Glutamate receptor IIA and Glutamate receptor IIB
In almost all nervous systems, rapid excitatory synaptic communication is mediated by a diversity of ionotropic glutamate
receptors. In C. elegans, 10 putative ionotropic glutamate receptor subunits have been identified, a surprising
number for an organism with only 302 neurons. Sequence analysis of the predicted proteins has identified two NMDA and eight
non-NMDA receptor subunits. The relationship between the putative C. elegans and known vertebrate glutamate receptor subunits was analyzed by generating a neighbor-joining tree of receptor
subunits. Clear groupings of the subunits can be distinguished. Thus, GLR-1 and GLR-2 group together and are most similar to rat AMPA receptors. GLR-3
and GLR-4, and more weakly GLR-5 and GLR-6, also group together. GLR-3-GLR-7 are clearly non-NMDA receptor subunits but cannot be obviously classified
into pharmacological subtypes. GLR-8 is the farthest outlier and is even more divergent than glutamate receptors identified in Arabidopsis. NMR-1 is grouped with rat
NR1 and Drosophila Nmdar1, and NMR-2 is grouped with rat NR2 receptors. The complete distribution of these subunits in the nervous system of C. elegans is described in this study.
Receptor subunits were found almost exclusively in interneurons and motor neurons, but no expression was detected in muscle
cells. Interestingly, some neurons express only a single subunit, suggesting that these may form functional homomeric channels. Conversely, interneurons of the
locomotory control circuit (AVA, AVB, AVD, AVE, and PVC) coexpress up to six subunits, suggesting that these subunits interact to generate a diversity of
heteromeric glutamate receptor channels that regulate various aspects of worm movement. Expression of these subunits in this circuit is
differentially regulated by the homeodomain protein UNC-42 (Note: there is no known Drosophila homolog) and UNC-42 is also required for axonal pathfinding of neurons in the circuit. In wild-type worms,
the axons of AVA, AVD, and AVE lie in the ventral cord, whereas in unc-42 mutants, the axons are anteriorly, laterally, or dorsally displaced, and the mutant worms
have sensory and locomotor defects (Brockie, 2001).
Recording of glutamate-activated currents in membrane patches was combined with RT-PCR-mediated AMPA receptor
(AMPAR) subunit mRNA analysis in single identified cells of rat brain slices. Analysis of AMPARs in principal neurons and
interneurons of hippocampus and neocortex and in auditory relay neurons and Bergmann glial cells indicates that the GluR-B
subunit in its flip version determines formation of receptors with relatively slow gating, whereas the GluR-D subunit promotes
assembly of more rapidly gated receptors. The relation between Ca2+ permeability of AMPAR channels and the relative
GluR-B mRNA abundance is consistent with the dominance of this subunit in determining the Ca2+ permeability of native
receptors. The results suggest that differential expression of GluR-B and GluR-D subunit genes, as well as the splicing and
editing of their mRNAs, account for the differences in gating and Ca2+ permeability of native AMPAR channels (Geiger, 1995).
The hypothesis that subtypes of glutamate receptors (GluRs) are differentially expressed during corticogenesis was tested.
The neocortex of fetal sheep (term = approximately 145 days) was evaluated by immunoblotting and immunohistochemistry
to determine the protein expression of AMPA receptors
(GluR1, GluR2/GluR3 [GluR2/3], and GluR4); kainate (KA) receptors (GluR6/GluR7 [GluR6/7]), and a metabotropic
GluR (mGluR5). AMPA/KA receptors and mGluR5 are expressed in neocortex by midgestation. GluR1 and mGluR5
expression increases progressively, with expression being maximal just before birth and then decreasing postnatally.
GluR2/3 and GluR6/7 levels increase progressively during corticogenesis to reach adult levels near term. GluR4 is
expressed at low levels during corticogenesis and in adult neocortex. The localizations of GluRs in the developing
neocortex are distinct. Each GluR has a differential localization within the marginal zone, cortical plate, and subplate.
GluR subtypes are expressed in laminar patterns before major cytoarchitectonic segregation occurs based on Nissl
staining, although connectional patterns are emergent by midgestation based on labeling of corticostriatal projections with
DiI. The GluR localizations change during cortical plate segregation, resulting in highly differential distributions in the
neocortex at term. AMPA/KA receptors are expressed transiently in proliferative zones and in developing white matter.
Oligodendrocytes in fetal brain express AMPA receptors. The expression of ion channel and metabotropic GluR subtypes
is dynamic during corticogenesis, with subtype- and subunit-specific regulation occurring during the laminar segregation of
the cortical plate and differentiation of the neocortex (Furuta, 1999).
Early in postnatal development, glutamatergic synapses transmit primarily through NMDA receptors. As development
progresses, synapses acquire AMPA receptor function. The molecular basis of these physiological observations is not
known. Single excitatory synapses were examined with immunogold electron-microscopic analysis of AMPA and
NMDA receptors along with electrophysiological measurements. Early in postnatal development, a significant fraction of
excitatory synapses have NMDA receptors and lack AMPA receptors. As development progresses, synapses acquire
AMPA receptors with little change in NMDA receptor number. Thus, synapses with NMDA receptors but no AMPA
receptors can account for the electrophysiologically observed 'silent synapse' (Petralia, 1999).
It has been suggested that some glutamatergic synapses lack functional AMPA receptors. Quantitative immunogold
localization was used to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of
synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses
on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as
synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3
pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the
immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, the AMPA
receptor content of C/A synapses on CA3 pyramidal spines is estimated to ranges from less than 3 to 140. A similar range is found in adult
Schaffer collateral and C/A synapses (Nusser, 1998).
