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Molecular Pharmacology, Volume 52, Issue 5, 861-873
Glutamate Receptor Laboratory, Max-Planck-Institute for Experimental Medicine, D-37075 Göttingen, Germany
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Summary
Introduction
Materials & Methods
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Discussion
References
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All ionotropic glutamate receptor (iGluR) subunits analyzed so far are
heavily N-glycosylated at multiple sites on their
amino-terminal extracellular domains. Although the exact functional
significance of this glycosylation remains to be determined, it has
been suggested that N-glycosylation may be a
precondition for the formation of functional ion channels. In
particular, it has been argued that N-glycosylation is
required for the formation of functional ligand binding sites. We
analyzed heterologously expressed recombinant glutamate receptors
(GluRs) of all three pharmacological subclasses of glutamate receptors,
N-methyl-D-aspartate (NMDA),
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and kainate
receptors. By expressing the GluR subunits in tunicamycin-treated,
nonglycosylating Xenopus laevis oocytes, we determined
that in neither case is N-glycosylation required for ion
channel function, although for NMDA receptors, functional expression in
the absence of N-glycosylation is very low. Furthermore,
we analyzed and compared the interaction of the
desensitization-inhibiting lectin concanavalin A (ConA) with all
functional GluR subunits. We show that although ConA has its most
pronounced effects on kainate receptors, it potentiates currents at
most other receptor subtypes as well, including certain NMDA receptor
subunits, although to a much lesser extent. One notable exception is
the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor
GluR2, which is not affected by ConA. Furthermore, we show that ConA
acts directly via binding to the carbohydrate side chains of the
receptor protein.
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Introduction |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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iGluRs are the prevalent excitatory neurotransmitter receptors in the central nervous system of vertebrates (1). They can be classified into three major subfamilies on the basis of pharmacological and electrophysiological profiles: NMDA, AMPA, and KA receptors. Being the main mediators of cell-to-cell signaling, GluRs are regulated and functionally modulated by a multitude of post-transcriptional and post-translational mechanisms such as alternative splicing, RNA editing, protein phosphorylation, palmitoylation, and N-glycosylation (for a review, see Ref. 2). iGluRs are thought to consist of five subunits and may form heteromeric receptors containing different subunits, in many cases resulting in a receptor complex with different properties than their constituent subunits. For some receptor subunits, function can be demonstrated only on coexpression with another subunit of the same subfamily (for a review, see Ref. 2).
The amino acid sequences of iGluR subunits contain 4-12 potential
extracellular sites for N-glycosylation, which conform to the universal consensus sequence N-X-S/T, with X 
P. These
sites are marked in Fig. 1 and occur in
the two large domains of iGluRs that according to the recently proposed
three-transmembrane domain model, are located in the two putatively
extracellular domains, the amino terminus and the loop between
transmembrane domains B and C (3, 4). Many but not all of the sites are
conserved within or even across subfamilies; however, no site is
conserved across all iGluRs. The use of at least some of these
consensus sites has been confirmed (5). For GluR1 alone, a systematic investigation of all existing consensus sites demonstrated that each of
the six sites is glycosylated, at least in oocytes (3); however, the
functional role of the carbohydrate moieties of iGluRs is not clear.
Carbohydrate side chains have been implicated in many diverse
functions, including protection from proteases, correct assembly of the
subunits, formation of ligand binding sites, receptor targeting to the
cell surface, and recognition by extracellular modulators (for a
review, see Ref. 6). Importantly, the role of
N-glycosylation in receptor function seems to vary widely
among different receptors.
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There have been contradictory reports regarding whether N-glycosylation is essential for GluR function. Several groups have claimed that glycosylation is required for channel function (7, 8) or ligand binding (9-11), whereas other studies found no such requirements (3, 12). We therefore reinvestigated this question to determine whether N-glycosylation is necessary for channel function.
Carbohydrate side chains attached to N-glycosylation consensus sites of iGluRs most likely are the sites of interaction with lectins such as ConA. ConA has been found to potentiate current responses of native (13-17) and recombinant (18-21) iGluRs, with a strong selectivity for KA receptors over AMPA receptors. It is assumed that ConA acts by inhibiting receptor desensitization (14, 15). Several studies have reported the potentiation of selected iGluR subunits by ConA (18-20, 22), but not all of the functional subunits have been examined, and no study has compared the effects of ConA at all members of the iGluR family. Specifically, NMDA receptors have not been investigated in great detail, and the two existing reports on ConA modulation of these receptors are contradictory (18, 19). NMDA receptor splice variants have not been examined at all. Because the potentiating effect of ConA is increasingly used as a pharmacological tool to identify subsets of iGluRs in tissue slices or cell culture, it is important to know which effects ConA exerts on each of the known functional iGluR subunits or combinations; therefore, we systematically investigated the modulatory properties of ConA for all cloned subtypes of iGluRs.
