinal layer, before the excitatory synaptic innervation of granule cells (Ripellino et al., 1998).
In the case of hippocampal neurons, single-cell PCR of unidentified
cells in short-term culture, which showed rapidly desensitizing responses to kainate, revealed the expression of GluR6 in 11of 11 cells
that were examined, with coexpression of GluR5 in only 3 of the 11 cells (Ruano et al., 1995). However, as noted above, although mossy
fiber kainate receptor-mediated EPSCs in CA3 neurons are absent in
GluR6/ mice, the functional properties
of these synaptic currents are not well explained by those of homomeric
GluR6. Most likely this is because additional subunits are required to
generate or correctly target the postsynaptic kainate receptors in CA3
neurons. These additional subunits could be GluR5, KA1, or KA2, because
the coassembly of GluR6 with either GluR5 or KA2 confers sensitivity to
ATPA or I-will (Swanson et al., 1998). Despite this, experiments with GluR6/ mice indicate that GluR6 plays
a unique role in generating synaptic kainate receptors in CA3 neurons,
perhaps by stabilizing or enhancing the functional response of other
kainate receptor subunits. In contrast, trigeminal ganglion neurons
(Sahara et al., 1997) and DRG neurons (Partin et al., 1993) express
mRNA for GluR5 at much higher levels than for GluR6 or other kainate
receptor subunits, suggesting considerable diversity of kainate
receptor subunit composition in different areas of the CNS. This is
reinforced by the results of in situ hybridization that
reveal a large number of potential subunit combinations (Wisden and
Seeburg, 1993; Bahn et al., 1994; Bischoff et al., 1997). Clearly, much
remains to be learned about kainate receptors at many levels, from
basic properties to their role in synaptic circuitry and behavior.
| |
FOOTNOTES |
Received June 2, 1999; revised July 19, 1999; accepted July 21, 1999.
We thank Drs. P. Seeburg and S. Heinemann for cDNAs; Drs. J. C. Watkins, D. E. Jane, and P. Krogsgaard-Larsen for gifts of (S)-5-iodowillardiine and ATPA; Dr. V. Panchenko
for Igor templates; Ms. Carla Glasser for technical assistance; and Dr.
C. McBain for comments on this manuscript.
Correspondence should be addressed to Dr. M. L. Mayer, Building
49, Room 5A78, National Institutes of Health, 49 Convent Drive, Bethesda MD 20892-4495.
| |
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ABSTRACT
INTRODUCTION