The superficial dorsal horn is a major site of termination of nociceptive primary afferents. Fast excitatory synaptic
transmission in this region is mediated mainly by release of glutamate onto postsynaptic AMPA and NMDA receptors.
NMDA receptors are known to be Ca2+-permeable and to provide synaptically localized Ca2+ signals that mediate
short-term and long-term changes in synaptic strength. Less well known is a subpopulation of AMPA receptors that is
Ca2+-permeable and has been shown to be synaptically localized on dorsal horn neurons in culture and
expressed by dorsal horn neurons in situ. Kainate-induced cobalt
uptake was used as a functional marker of neurons expressing Ca2+-permeable AMPA receptors and this was combined with markers of
nociceptive primary afferents in the postnatal rat dorsal horn. Cobalt-positive neurons are located in
lamina I and outer lamina II, a region strongly innervated by nociceptors. These cobalt-positive neurons colocalize with
afferents labeled by LD2, and with the most dorsal region of capsaicin-sensitive and IB4- and LA4-positive afferents. In
contrast, inner lamina II has a sparser distribution of cobalt-positive neurons. Some lamina I neurons expressing the NK1
receptor, the receptor for substance P, are also cobalt positive. These neurons are likely to be projection neurons in the
nociceptive pathway. On the basis of all of these observations, it is proposed that Ca2+-permeable AMPA receptors are
localized to mediate transmission of nociceptive information (Engelman, 1999).
The GluR2 subunit controls three key features of ion flux through the AMPA subtype of glutamate receptors-calcium
permeability, inward rectification, and channel block by external polyamines, but whether each of these features is equally
sensitive to GluR2 abundance is unknown. The relations among these properties were compared in native AMPA receptors
expressed by acutely isolated hippocampal interneurons and in recombinant receptors expressed by Xenopus oocytes. The
shape of current-voltage (I-V) relations between -100 and +50 mV for either recombinant or native AMPA receptors is well
described by a Woodhull block model in which the affinity for internal polyamine varies over a 1000-fold range in different
cells. In oocytes injected with mixtures of GluR2:non-GluR2 mRNA, the relative abundance of GluR2 required to reduce the
log of internal blocker affinity by 50% is two- to fourfold higher than that needed to half-maximally reduce divalent
permeability or channel block by external polyamines. Likewise, in interneurons the affinity of externally applied argiotoxin
for its blocking site is a steep function of internal blocker affinity. These results indicate that the number of GluR2 subunits
in AMPA receptors is variable in both oocytes and interneurons. More GluR2 subunits in an AMPA receptor are required to maximally reduce internal blocker affinity than to abolish calcium permeability or external polyamine channel block. Accordingly, single-cell RT-PCR shows that approximately one-half of the physiologically characterized interneurons exhibiting inwardly rectifying AMPA receptors express detectable levels of edited GluR2. Thus, the physiological effects of a moderate change in GluR2 relative abundance, such as occurs after ischemia or seizures or after chronic exposure to morphine, will be dependent on the ambient GluR2 level in a cell-specific manner (Washburn, 1997).
Formation of glutamatergic synapses entails development of 'silent' immature contacts into mature functional synapses. To determine how this transformation occurs, the development of neurotransmission was investigated at single synapses in vitro. Maturation of presynaptic function, assayed with endocytotic markers, follows accumulation of synapsin I. During this period, synaptic transmission is primarily mediated by activation of NMDA receptors, suggesting that most synapses are functionally silent. However, local glutamate application to silent synapses indicates that these synapses contain functional AMPA receptors, suggesting a possible presynaptic locus for silent transmission. Interference with presynaptic vesicle fusion by exposure to tetanus toxin reverts functional to silent transmission, implicating SNARE-mediated fusion as a determinant of the ratio
of NMDA:AMPA receptor activation. This work reveals that functional maturation of synaptic transmission involves transformation of presynaptic silent secretion into mature synaptic transmitter release (Renger, 2001).
It is difficult to conclude that silent or AMPA-quiet synaptic transmission events were solely due to the absence or highly dynamic nature of AMPA receptors. Therefore, alternative explanations were explored that take into account both the immature nature of presynaptic terminals and the postsynaptic inclusion of AMPA and NMDA receptors during AMPA-quiet synaptic transmission. One possible mechanism is a change in the flux of transmitter release. It was found that the activation of AMPA receptors is significantly reduced when the speed of transmitter release is slowed. However, the concentration profile of neurotransmitter does not alter the amount of NMDA receptor activation. Although there are multiple possibilities that might affect the flux of transmitter, the preferred explanation is a change in the process of vesicular fusion during synaptic transmission. If the fusion pore conductance is reduced, it would slow the release of glutamate, and for presynaptic functional marker molecules like FM1-43, which are 4-fold larger than glutamate, it could prevent passage into the vesicle. This would explain the lack of FM staining, the relatively small amount of AMPA receptor-mediated current, and the increased rise time of NMDA currents at young synapses. The perturbation of vesicle fusion through TeNTx-treatment provided a strong test of this possibility. Following toxin treatment, mature synapses generally fail to label with FM dye, have higher frequencies of miniature and evoked AMPA-quiet transmission events, higher failure rates of transmission, and slowed NMDA receptor activation, reminiscent of young synapses. To conclude that maturation of the synaptic vesicle fusion process does underlie the conversion of AMPA-quiet to functional transmission, future experiments will have to directly monitor the developmental changes in the proteins involved in the formation of the synaptic vesicle fusion complex (Renger, 2001).