This study shows that (a) N-glycosylation is not strictly essential for functional expression of any iGluR subunit combination but may affect current amplitudes and (b) receptor modulation by ConA is a direct effect of the lectin on the receptor protein and is subunit and splice variant dependent.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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ConA (grade VI) was obtained from Sigma Chemie (Munich, Germany). Tunicamycin was purchased from Boehringer-Mannheim Biochemica (Mannheim, Germany). Other drugs were purchased from Sigma unless noted otherwise. Adult female frogs (Xenopus laevis) were obtained from Nasco (Fort Atkinson, WI).
cRNA synthesis. Template was prepared from circular plasmid cDNA by linearization of each clone with a suitable restriction enzyme. cRNA was prepared from 1 µg of linearized template using an in vitro transcription kit (Stratagene, Heidelberg, Germany) with a modified standard protocol that uses each of the nucleotides at 800 µM (except GTP, for which 200 µM was used), 800 µM m7GpppG (Pharmacia, Vienna, Austria) for capping, and an extended reaction time of 3 hr with T3 or T7 RNA polymerase. All cRNAs were trace-labeled with [32P]UTP (Amersham, Braunschweig, Germany) to allow quality checks by gel electrophoresis and calculation of the yield.
Electrophysiological recordings from X. laevis
oocytes.
Frog oocytes of stages V or VI were obtained by surgical
removal of parts of the ovaries of X. laevis anesthetized
with 3-aminobenzoic acid ethyl ester (2 g/liter). The removed ovaries
were chopped and incubated with 815 units/ml (2.8 mg/ml) collagenase
type I (Worthington Biochemicals, Freehold, NJ) and 2200 units/ml (0.15 mg/ml) trypsin at 20° for 2 hr in calcium-free Barth's solution (see
below) with slow agitation to remove the follicular cell layer and then
washed extensively with Barth's solution [88 mM NaCl, 1.1 mM KCl, 2.4 mM NaHCO3,
0.3 mM Ca(NO)3, 0.3 mM
CaCl2, 0.8 mM
MgCl2, 15 mM HEPES, pH 7.6 with
NaOH]. Oocytes were maintained in Barth's solution supplemented with
100 µg/ml gentamycin, 40 µg/ml streptomycin, and 63 µg/ml
penicillin. Oocytes were injected with 10 ng of cRNA for homomeric
receptors and 5 ng of cRNA for each subunit of heteromeric receptors 24 hr after collagenase treatment using a 10-µl Drummond (Broomall, PA)
microdispenser. At 5-6 days after RNA injection, oocytes were recorded
in amphibian Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.2 with NaOH) under voltage clamp at a
holding potential of 
70 mV, with a Turbo Tec-10CD amplifier (NPI,
Tamm, Germany). Voltage electrodes had resistances of 1-3 M
and
were filled with 3 M KCl; current electrodes had
resistances of ~1 M and were filled with 3 M CsCl. Agonist was applied by superfusion at a flow rate
of 6 ml/min in a 50-µl recording chamber. For each oocyte, steady
state currents were recorded before and after ConA treatment. Lectin
treatment was carried out by pipetting 100 µl of 10 µM ConA in amphibian Ringer's solution (calculated for the tetramer of
102 kDa) directly into the recording chamber while perfusion was
stopped. Oocytes were incubated for 8 min in the lectin solution; then,
perfusion was restarted for 1 min before the next agonist application.
To estimate EC50 values, eight different agonist concentrations (A) were applied, and steady state values of the evoked
currents (I) were measured and fitted with the SigmaPlot program
(Jandel Scientific, San Rafael, CA) to the equation I = Imax/[1 + (EC50/A)nH], where
Imax is the maximal current,
EC50 is the agonist concentration giving
half-maximal currents, and nH is the
Hill coefficient.
Inhibition of N-glycosylation by tunicamycin. To express nonglycosylated receptors in oocytes, we used tunicamycin, a potent inhibitor of N-glycosylation (23). At 1 day before injection with cRNA, oocytes were preinjected with 50 nl of 400 µg/ml tunicamycin (20 ng/oocyte, or ~22 µg/ml for an oocyte of 1.2-mm diameter). Immediately before injection, a stock solution of 10 mg/ml tunicamycin in DMSO was diluted to 4% DMSO with amphibian Ringer's solution; 4% DMSO did not adversely affect the oocytes. Bath treatment of oocytes with tunicamycin turned out to be largely ineffective in inhibiting N-glycosylation (data not shown).
