I. Introduction

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The ionotropic glutamate receptors are ligand-gated ion channels that mediate the vast majority of excitatory neurotransmission in the brain. The cloning of cDNAs encoding glutamate receptor subunits, which occurred mainly between 1989 and 1992 (Hollmann and Heinemann, 1994), stimulated this field like no other event since the recognition in the early 1980s that the N-methyl-D-aspartate (NMDA)² receptor antagonist, D-AP5, has neuroprotective and anticonvulsant properties (reviewed by Choi, 1998; Dingledine et al., 1990), and that calcium entry through glutamate receptor channels plays important roles in development and in forms of synaptic plasticity that may underlie higher order processes such as learning and memory (Maren and Baudry, 1995; Asztely and Gustafsson, 1996). These earlier findings implicated NMDA receptors in a variety of neurologic disorders that include epilepsy, ischemic brain damage, and, more speculatively, neurodegenerative disorders such as Parkinson's and Alzheimer's diseases, Huntington's chorea, and amyotrophic lateral sclerosis.

Glutamate receptors are expressed mainly in the central nervous system, but several potentially important exceptions are worth mentioning. The realization that pancreatic islet cells express glutamate receptors that modulate insulin secretion (Inagaki et al., 1995; Weaver et al., 1996, 1998) and that antagonists of NMDA receptors expressed by osteoclasts and osteoblasts slow bone resorption (Chenu et al., 1998; Patton et al., 1998) raise the possibilities that antagonists restricted to the periphery might find uses in the treatment of diabetes and osteoporosis. Moreover, there is evidence for the presence of NMDA and non-NMDA receptors in small, unmyelinated sensory nerve terminals in the skin (Ault and Hildebrand, 1993; Carlton et al., 1995). Subcutaneous injection of as little as 300 pmol of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 30 pmol of MK-801 produced analgesia for a subsequent injection of formalin into the same site. These findings raise the possibility that peripheral glutamate receptors residing on nerve terminals in the skin may be a target for certain forms of pain associated with inflammation. NMDA receptor antagonists can also reduce histamine secretion from mast cells collected from the rat peritoneal cavity (Purcell et al., 1996), and NMDA depolarizes and elevates intracellular Ca²⁺ in mouse taste receptor cells in taste buds (Hayashi et al., 1996). Numerous ionotropic glutamate receptor subunits appear to be expressed by cardiac ganglia, but their functions are unknown (Gill et al., 1998). Thus, the potential therapeutic realm of drugs targeted to glutamate receptors is expanding to include cells (neural and nonneural) in the periphery. Most recently, evidence for a role for ionotropic glutamate receptors expressed by plant cells in light signal transduction has been reported (Lam et al., 1998), suggesting that mammalian receptors may have evolved from a more primitive signaling mechanism.

The cloning of the glutamate receptors in the early 1990s has taken the study of glutamate receptor pharmacology, physiology, and pathophysiology to the molecular level. Several major reviews of the initial fruits of cloning appeared in 1994 (Hollmann and Heinemann, 1994; McBain and Mayer, 1994; Nakanishi and Masu, 1994; Gill, 1994). This review focuses primarily on the functional insights and new pharmacological targets identified by molecular biological approaches since 1994. Although synaptic functions of NMDA and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been well understood, until recently the physiological roles of kainate receptors have been elusive. The recent applications of new drugs and genetic knockout technology are finally providing clues to the role of kainate receptors in synaptic transmission (e.g., Clarke et al., 1997; Rodriguez-Moreno and Lerma, 1998). However, to focus this review on the properties of the receptors themselves, we do not provide detailed information on the physiological roles of the various receptors, or their regional distribution, or extensive evaluation of genetically modified mice. Several recent reviews complement this one (Edmonds et al., 1995; Bettler and Mulle, 1995; Steinhauser and Gallo, 1996; Fletcher and Lodge, 1996; Sucher et al., 1996; Ben-Ari et al., 1997; Borges and Dingledine, 1998; Dingledine and McBain, 1998; Ozawa et al., 1998; Myers et al., 1999).

	II. Gene Families

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The three pharmacologically defined classes of ionotropic glutamate receptor were originally named after reasonably selective agonists NMDA, AMPA, and kainate. It turned out that NMDA, AMPA, and kainate receptor subunits are encoded by at least six gene families as defined by sequence homology: a single family for AMPA receptors, two for kainate, and three for NMDA (Table 1). Sequence similarity and, in some cases, similarity in intron-exon structure (Suchanek et al., 1995) suggests a common evolutionary origin for all of the ionotropic glutamate receptor genes. These genes are scattered over numerous chromosomes (Table 1), although the GRIA4 and GRIK4 genes are located near one another on the long arm of chromosome 11 and GRIK5 and GRIN2D may be close together on the long arm of chromosome 19. The protein products of these two pairs of genes do not appear to interact functionally, and it is not known whether these gene pairs are coordinately regulated similar to the gene clusters of the nicotinic acetylcholine receptor (Boulter et al., 1990a).