AMPA receptor channels mediate the fast component of
excitatory postsynaptic currents in the central nervous system. Site-selective nuclear RNA editing controls the calcium
permeability of these channels, and RNA editing at a second site is shown here to affect the kinetic aspects of these channels in
rat brain. In three of the four AMPA receptor subunits (GluR-B, -C, and -D), intronic elements determine a codon switch
[AGA (arginine) to GGA (glycine)] in the primary transcripts in a position termed the R/G site, which immediately precedes the
alternatively spliced modules 'flip' and 'flop'. The extent of editing at this site progresses with brain development in a manner
specific for subunit and splice form, and edited channels possess faster recovery rates from desensitization (Lomeli, 1994).
Glutamatergic transmission converging on calcium signaling plays a key role in dendritic differentiation. In early development, AMPA receptor (AMPAR) transcripts are extensively spliced and edited to generate subunits that differ in their biophysical properties. Each GluA gene is subject to alternative splicing into the flip and flop isoforms. Flip-containing receptors are more efficiently activated and desensitize with slower kinetics. During early development, flip variants are prominently expressed, but towards adulthood they become replaced by flop-containing subunits. Whether the various AMPAR subunits have specific roles in the context of structural differentiation is unclear. This study investigated the role of nine GluA variants and revealed a correlation between the expression of flip variants and the period of major dendritic growth. In interneurons, only GluA1(Q)-flip increased dendritic length and branching. In pyramidal cells, GluA2(Q)-flop, GluA2(Q)-flip, GluA3(Q)-flip and calcium-impermeable GluA2(R)-flip promoted dendritic growth, suggesting that flip variants with slower desensitization kinetics are more important than receptors with elevated calcium permeability. Imaging revealed significantly higher calcium signals in pyramidal cells transfected with GluA2(R)-flip as compared with GluA2(R)-flop, suggesting a contribution of voltage-activated calcium channels. Indeed, dendritic growth induced by GluA2(R)-flip in pyramidal cells was prevented by blocking NMDA receptors (NMDARs) or voltage-gated calcium channels (VGCCs), suggesting that they act downstream of AMPARs. Intriguingly, the action of GluA1(Q)-flip in interneurons was also dependent on NMDARs and VGCCs. Cell class-specific effects were not observed for spine formation, as GluA2(Q)-flip and GluA2(Q)-flop increased spine density in pyramidal cells as well as in interneurons. The results suggest that AMPAR variants expressed early in development are important determinants for activity-dependent dendritic growth in a cell type-specific and cell compartment-specific manner (Hamad, 2011).
RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore
protein function. ADAR2 (Drosophila homolog: ADAR) is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its
RNA substrates are unknown. ADAR2-mediated RNA editing has been studied by generating mice that are homozygous for a
targeted functional null allele. Editing in ADAR2-/- mice is substantially reduced at most of 25 positions in diverse transcripts; the
mutant mice become prone to seizures and die young. The impaired phenotype appears to result entirely from a single underedited
position, as it reverts to normal when both alleles for the underedited transcript are substituted exonically, with alleles encoding the edited
version. The critical position specifies an ion channel determinant, the Q/R site, in AMPAreceptor GluR-B pre-messenger RNA. It is concluded that this transcript is the physiologically most important substrate of ADAR2 (Higuchi, 2000).
Mammalian transcripts that are known to be edited by site-selective adenosine deamination are expressed largely in brain:
most encode subunits of ionotropic glutamate receptors (GluRs) that mediate fast excitatory neurotransmission. The
only position edited to nearly 100% is the Q/R site of GluR-B, for which the mRNA contains an arginine (R) codon (CIG)
in place of the genomic glutamine (Q) codon (CAG). The physiological importance of this codon substitution wrought by
RNA editing has been revealed by early onset epilepsy and premature death of mice heterozygous for an intron-11-modified
GluR-BECS allele with Q/R site-uneditable transcripts (Higuchi, 2000).
Heterozygous ADAR2+/- mice are phenotypically normal, but ADAR2-/- mice die between P0 and P20 and become
progressively seizure-prone after P12, akin to GluR-B+/delta ECS mice. Therefore, this investigation focussed on the effect of ADAR2
deficiency on Q/R site editing of GluR-B pre-mRNA, the substrate for a nuclear RNA-dependent adenosine deaminase
activity. As determined from cloned polymerase chain reaction with reverse transcription (RT-PCR) products from brain
RNA6, Q/R site editing in primary GluR-B transcripts is tenfold lower in ADAR2-/- than in wild-type mice (10%
compared with 98%). This identifies ADAR2 as the principal RNA-editing enzyme at the Q/R site. The remaining
low level of Q/R site editing in GluR-B pre-mRNA cannot be mediated by the residual, enzymatically inactive, truncated
ADAR2 protein, but is mediated by another ADAR, perhaps ADAR1, for which gene expression appeared
unchanged in ADAR2 -/- mice (Higuchi, 2000).