Site-directed mutagenesis.
Single nucleotide exchanges were
introduced by polymerase chain reaction-mediated site-directed
mutagenesis using mutagenetic primers as described previously (3). We
mutated wild-type GluR2 at the Q/R editing site to GluR2(R586Q)
[hereafter referred to as GluR2(Q)] for both the flip and flop splice
variants. To create N-glycosylation site mutants of GluR1,
we deleted the first N-glycosylation consensus site in the
GluR1 sequence to obtain GluR1(N45S), which we called GluR1-
NG1. We
also created the double-mutant GluR1-NG1/3 by also deleting the
third N-glycosylation consensus site [mutation GluR1(N239S)], and we engineered the mutant GluR1-NG3 by
introducing solely the mutation N239S. GluR1-NG2/4/5/6 was
engineered by deleting N-glycosylation sites 2 and 4-6 by
introducing the mutations N231S, N345S, N383S, and N388S, respectively.
GluR2(Q) with two ectopic N-glycosylation sites
(GluR2-EG1/2) was engineered by introducing the mutations E45N and A47T
(to create EG1) and D243N and D245T (to create EG2). All mutations were
verified by chain-termination method sequencing using the Sequenase kit
from United States Biochemical Corp. (Cleveland, OH).
Labeling of cell surface glycoproteins with biotinylated
ConA.
To identify only the fraction of receptor protein inserted
in the plasma membrane, surface proteins were tagged with biotin and
isolated by streptavidin/Sepharose-mediated precipitation of the
labeled protein. Briefly, intact oocytes were incubated in 1 mg/ml
NHS-SS-Biotin (Pierce, Indianapolis, IN) solution for 2 hr at 4°.
After five 10-min washes in frog Ringer's solution, 10 oocytes were
homogenized with a Teflon pestle in 200 µl of buffer H [100
mM NaCl, 20 mM Tris·HCl, pH 7.4, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, and a cocktail
of additional proteinase inhibitors (2.5 µg/ml leupeptin, 20 µg/ml
aprotinin, 2.5 µg/ml pepstatin, and 20 µg/ml benzamidine
hydrochloride)]. The homogenate was kept on ice for 60 min. After
centrifugation for 60 sec at 16,000 × g to remove yolk
platelets and the melanin pigment granula, the supernatants were
supplemented with 20 µl of streptavidin/Sepharose beads (Sigma) and
incubated for 3 hr at 4° on a rotator. The streptavidin/Sepharose
beads were pelleted by a 60-sec spin and washed three times with buffer
H, and the washed pellets were boiled in 40 µl of SDS-polyacrylamide
gel loading buffer (0.8 M 
-mercaptoethanol, 6% SDS,
20% glycerol, 25 mM Tris·HCl, pH 6.8, 0.1% bromphenol
blue).
Crude membrane preparation, gel electrophoresis, and Western blotting. At 3-6 days after RNA injection, a crude membrane fraction was prepared from oocytes. Briefly, 20 oocytes were homogenized with a Teflon pestle in 400 µl of buffer H as described above. The homogenate was kept in a shaker at 4° for 15 min and then spun down for 1 min at 16,000 × g. The supernatants containing the cytosolic proteins as well as the solubilized membrane proteins were separated at 4° on discontinuous SDS-polyacrylamide gels (3) with a 5% stacking gel and a 7.5% separating gel. The gel was blotted (3) onto Hybond ECL nylon membranes (Amersham). Membranes were blocked with 1X Roti-block (Roth) and probed (4° overnight) with the primary anti-GluR antibody diluted in antibody incubation buffer (0.1X Roti-block, 0.1% Triton X-100, 20 mM Tris·HCl, pH 7.6, 140 mM NaCl). Immunoreactive bands were detected by peroxidase-labeled donkey anti-rabbit IgG antibodies (Jackson Laboratories, Bar Harbor, ME) in the case of polyclonal primary antibodies or by peroxidase-labeled donkey anti-mouse IgG antibodies (Jackson Laboratories, Bar Harbor, ME) in the case of monoclonal primary antibodies. All antibodies were diluted in antibody incubation buffer and visualized with the enhanced chemiluminescence method (ECL Kit, Amersham).
Deglycosylation. Crude membranes were enzymatically deglycosylated with N-Glycosidase F (Boehringer-Mannheim) at 37° for 4 hr in the presence of protease inhibitors (see above) after denaturation for 10 min at 100° in 1% SDS, following the manufacturer's protocol.