The low extent of Q/R site editing of GluR-B pre-mRNA led to nuclear accumulation of incompletely processed primary
GluR-B transcripts and to a fivefold reduction in GluR-B mRNA, as assessed by RNase protection and quantitative RT-PCR. The increased level of intron 11-containing
GluR-B transcripts and the decrease in GluR-B mRNA are easily visualized by in situ hybridization. Editing is
thus a prerequisite for efficient splicing and processing of the pre-mRNA. The edited GluR-B transcripts are preferentially
spliced, as revealed by a shift in Q/R site editing from 10% to 40% when comparing intron-11-containing transcripts with
GluR-B mRNA. A defect in transcript processing caused by the interaction of the residual truncated ADAR2
protein with RNA can be excluded because GluR-B pre-mRNA accumulation is also observed in ADAR2+/+ mice
expressing the Q/R site-uneditable GluR-BdeltaECS allele (Higuchi, 2000).
AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop.
Editing at the GluR2 Q/R site is specific for GluR2 since GluR1, -3, and -4 carry a Gln (Q) at this critical, pore-lining position. The vast majority of GluR2 (>99%) in adult brain is edited to Arg; editing at this site is very efficient and crucial for brain development and function. Editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain (Greger, 2003).
What are the functional implications of these finds for AMPAR transmission? Subunit composition is a major determinant for AMPAR conductance properties. In brain, the majority of AMPAR transmission is functionally dominated by the GluR2 subunit, which renders receptors Ca2+ impermeable, alters their voltage sensitivity, and reduces conductance. Only a subset of interneurons express low levels of GluR2, and therefore, Ca2+-permeable AMPARs. It has also been shown that the number of GluR2 subunits in AMPAR tetramers can vary (at least in interneurons), and that transmission properties are differently affected by GluR2 abundance. GluR2 subunit numbers in tetramers may be subject to regulation. Repetitive synaptic activity results in a switch from GluR2-lacking to GluR2-containing AMPARs in cerebellar stellate cells. Similarly, activity may cause increased inclusion of GluR2 subunits into tetramers. Such a mechanism would protect neurons from excessive Ca2+ influx through AMPARs. However, only a limited number of GluR2 subunits can be included into a tetramer. The assembly rules described here will impair the formation of GluR2 homomers, and, by extension, restrict GluR2 numbers in AMPAR tetramers. Occlusion of GluR2 homomers could be crucial as such channels display a very small single-channel conductance, and conduct anions. The existence of such channels would decrease the efficiency of a synapse, and barely contribute to the generation of an EPSP (excitatory postsynaptic potential) (Greger, 2003).
AMPA-type glutamate receptors are specifically inhibited by the noncompetitive antagonists GYKI-53655 and CP-465,022, which act through sites and mechanisms that are not understood. Using receptor mutagenesis, it was found that these antagonists bind at the interface between the S1 and S2 glutamate binding core and channel transmembrane domains, specifically interacting with S1-M1 and S2-M4 linkers, thereby disrupting the transduction of agonist binding into channel opening. It was also found that the antagonists' affinity is higher for agonist-unbound receptors than for activated nondesensitized receptors, further depending on the level of S1 and S2 domain closure. These results provide evidence for substantial conformational changes in the S1-M1 and S2-M4 linkers following agonist binding and channel opening, offering a conceptual frame to account for noncompetitive antagonism of AMPA receptors (Balannik, 2005).
AMPARs assemble as tetramers in various combinations of four homologous subunits, termed GluR1-4 (or GluR-A to -D). Like all iGluR subunits, the AMPAR subunits share a modular design consisting of an extracellular N-terminal oligomerization domain (NTD); an extracellular agonist-binding domain formed by two segments, S1 and S2; a channel-forming domain consisting of three transmembrane domains, M1, M3, and M4 and a reentrant loop M2; and an intracellular C-terminal trafficking and anchoring domain (CTD). Currently, structural data at atomic resolution are available only for the S1 and S2 domains, of which the first and most extensively characterized is that derived from the AMPAR GluR2. Collectively, it has been shown that S1 and S2, which in the intact receptors are separated by the membrane regions M1 to M3, fold in a special manner, creating two globular domains (D1 and D2). Glutamate first docks in D1, which then promotes the rotation of D2 and closure of the binding cleft. Full agonists like glutamate and AMPA induce a large movement resulting in full activation. Partial agonists like kainate induce an intermediate closure and partial activation, and competitive antagonists like DNQX promote only a small extent of domain closure that is insufficient to trigger ion channel gating. Several studies have provided evidence that the agonist binding domains assemble as dimers, and the mechanism of desensitization has been further defined as a rearrangement of the dimer interface. Therefore, the idea that agonist binding to S1 and S2 evokes significant conformational changes in the extracellular domains leading to channel opening is widely accepted. However, the mechanism by which these conformational changes are transduced to channel gating is still unclear. Gating is likely to involve the linker regions between the agonist-binding and channel-forming domains, namely the S1-M1, S2-M3, and S2-M4 linkers. These regions are postulated to contribute to an extended mass at the bottom of D2 and are therefore likely to be coupled to the movement of D2 upon agonist binding. So far, experimental evidence for such conformational rearrangements is limited to the M3 linker (Balannik, 2005).
There are a number of pharmacological agents that affect AMPAR function through interactions outside of the agonist-binding domain. Thus, investigating the means by which binding of these ligands modulate channel gating may provide additional insight into mechanisms of receptor function. Toward this end, the site of interaction with AMPAR of two selective noncompetitive AMPAR antagonists, GYKI-53655 (GYKI) and CP-465,022 (CP), was investigated. GYKI belongs to a family of 2,3-benzodiazepines, and it is a more potent and selective analog of GYKI-52466, the first identified AMPAR noncompetitive antagonist. CP is a derivative of piraquilone and is ~100-fold more potent than GYKI on hippocampal neurons. Radioligand-binding assays suggested that the binding sites of these two compounds overlap with one another but that this site is distinct from the agonist-binding site. These antagonists are not open-channel blockers nor do they affect channel desensitization, suggesting a mechanism of action not involving binding to the channel pore. However, there is an allosteric interaction between GYKI and the inhibitor of desensitization cyclothiazide (CTZ), suggesting that the binding site for GYKI is affected by gating, although the molecular mechanism for this interaction is not known (Balannik, 2005).
This study shows that GYKI and CP bind with different affinity to different gating states of the AMPAR. The highest affinity is for the closed state, most likely the resting rather than the desensitized state, and the lowest is for the open state, further depending on agonist efficacy to open the channel. Using AMPA/kainate chimeras, it was shown that GYKI and CP bind at the linker regions between S1 and S2 and the channel, specifically interacting with S1-M1 and S2-M4. Therefore, the change in antagonist binding affinity upon gating is indicative of substantial conformational changes in these linkers following agonist binding and channel opening. A model in which these noncompetitive inhibitors constrain the movements of these linkers provides the insight into the way agonist binding is transduced to channel gating through the linker regions. As such, this study provides a potential template for rational drug design (Balannik, 2005).
The phosphorylation of the glutamate receptor subunit GluR1 has been characterized using biochemical and
electrophysiological techniques. GluR1 is phosphorylated on multiple sites that are all located on the C-terminus of the
protein. Cyclic AMP-dependent protein kinase specifically phosphorylates SER-845 of GluR1 in transfected HEK cells and
in neurons in culture. Phosphorylation of this residue results in a 40% potentiation of the peak current through GluR1
homomeric channels. In addition, protein kinase C specifically phosphorylates Ser-831 of GluR1 in HEK-293 cells and in
cultured neurons. These results are consistent with the recently proposed transmembrane topology models of glutamate
receptors, in which the C-terminus is intracellular. In addition, the modulation of GluR1 by PKA phosphorylation of
Ser-845 suggests that phosphorylation of this residue may underlie the PKA-induced potentiation of AMPA receptors in
neurons (Roche, 1996).
Long-term potentiation (LTP), a cellular model of learning and memory, requires calcium-dependent protein kinases. Induction
of LTP increases the phosphorus-32 labeling of AMPA-type
glutamate receptors (AMPA-Rs), which mediate rapid excitatory synaptic transmission. This AMPA-R phosphorylation
appears to be catalyzed by Ca2+- and calmodulin-dependent protein kinase II (CaM-KII): (1) it correlates with the activation
and autophosphorylation of CaM-KII, (2) it is blocked by the CaM-KII inhibitor KN-62, and (3) its phosphorus-32 peptide
map is the same as that of GluR1 coexpressed with activated CaM-KII in HEK-293 cells. This covalent modulation of
AMPA-Rs in LTP provides a postsynaptic molecular mechanism for synaptic plasticity (Barria, 1997a).
Ca2+/CaM-dependent protein kinase II (CaM-KII) can phosphorylate and potentiate responses of
AMPA-type glutamate receptors in a number of systems: recent
studies implicate this mechanism in long term potentiation, a cellular model of learning and memory. In this study, this CaM-KII regulatory site has been identified using deletion and site-specific mutants of glutamate receptor 1 (GluR1). Only
mutations affecting Ser831 alter the 32P peptide maps of GluR1 from HEK-293 cells co-expressing an activated
CaM-KII. Likewise, when CaM-KII is infused into cells expressing GluR1, the Ser831 to Ala mutant fails to show
potentiation of the GluR1 current. The Ser831 site is specific to GluR1, and CaM-KII does not phosphorylate or potentiate
current in cells expressing GluR2, emphasizing the importance of the GluR1 subunit in this regulatory mechanism. Because
Ser831 has previously been identified as a protein kinase C phosphorylation site, this raises the possibility of synergistic
interactions between CaM-KII and protein kinase C in regulating synaptic plasticity (Barria, 1997b).
Brief bath application of N-methyl-D-aspartate (NMDA) to hippocampal slices produces long-term synaptic depression
(LTD) in CA1 that is (1) sensitive to postnatal age, (2) saturable, (3) induced postsynaptically, (4) reversible, and (5) not
associated with a change in paired pulse facilitation. Chemically induced LTD (chem-LTD) and homosynaptic LTD are
mutually occluding, suggesting a common expression mechanism. Using phosphorylation site-specific antibodies, induction of chem-LTD is found to produce a persistent dephosphorylation of the GluR1 subunit of AMPA receptors at
serine 845, a cAMP-dependent protein kinase (PKA) substrate, but not at serine 831, a substrate of protein kinase C (PKC)
and calcium/calmodulin-dependent protein kinase II (CaMKII). These results suggest that dephosphorylation of AMPA
receptors is an expression mechanism for LTD and indicate an unexpected role for PKA in the postsynaptic modulation of
excitatory synaptic transmission (Lee, 1998).
Modulation of AMPA-type glutamate channels is important for synaptic plasticity. Physiological evidence is provided
that the activity of AMPA channels is regulated by protein phosphatase 1 (PP-1) in neostriatal neurons. Two
distinct molecular mechanisms of this regulation are identified. One mechanism involves control of PP-1 catalytic activity by DARPP-32,
a dopamine- and cAMP-regulated phosphoprotein highly enriched in neostriatum. The other involves binding of PP-1 to
spinophilin, a protein that colocalizes PP-1 with AMPA receptors in postsynaptic densities. The results suggest that
regulation of anchored PP-1 is important for AMPA-receptor-mediated synaptic transmission and plasticity (Yan, 1999).