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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N-Glycosylation is not required for receptor function. To investigate the requirement for N-glycosylation of GluRs, we expressed recombinant rat GluR subunits in X. laevis oocytes as homomeric proteins as well as in various heteromeric combinations. Oocytes were either not pretreated at all or preinjected with the antibiotic tunicamycin. Tunicamycin, a hydrophobic analog of UDP-N-acetylglucosamine, specifically inhibits N-glycosylation by blocking the addition of N-acetylglucosamine to the sugar carrier dolicholphosphate, which is the first step in the formation of the core oligosaccharide in N-linked glycosylation events. We then compared the steady state current amplitudes of receptor subunits expressed in untreated, glycosylating oocytes with those in tunicamycin-preinjected, nonglycosylating oocytes.
For AMPA receptors, function is preserved in the nonglycosylated state for all homomeric and heteromeric subunit combinations tested (Table 1). Interestingly, the flop and flip versions of each of the four AMPA receptor subunits behave quite differently: although lack of N-glycosylation increases Glu-evoked currents at all flip versions except for GluR3flip, amplitudes at flop versions are generally reduced, except in the case of GluR4flop (Fig. 2, A and B, and Table 1). The behavior of heteromeric AMPA receptor combinations cannot be easily predicted on the basis of that of the constituting subunits, except for a tendency to follow the behavior of the flop subunit in the combination rather than the flip subunit.
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99%) in steady state currents in heteromeric subunit combinations containing NR2A or NR2B as the structural subunit but does
not seem to abolish channel function entirely (Fig. 2F and Table 1).
For all homomeric subunits tested and for heteromeric combinations
containing either NR2C or NR2D, we failed to detect any currents in
nonglycosylating oocytes (Fig. 2E and Table 1). However, because these
particular subunits and subunit combinations comprise the NMDA
receptors with the smallest currents, it is possible that channel
function was not entirely abolished but merely dropped below our
detection level. If we assume that for the subunits with low levels of
expression, lack of N-glycosylation reduces currents to
1% of control values measured in glycosylating oocytes, as it is for
the better-expressing NMDA receptor subunits, then maximal amplitudes
would have dropped to ~1 nA and thus could not have been detected
in our expression system.
To rule out that the small NMDA receptor currents observed in
tunicamycin-treated oocytes are due to incomplete inhibition of
N-glycosylation, the absence of carbohydrate side chains
must be confirmed. Two lines of evidence argue against residual
N-glycosylation. First, we found a total lack of
ConA-mediated current potentiation on tunicamycin treatment for all
subunits tested, including NR1-4a/NR2B, which normally will be
potentiated when glycosylated (Table 1). A second line of evidence was
provided by Western blots of Triton X-100-solubilized crude oocyte
membranes separated on polyacrylamide gels. Because all receptor
subunits have at least four consensus sites for
N-glycosylation (Fig. 1) and all or most of these sites are
likely to be used (3), a substantial difference in molecular weight can
be expected between N-glycosylated and
non-N-glycosylated proteins, which is easily resolved on a
polyacrylamide gel. Western blots of such gels revealed a downward
shift in the molecular weight of receptor proteins expressed in
tunicamycin-treated oocytes. No bands equivalent to the
N-glycosylated protein could be detected in
N-glycosylation-incompetent oocytes (Fig.
3). This confirms our previous findings
for GluR1 that up to 12 days after cRNA injection into
tunicamycin-treated oocytes, no N-glycosylated receptor
protein is being synthesized (3). Furthermore, N-glycosidase F treatment of membrane preparations demonstrated that the molecular weight of nonglycosylated receptor protein obtained from
tunicamycin-pretreated oocytes was identical to that obtained by
enzymatic deglycosylation of receptor protein from control oocytes
(Fig. 3B). Moreover, N-glycosidase F treatment of receptor
protein from tunicamycin-pretreated oocytes did not show any further
downward shift in the molecular weight (Fig. 3B).
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Lectins potentiate current amplitudes at GluRs in a highly
subunit-specific manner.
To analyze lectin-mediated modulation of
AMPA receptors, we examined all eight homomeric flop/flip splice
variants (flop and flip variants each of GluR1 to GluR4) as well as six
heteromeric combinations of these subunits. With 300 µM
Glu as the agonist, all receptors except homomeric GluR2 subunits
showed small but significant potentiations of steady state currents
after an 8-min pretreatment with 10 µM ConA.