Hippocampal N-methyl-D-aspartate (NMDA) receptor-dependent long-term synaptic depression (LTD) is associated with a
persistent dephosphorylation of the GluR1 subunit of AMPA receptors at a site (Ser-845) phosphorylated by
cAMP-dependent protein kinase (PKA). In the present study, it is shown that dephosphorylation of a postsynaptic PKA
substrate may be crucial for LTD expression. PKA activators inhibit both AMPA receptor dephosphorylation and LTD.
Injection of a cAMP analog into postsynaptic neurons prevents LTD induction and reverses previously established
homosynaptic LTD without affecting baseline synaptic transmission. Moreover, infusing a PKA inhibitor into postsynaptic
cells produces synaptic depression that occludes homosynaptic LTD. These findings suggest that dephosphorylation of a
PKA site on AMPA receptors may be one mechanism for NMDA receptor-dependent homosynaptic LTD expression (Kameyama, 1998).
Modulation of postsynaptic AMPA receptors in the brain by phosphorylation may
play a role in the expression of synaptic plasticity at central excitatory
synapses. It is known from biochemical studies that GluR1 AMPA receptor subunits
can be phosphorylated within their C terminal by cAMP-dependent protein kinase A
(PKA), which is colocalized with the phosphatase calcineurin (i.e., phosphatase
2B). The effect of PKA and calcineurin has been studied on the time course, peak
open probability, and single-channel properties of glutamate evoked
responses for neuronal AMPA receptors and homomeric GluR1(flip) receptors
recorded in outside-out patches. Inclusion of purified catalytic subunit
Calpha-PKA in the pipette solution increases neuronal AMPA receptor P(O,PEAK) compared with recordings made with calcineurin included in the pipette. Similarly, Calpha-PKA increases peak
open probability for recombinant
GluR1 receptors compared with patches excised from cells cotransfected
with a cDNA encoding the PKA peptide inhibitor PKI or patches
with calcineurin included in the pipette. Neither PKA nor
calcineurin alters the amplitude of single-channel subconductance levels,
weighted mean unitary current, mean channel open period, burst length, or
macroscopic response waveform for recombinant GluR1 receptors. Substitution of
an amino acid at the PKA phosphorylation site (S845A) on GluR1 eliminates the
PKA-induced increase in peak
open probability, whereas the mutation of a Ca(2+),
calmodulin-dependent kinase II and PKC phosphorylation site (S831A) is without
effect. These results suggest that AMPA receptor peak response open probability
can be increased by PKA through phosphorylation of GluR1 Ser845 (Banke, 2000).
Bidirectional changes in the efficacy of neuronal synaptic transmission, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD), are thought
to be mechanisms for information storage in the brain. LTP and LTD may be mediated by the modulation of AMPA
receptor phosphorylation. LTP and LTD reversibly modify the
phosphorylation of the AMPA receptor GluR1 subunit. However, contrary to the hypothesis that LTP and LTD are the functional inverse of each other, LTP and LTD
are associated with the phosphorylation and dephosphorylation, respectively, of distinct GluR1 phosphorylation sites. Moreover, the site modulated depends on the
stimulation history of the synapse. LTD induction in naive synapses dephosphorylates the major cyclic-AMP-dependent protein kinase (PKA) site, whereas in
potentiated synapses the major calcium/calmodulin-dependent protein kinase II (CaMKII) site is dephosphorylated. Conversely, LTP induction in naive synapses and
depressed synapses increases phosphorylation of the CaMKII site and the PKA site, respectively. LTP is differentially sensitive to CaMKII and PKA inhibitors
depending on the history of the synapse. These results indicate that AMPA receptor phosphorylation is critical for synaptic plasticity, and that identical stimulation
conditions recruit different signal-transduction pathways depending on synaptic history (Lee, 2000).
A chemically induced form of LTD (chemLTD) is associated with persistent dephosphorylation of GluR1 at a PKA
phosphorylation site, Ser 845. The chemLTD approach was designed to maximize the number of affected synapses, thereby increasing the probability of
detecting biochemical changes. The sensitivity of the chemLTD assay could be increased to detect small changes in GluR1 phosphorylation on Ser 831 and Ser 845 in
single hippocampal slices after the synaptic induction of LTP and LTD.
To analyse phosphorylation of GluR1 during synaptic plasticity, two hippocampal slices were placed in the same recording chamber and extracellular field
potentials were recorded simultaneously in the CA1 dendritic region of both slices upon stimulation of the Schaffer collaterals. After collecting a stable baseline, stimulation to one slice
was turned off while the other slice received low-frequency stimulation (LFS; 1 Hz, 900 pulses). After the LFS, the stimulation was returned to the baseline frequency,
and stimulation to the control slice was resumed. The slice that received LFS showed homosynaptic LTD, but there was no significant change in synaptic strength in the control slice. One hour
after the induction of LTD the slices were collected and frozen immediately on dry ice. Phosphorylation states of GluR1 at Ser 831 and Ser 845 in the control and LTD slices were then examined
by quantitative immunoblotting (Lee, 2000).
Like chemLTD, homosynaptic LTD produces specific dephosphorylation of Ser 845, whereas there is no significant change in phosphorylation of Ser 831. The dephosphorylation at Ser 845 could be
detected as early as 30 min after the onset of LFS. As expected, the magnitude of the change in GluR1 phosphorylation
following homosynaptic LTD is smaller than that reported for chemLTD, because only a small percentage of synapses in the slice are depressed during LFS-induced
homosynaptic LTD. However, this effect is reproducible and statistically significant. In addition, there is no dephosphorylation in slices that do not exhibit LTD
after LFS, either because induction of LTD fails or because the NMDA receptor antagonist AP5
was applied. Collectively, these results confirm that chemLTD and homosynaptic LTD share similar downstream expression mechanism(s) (Lee, 2000).
Homosynaptic LTD is dependent on postsynaptic protein phosphatase activity. To test whether the dephosphorylation of GluR1 following LTD is also sensitive to
protein phosphatase inhibitors, the control and experimental slices were incubated in okadaic acid for 2-3 h
and transferred to the recording chamber. Okadaic acid abolishes LTD. Moreover, pretreatment of slices with okadaic acid blocks the LFS-induced dephosphorylation of
Ser 845. In contrast, induction of LTD in slices kept in vehicle solution still produces significant
dephosphorylation at Ser 845. This result indicates that protein phosphatase
1/2A may be critical for the LFS-induced dephosphorylation of GluR1 at Ser 845 (Lee, 2000).
LTP is probably associated with an increase in phosphorylation of GluR1 by CaMKII. A test was performed to see whether the same changes could be detected using
phosphorylation-site-specific antibodies. Slices that received theta burst stimulation (TBS) show LTP,
and a significant increase in GluR1 phosphorylation at Ser 831 (CaMKII/PKC site) at both 30 min and 1 h after TBS. Phosphorylation of Ser 845 (PKA site) is not significantly increased 30 or 60 min
after LTP induction. In slices that do not exhibit LTP, GluR1 phosphorylation does not change. These results indicate that the early phase of LTP is dependent on postsynaptic CaMKII activation and that phosphorylation of GluR1 by CaMKII increases
following LTP5 (Lee, 2000).
Thus the data indicate that LTP and LTD may be associated with changes in phosphorylation of GluR1 at different sites. LTD is associated
with dephosphorylation at Ser 845, a PKA site, whereas LTP is associated with increased phosphorylation of Ser 831, a CaMKII site. This indicates that although
induction of LTP and LTD results in the bidirectional control of AMPA-receptor phosphorylation, LTP and LTD do not regulate phosphorylation of the same site. Since
LTP and LTD are reversible processes, a test of what happens to GluR1 phosphorylation after the reversal of LTP with LFS ('depotentiation') and the reversal of
LTD with TBS ('de-depression') was performed (Lee, 2000).
To examine depotentiation both slices in the recording chamber were subjected to TBS simultaneously: this results in LTP. After 30 min of recording, LFS was given
to one of the slices. This stimulation depresses the synaptic strength back to the baseline level while the synaptic response stays potentiated in slices that only receive TBS.
There is significant dephosphorylation of Ser 831 in depotentiated slices but no
significant change in Ser 845. This result indicates that the same stimulation protocol (LFS) results in the
dephosphorylation of different sites on GluR1 depending on the previous experience of the synapse (whether it had previously undergone LTP) (Lee, 2000).
To examine de-depression, LTD was induced in two slices in the recording chamber; 30 min later TBS was delivered to one of the slices. The synaptic response in slices
that received TBS was potentiated back to around the baseline response whereas the synaptic strength in slices that
received only LFS stayed depressed throughout the experiment. There is no significant change in phosphorylation of Ser 831
(CaMKII/PKC site) following de-depression. Interestingly, the de-depression increases phosphorylation at
Ser 845 (PKA site). These results indicate that, like LTD-inducing stimuli, LTP-inducing stimuli
result in the differential regulation of GluR1 phosphorylation depending on the previous experience of the synapse. TBS delivered to 'naive' synapses causes
phosphorylation of a CaMKII site, but TBS delivered to previously depressed synapses causes phosphorylation of a PKA site (Lee, 2000).
From these results it has been predicted that the synaptic potentiation due to changes in AMPA receptor phosphorylation following TBS-induced de-depression or TBS-induced
LTP should be differentially sensitive to CaMKII and PKA inhibitors. To test this hypothesis, two-pathway experiments were performed in which the effects of TBS could be compared on naive and previously depressed synaptic inputs. Under control conditions, TBS produces the same amount
of synaptic potentiation regardless of the initial state of the synapses, and both LTP and de-depression are prevented when NMDA receptors are
blocked. However, when slices are incubated in the selective CaMKII inhibitor KN-93, TBS produces significantly less de
novo potentiation than de-depression (measured 50 min after TBS). Conversely, when PKA is transiently inhibited at the time
of tetanic stimulation using the selective inhibitor KT5720, TBS produced significantly less de-depression than de novo LTP (measured 50 min
after TBS). Thus, the relative contributions of CaMKII and PKA to TBS-induced potentiation vary depending on the initial
state of the synapse, as predicted. This conclusion agrees with a previous report suggesting that there are different LTP-expression mechanisms in naive versus
depressed synapses in PKC- knockout mice (Lee, 2000).
These results show that bidirectional changes in synaptic function are associated with the reversible regulation of AMPA receptor phosphorylation. However, the
phosphorylation or dephosphorylation of GluR1 occurs on distinct sites, depending on the past experience of the synapse. Although the mechanism of this
differential phosphorylation remains to be determined, it may involve differential activation of protein kinases and protein phosphatases in the potentiated, naive and
depressed synapses. This differential bidirectional regulation of the phosphorylation of AMPA receptors has significant computational implications for synaptic plasticity,
since the results indicate that there may be at least three states of AMPA receptor activity with limited and regulated transitions between the states (Lee, 2000).