Potentiations of Glu-evoked currents ranged from 3.6 ± 1.2 for
GluR4flop/GluR2flop to
13 ± 4.0 for GluR4flip and were always
larger than potentiations of KA-evoked currents (Table
1). Notably, we found no significant differences in potentiation between flop and flip splice variants, regardless of
whether homomeric or heteromeric receptors were analyzed (Fig. 5A). Heteromeric combinations of GluR1,
GluR3, or GluR4 with GluR2 (flip or flop) generally displayed
ConA-mediated current potentiations similar to those of the respective
homomeric receptors (Table 1), indicating that the nonpotentiated GluR2
subunit does not dominate the ConA effect the way it dominates the
rectification properties and the calcium permeability when expressed in
heteromeric assemblies (2).
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NG1) or the third site (GluR1-NG3) and a double-mutant lacking both the first and third sites (GluR1-NG1/3). When these mutants were tested for ConA-mediated current potentiation, GluR1-NG1 and GluR1-NG3 still
showed potentiation, although Glu-evoked currents were significantly less increased than in wild-type GluR1 (Fig.
6). However, the double-mutant
GluR1-NG1/3 could not be lectin-potentiated any more, regardless of
the agonist used for channel activation (Fig. 6). The complementary
quadruple mutant GluR1-NG2/4/5/6, which lacks
N-glycosylation sites 2 and 4-6, on the other hand, was still potentiated (Fig. 6), demonstrating that sites 1 and 3 are sufficient to allow potentiation. This mutant was difficult to analyze,
however, because the currents were quite small; in some cases, currents
could be seen only after ConA treatment. These data suggest that the
GluR2-like N-glycosylation pattern of GluR1-NG1/3 is
linked to a GluR2-like phenotype in terms of lectin interaction and the
two sites are both necessary and sufficient. We conclude that the lack
of ConA-mediated current potentiation of GluR2 is a consequence of its
particular configuration of N-glycosylation sites rather
than differences in the amino acid sequence between GluR2 and the other
AMPA receptors elsewhere in the protein. To further support this
conclusion, we engineered a mutant GluR2(Q), which has two additional,
ectopic N-glycosylation sites at positions equivalent to
sites 1 and 3 of GluR1 (see Fig. 1). This "GluR1-like" mutant of
GluR2(Q) indeed regained some ConA-mediated current potentiation,
although it did not approach wild-type GluR1 levels (Fig. 6).
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8 min at 10 µM
ConA (5 min gave half-maximal potentiation; data not shown).
Unexpectedly, we found a marked difference in ConA potentiation of
GluR6 receptors of different Q/R-site editing status. Although the
unedited Q form gave huge potentiations (>1000-fold, Fig. 5C), the
effect was much more modest with the edited R form (~100-fold). This
was counterintuitive because the extracellular ligand binding sites as
well as the N-glycosylation sites presumably targeted by
ConA should be identical in both these editing variants (see
Discussion).
An additional difference between Q and R editing variants was revealed
by the agonist-dependency of the degree of ConA potentiation: Glu-evoked currents at Q variants are potentiated to a larger degree
than KA-evoked currents, whereas at R variants, there is no obvious
difference. The agonist-dependent differences in ConA potentiation of
KA receptor variants extended to other agonists as well, such as
domoate and methylglutamate. Interestingly, there is an inverse
relation between ConA-mediated current potentiation and the amplitude
of agonist-evoked basal currents. Although agonist-evoked steady state
currents at GluR6(Q) follow the sequence domoate 
KA > methylglutamate > Glu, ConA-mediated current potentiation follows
the reverse sequence Glu > methylglutamate > KA domoate (data not shown).
The GluR7 subunit of the KA receptor subfamily could not be analyzed
because it did not give any measurable currents, whether expressed
homomerically nor in heteromeric combinations with KA1, KA2, or both
(Table 1). This confirms previous observations by other researchers
(26, 27) .
NMDA receptors revealed an interesting pattern of small (2-fold)
ConA-mediated current potentiations of certain subunits (Fig. 5, D and
E, and Table 1). We compared all eight splice variants of the homomeric
NR1 subunit, comprising four progressively shorter carboxyl-terminally
spliced forms (NR1-1-4) that each can occur without ("a") or with
("b") an amino-terminal insertion of 21 amino acids (28, 29). All
splice variants lacking the amino-terminal insertion (NR1-1a, NR1-2a,
NR1-3a, and NR1-4a) showed potentiation, whereas the corresponding
"b" splice variants (NR1-1b, NR1-2b, NR1-3b, and NR1-4b) did
not (Fig. 5, D and E, Table 1). Instead, there was a small apparent
reduction in maximal amplitudes in "b" splice variants. This
reduction, however, was due to a slow run-down of NMDA receptor
currents seen in most oocytes over time. This run-down occurred during
the 8-min ConA incubation period and did not depend on the presence of
ConA (data not shown).
On examination of heteromeric combinations of NR1-1a or NR1-1b with
each of the four structural NMDA receptor subunits, NR2A, NR2B, NR2C,
and NR2D (30), no potentiation was seen in any combination containing a
"b" splice variant of NR1 (Table 1). However, when heteromeric
combinations included an "a" splice variant of NR1, a small
ConA-mediated potentiation was observed in cases in which the
structural subunit was either NR2B or NR2D but not NR2A or NR2C. Thus,
ConA-mediated current potentiation at NMDA receptors is subunit
specific as well as splice variant dependent.
ConA acts via direct binding to the carbohydrate side chains of
GluRs but does not affect the ligand binding site.
Current
potentiation seen after ConA treatment is not due to an increase in the
affinity for the agonists KA and Glu; EC50 values
were virtually unchanged by ConA treatment. On treatment with lectin,
they were entirely unaffected (NR1-1b/NR2B; Fig. 7C), marginally decreased [GluR6(Q);
Fig. 7B], or even slightly increased (GluR1flop;
Fig. 7A). This finding was quite unexpected because binding of a large
molecule such as ConA to the extracellular domain of a receptor protein
might be expected to severely affect the ligand binding site; after
all, ConA is a tetramer with a molecular weight of ~102 kDa, which is
in the same range as that of one glycosylated GluR subunit (~105 kDa
for GluR1, ~118 kDa for GluR6, and ~117 kDa for NR1).
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The functional importance of N-glycosylation. Our data show that N-glycosylation in general is not required for GluR function. The analysis did not include O-glycosylation, which is known to occur in GluRs (5) and persists in tunicamycin-treated oocytes. For AMPA and KA receptors, the lack of N-glycosylation does not impair the formation of fully functional ion channels. Certain subunits, such as the flip splice variants of the AMPA receptor or heteromeric KA receptors, pass even larger currents in the absence of N-glycosylation. The lack of N-glycosylation does not significantly alter the EC50 values for agonists at AMPA or KA receptors, indicating that carbohydrate side chains are not part of the ligand binding pocket and do not play a significant role in establishing the protein conformation required for the formation of that pocket. We observed that at many AMPA and KA receptor subunits, inhibition of N-glycosylation causes an increase in Glu-evoked relative to KA-evoked steady state currents regardless of how the absence of N-glycosylation affects the absolute amplitudes of the currents. Because the smaller steady state currents seen with Glu are usually attributed to Glu being a more strongly desensitizing agonist than KA (22), it seems that Glu is less desensitizing at nonglycosylated receptors. This suggests that N-glycosylation has a role in setting the maximal desensitization levels of AMPA and KA receptors.
The lack of N-glycosylation does not prevent AMPA or KA receptor protein synthesis, transport, subunit assembly, or insertion into the plasma membrane, as demonstrated by the perfectly functional ion channels generated in nonglycosylating oocytes. Our data on recombinant receptors are in agreement with the report by Sumikawa et al. (12), who found that KA currents expressed from total brain RNA were not abolished by tunicamycin. Our data partially contradict those of Kawamoto et al. (9, 10), who claimed that N-glycosylation is essential for ligand binding of GluR1 and GluR2. The fact that we observed functional receptors that could be activated by various glutamatergic agonists (Glu and KA; Fig. 2, A and B) in tunicamycin-treated oocytes that showed no biochemical (Fig. 3) or pharmacological (Table 1) evidence of residual N-glycosylated receptors demonstrates conclusively that ligands must have bound to the nonglycosylated receptor. This is especially obvious for the flip splice variants of GluR1 and GluR2, which are even potentiated by lack of N-glycosylation. Muhoff et al. (7) used tunicamycin-treated oocytes to
investigate the effect of inhibition of N-glycosylation on
AMPA, KA, and quisqualate responses expressed from total rat brain RNA
and reported a total lack of responses in the absence of
N-glycosylation. This finding contradicts our data as well
as those of Sumikawa et al. (12). The reason for this
discrepancy remains unclear; however, it might be speculated that the
RNA preparation that Muhoff et al. used selectively
expressed the more N-glycosylation-sensitive GluR subunits
(Table 1) or an N-glycosylation-dependent subunit that has
not yet been cloned.
Non-N-glycosylated NMDA receptors, unlike AMPA and KA
receptors, undergo large reductions in their current amplitudes but no
change in the EC50 values for the agonist,
demonstrating that ligand binding site formation is not dependent on
the direct or indirect participation of N-linked
carbohydrate side chains. Western blots reveal a significant reduction
in NR1 (but, interestingly, not NR2) protein expression (Fig. 3, D-H).