Although phosphorylation of the GluR1 subunit on either Ser 831 or Ser 845 potentiates AMPA receptor function, it appears to do so through distinct biophysical
mechanisms. PKA phosphorylation of Ser 845 regulates the open channel probability of AMPA receptors, whereas CaMKII phosphorylation regulates the
apparent single-channel conductance of the receptor. Note that single-channel conductance increases with LTP. Regulation of phosphorylation at these two sites
should contribute to the changes in the efficacy of synaptic transmission that are observed during LTP and LTD. However, the trafficking and synaptic targeting of
AMPA receptors may also be important in LTP and LTD. The role of phosphorylation of the GluR1 subunit in the regulation of AMPA receptor synaptic targeting
is unclear. It is possible that phosphorylation of these sites regulates synaptic trafficking of the AMPA receptor in addition to regulating channel function. Alternatively,
the synaptic trafficking of AMPA receptors may be regulated through a distinct mechanism, occurring with and complementing the modification of channel properties.
The findings that different phosphorylation sites and signal-transduction pathways contribute to bidirectional synaptic modifications in the hippocampus provide evidence
for unexpected complexity in the control of the gain of excitatory synaptic transmission (Lee, 2000).
Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP (See Drosophila Grip) and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuate LTD induction by glutamate/depolarization pairing, as do antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins (Xia, 2000).
Modification of AMPA receptor function is a major mechanism for the regulation of synaptic transmission and underlies several forms of synaptic plasticity. Post-translational palmitoylation is a reversible modification that regulates localization of many proteins. Palmitoylation of the AMPA receptor regulates receptor trafficking. All AMPA receptor subunits are palmitoylated on two cysteine residues in their transmembrane domain (TMD) 2 and in their C-terminal region. Palmitoylation on TMD 2 is upregulated by the palmitoyl acyl transferase GODZ and leads to an accumulation of the receptor in the Golgi and a reduction of receptor surface expression. C-terminal palmitoylation decreases interaction of the AMPA receptor with the 4.1N protein and regulates AMPA- and NMDA-induced AMPA receptor internalization. Moreover, depalmitoylation of the receptor is regulated by activation of glutamate receptors. These data suggest that regulated palmitoylation of AMPA receptor subunits modulates receptor trafficking and may be important for synaptic plasticity (Hayashi, 2005).
The precise subunit composition of synaptic ionotropic receptors in the brain is poorly understood. This information is of particular importance with regard to AMPA-type glutamate receptors, the multimeric complexes assembled from GluA1-A4 subunits; the trafficking of these receptors into and out of synapses is proposed to depend upon the subunit composition of the receptor. This study reports a molecular quantification of synaptic AMPA receptors (AMPARs) by employing a single-cell genetic approach coupled with electrophysiology in hippocampal CA1 pyramidal neurons. In contrast to prevailing views, it was found that GluA1A2 heteromers are the dominant AMPARs at CA1 cell synapses (approximately 80%). In cells lacking GluA1, -A2, and -A3, synapses are devoid of AMPARs, yet synaptic NMDA receptors (NMDARs) and dendritic morphology remain unchanged. These data demonstrate a functional dissociation of AMPARs from trafficking of NMDARs and neuronal morphogenesis. This study provides a functional quantification of the subunit composition of AMPARs in the CNS and suggests novel roles for AMPAR subunits in receptor trafficking (Lu, 2009).
The subunit composition of most ionotropic neurotransmitter receptors in the CNS has not been precisely determined. For the AMPA subtype of glutamate receptor, this is a particularly important problem. Recent evidence suggests that the subunit composition of AMPARs determines not only their biophysical properties but their activity-dependent trafficking to the synapse as well. Thus a rigorous quantitative description of the subunit composition of AMPARs is a prerequisite for understanding their roles in both the maintenance of synaptic transmission and synaptic plasticity. By using a conditional KO approach, each of the AMPAR subunits, both individually and in combination, were selectively deleted in a subset of CA1 hippocampal pyramidal cells. Simultaneous whole-cell recording from a gene-deleted cell and a neighboring control cell was used to quantify the changes induced by these genetic manipulations. The main results of this study are as follows. (1) All surface AMPARs contain GluA2 on CA1 pyramidal neurons. (2) GluA1, GluA2, and GluA3 fully account for the AMPARs on these neurons. (3) About 80% of synaptic AMPARs and >95% of extrasynaptic AMPARs are GluA1A2 heteromers, and most of the remaining receptors are GluA2A3 heteromers. (4) Aberrant homomeric GluA1, GluA2, and GluA3 receptors are capable of forming, depending on the deletion, but are unlikely to contribute significantly to normal AMPAR EPSCs on these neurons. This indicates that there is a hierarchy in the subunit assembly process, with GluA2-containing receptor complexes strongly preferred over other combinations. (5) No detectable changes in NMDAR EPSCs, spine morphology, or presynaptic properties were observed following the removal of all surface AMPARs. These findings provide new insight concerning the roles of AMPARs in neuronal physiology and morphology (Lu, 2009).
Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist
kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with
the extent of domain separation decreasing in the order of apo>DNQX>kainate>glutamate approximately equal to AMPA. These results suggest that agonist-induced
domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also
promote a 180° flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in
the intact receptor and mechanisms by which allosteric effectors modulate receptor activity (Armstrong, 2000).
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Glutamate receptor IIA and Glutamate receptor IIB:
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