This reduction is not an artifact caused by our use of a monoclonal
anti-NR1 antibody that might not bind to the nonglycosylated form of
the receptor because we obtained similar results with a polyclonal
anti-NR1 antiserum (data not shown). The reduction in NR1 expression
seems to be the likely cause of the marked reduction (>99%) in
maximal current amplitudes. The small fraction of residual channel
activity is not due to an incomplete block of glycosylation, as
indicated by two independent lines of evidence. First, on Western blots of total membranes from tunicamycin-treated oocytes, we do not detect
the N-glycosylated receptor, whereas we see a distinct band
at a lower molecular weight, which is consistent with the nonglycosylated receptor protein (Fig. 3). Second, the small residual currents detected for the subunit combinations NR1-1a/NR2B and NR1-4a/NR2B in tunicamycin-treated oocytes were not potentiated by
ConA, which they should have been if they had somehow escaped tunicamycin inhibition.
The observed general decrease in NMDA receptor function on block of
N-glycosylation is consistent with the reported lack of binding of the glycine site antagonist 5,7-dichlorokynurenate to NR1
subunits expressed in tunicamycin-treated Sf9 insect cells (11).
However, Kawamoto et al. (11) observed no concomitant decrease in protein levels. Our Western blot data are also at odds with
reports that the number of human embryonic kidney 293 cells expressing
nonglycosylated NR1/NR2A receptors on their surface is similar to the
number expressing glycosylated receptors (8). However, the immunoblot
data shown by these authors indicate a large decrease in the amount of
NR1 protein (8). Furthermore, although the same number of cells may be
labeled, the expression level in each cell might be reduced. The
drastic decrease in functional receptors as measured by a decrease in
NMDA receptor-mediated excitotoxic cell death by these authors matches
our finding that current amplitudes are reduced to 0.3% of
glycosylated controls (Table 1). Based on our observation that
EC50 values do not increase in nonglycosylated
NMDA receptors but that NR1 expression levels drop precipitously, we
conclude that although the channel structure remains intact, the
decrease in receptor function is due to the lack of sufficient NR1
subunits.
We do not know whether the observed large reduction in protein
expression of NR1 subunits is due to a down-regulation of protein synthesis or increased degradation of nonglycosylated NR1 subunits, but
we suspect the latter to be the case because protein synthesis itself
clearly is not compromised by tunicamycin treatment, as demonstrated by
our data for KA or AMPA receptor expression as well as the fact that
NR2 or GluR6 subunits, when coexpressed with NR1 in the same oocyte,
are not reduced (Fig. 3, H and C). Such an increase in degradation
might be caused by failure of nonglycosylated subunits to assume their
correct folding, assemble correctly with other subunits, or be
transported correctly to the Golgi apparatus and/or the plasma
membrane. Our data do not allow us to distinguish among these
possibilities. However, the similarity between the observed overall
decrease in NR1 receptor protein in total cell homogenates (Fig. 3E)
and the reduction seen in NR1 surface protein (Fig. 3F) rules out that
nonglycosylated receptor subunits are merely prevented from reaching
the plasma membrane and instead accumulate intracellularly without
degradation.
Previous studies on the functional importance of
N-glycosylation for various receptor proteins using
tunicamycin as a tool have uncovered a wide variety of effects. In the
case of acetylcholine receptors, N-glycosylation was found
to be required for correct subunit assembly (32) and for protection
from intracellular degradation (33, 34). Similarly, subunit assembly
and membrane insertion of Na+ channels were shown
to be dependent on proper N-glycosylation (35). On the other
hand, expressions of the two-subunit protein Na+/K+-ATPase (36), human
erythrocyte anion transporter (37), m2 muscarinic acetylcholine
receptor (38), and voltage-gated potassium channel RCK1 (7, 12) were
found to be independent of N-glycosylation.
This multitude of seemingly contradictory observations indicates that
N-glycosylation has distinct effects on specific receptor systems and that no general predictions can be made for the functional consequences (if any) of a lack of N-glycosylation. As our
data show, this multifaceted significance of N-glycosylation
is true not only for membrane proteins in general but also within the closely related family of iGluRs, in which the
N-glycosylation state can have profoundly different
functional consequences, depending on the subunit composition. This
mechanism adds one more tool to the modulatory arsenal of neuronal
cells.
ConA-mediated current potentiation. We cannot confirm the conclusion by Partin et al. (20, 22) that ConA potentiates AMPA receptors only weakly (<2-fold). Their conclusion was based solely on the examination of KA-evoked currents. With KA as the agonist, we also failed to see significant ConA-mediated potentiation, except for GluR1flop. When Glu is used as the agonist, a significant potentiation is revealed, ranging from 5.7- to 13-fold (Table 1). ConA did not differentially potentiate either one of the AMPA receptor splice variants, regardless whether Glu or KA was used as the agonist. Interestingly, GluR2 could not be potentiated, a feature we showed to be due to its particular arrangement of N-glycosylation sites (Fig. 6). However, this property is restricted to homomeric GluR2 receptors, and it is thus questionable whether it could have any physiological significance.
KA receptors in our study showed huge ConA-mediated potentiation (ltequ]6000-fold). The considerably smaller potentiation factors reported by Partin et al. (20) and Yue et al. (19) most likely stem from shorter incubation periods (2-3 min) and the lower concentrations of ConA used in those studies. Unlike Partin et al. (20), we found that ConA-mediated potentiation of homomeric GluR6 as well as heteromeric combinations of GluR6 with KA1 or KA2 depends on the editing status of the receptor. The highly potentiated Q variant and the modestly potentiated R variant differ merely by one amino acid located in the middle of the ion channel. Because ConA acts by inhibiting desensitization, this finding implies that desensitization may not simply be determined by a certain agonist-evoked conformational change of the extracellular ligand binding site but rather may reflect a complex interaction of that site with the ion pore itself. Before our study, NMDA receptor potentiation by ConA had been examined for only one splice variant of NR1 (NR1-1a) and for one heteromeric subunit combination (NR1-1a/NR2A), and no potentiation had been observed in either case (19). However, ConA treatment in the study by Yue et al. (1995) was carried out for only 2 min, which might have prevented full potentiation to be achieved. In the same study, succinyl-ConA was shown to cause a 2-fold potentiation of NR1-1a, presumably because the smaller, dimeric succinyl-ConA has a faster time course of action than the tetrameric ConA. In keeping with this interpretation, NMDA receptor currents expressed from total rat brain RNA have been shown to be potentiated by a very long (30 min) treatment with ConA (18). The data presented in our study demonstrate that all NMDA receptors made up of homomeric NR1 subunits of the "a" splice type can be modestly potentiated by ConA, and the same is true for certain heteromeric receptors containing NR1 "a" splice variants. Thus, the amino-terminal exon of 21 amino acids that is located between potential N-glycosylation sites 1 and 2 (see Fig. 1) and is absent from "a" splice variants but present in "b" splice variants seems to control the ConA effect. Plant lectins such as ConA do not occur in the mammalian central nervous system. Given the modulatory power of heterologous lectins, especially when acting on KA receptors, it is tempting to speculate that endogenous mammalian homologs of ConA might exist that could serve as highly potent and subunit- and even splice variant-specific modulators of GluR function under physiological conditions.| |
Acknowledgments |
|---|
We would like to thank Dr. Robert Wenthold (Laboratory of Neurochemisrty, National Institute on Deafness and Other Communication Disorders, Bethesda, MD) for his kind gift of affinity-purified anti-GluR1 and anti-GluR6 antibodies, Dr. Nils Brose (Dept. of Molecular Neurobiology, Max-Planck-Institute) for the monoclonal anti-NR1 antibody, and Dr. Peter Seeburg (Dept. of Molecular Neuroendocrinology, ZMBH, University of Heidelberg, Germany) for the GluR5(Q) cDNA clone.
| |
Footnotes |
|---|
Received April 7, 1997; Accepted July 16, 1997
This work was supported by Grant SFB 406 and a Heisenberg Fellowship of the Deutsche Forschungsgemeinschaft (M.H.).
Send reprint requests to: Dr. Michael Hollmann, Glutamate Receptor Laboratory, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany. E-mail: hollman{at}mail.mpiem.gwdg.de
| |
Abbreviations |
|---|
iGluR, inotropic glutamate receptor;
GluR, glutamate receptor;
DMSO, dimethylsulfoxide;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid;
Glu, glutamate;
SDS, sodium dodecyl sulfate;
KA, kainate, NMDA;
N-methyl-D-aspartate, ConA, concanavalin A;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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) and tunicamycin-treated (
) oocytes.
EC50 values, nH
values, and maximal currents (Imax) were estimated with KA
as the agonist for GluR1 and GluR6 and with Glu in the presence of 10 µM glycine as the agonist for NR1-1b/NR2B. Values
represent the mean ± standard error of three individually
measured dose-response curves.