The 1 and 2 genes are distant structural relatives (18-25% amino acid identity) of other glutamate receptor subunits (Lomeli et al., 1993). These orphan subunits do not form functional channels by themselves, nor have they been shown to modify the function of other subunit combinations. However, knockout of the 2 gene leads to loss of activity-related depression of the parallel fiber-Purkinje cell synapse (Kashiwabuchi et al., 1995), and the mouse Lurcher neurological mutant has recently been shown to be caused by a gain-of-function mutation in 2, which leads to a large constitutive inward current that may provide a genetic model for excitotoxicity (Zuo et al., 1997). The genetic knockout strategy has recently helped to delineate the potential functions of another protein distantly related to the NMDA receptor subunits, NR3A (previously named NMDA-RL). NR3A coimmunoprecipitates with NR1 and NR2B subunit proteins in homogenates of mouse cerebral cortex (but not with GluR2, GluR6, 1, or 2 subunit proteins). Coexpression of NR3A with NR1 and NR2A causes a reduction in both whole-cell currents (Ciabarra et al., 1995) and single-channel conductance (Das et al., 1998), and perhaps a lower Ca²⁺ permeability. Accordingly, NMDA-induced currents in cortical neurons were increased about 3-fold in NR3A knockout mice (Das et al., 1998). These findings strongly suggest that the NR3A subunit may serve a regulatory function in NMDA receptors; in particular for controlling the amplitude and Ca influx through synaptic NMDA receptor channels.

No genetic diseases in humans have yet been linked to mutations in any of the glutamate receptor subunits, although as noted above the mouse Lurcher mutant is caused by a mutation in 2. Additionally, the genotype at a polymorphic triplet repeat in the 3' untranslated region (UTR) of human GluR6 appears to have a minor influence on the age of onset of Huntington's disease (Rubenstein et al., 1997). A number of neurological disorders are accompanied by the appearance of antibodies to glutamate receptor subunits (e.g., to GluR3 in Rasmussen's encephalitisRogers et al., 1994; Twyman et al., 1995; Carlson et al., 1997; to GluR2 in nonfamilial olivopontocerebellar degenerationGähring et al., 1997; and to several AMPA and kainate receptor subunits in paraneoplastic neurodegenerative syndromeGähring et al., 1995), but the role of these antibodies in disease manifestation is unclear (e.g., He et al., 1998).

In addition to these mammalian genes, cDNAs encoding several kainate-binding proteins (KBPs) had been isolated in the early 1990s from frog, chick, and goldfish brain that exhibit weak sequence homology to the mammalian glutamate receptors (for review, Henley, 1994). These proteins have not been found in mammals and do not seem to form functional homo- or heteromeric complexes with other glutamate receptor channels. However, chimeric proteins consisting of the channel-forming domains of KBPs and the ligand recognition domains of GluR1 or GluR6 form functional ion channels (Villmann et al., 1997), suggesting that an undiscovered modulatory subunit may be required to form fully functional KBPs.

	III. Receptor Structure

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The mechanism by which a receptor protein is threaded through the membrane during synthesis determines which segments face the extracellular and cytoplasmic fluids; this in turn specifies the protein domains that are available for ligand recognition, cytoplasmic modification (phosphorylation etc.), and interactions between the receptor and cytoplasmic proteins. Against initial expectations, glutamate receptors proved to have only three transmembrane domains (M1, M3, and M4) plus a cytoplasm-facing re-entrant membrane loop (M2, Fig. 1). Thus, the N terminus is located extracellularly and the C terminus intracellularly. This was deduced first by localization of endogenous and introduced N-glycosylation sites in KBPs (Wo and Oswald, 1994, 1995), GluR1 (Hollmann et al., 1994), GluR3 (Bennett and Dingledine, 1995), and NR1 (Wood et al., 1995), and additionally by analysis of the protease sensitivity of a reporter group fused at different positions to GluR3 (Bennett and Dingledine, 1995). The M2 segment in NMDA receptors is also thought to be a re-entrant loop based on the pattern of accessibility from both sides of the membrane of charged sulfhydryl reagents to cysteines substituted for M2 residues (Kuner et al., 1996), and the same method supports three rather than four transmembrane segments in AMPA receptors (Kuner et al., 1997). The transmembrane topology of glutamate receptors thus appears different from the four-transmembrane model of nicotinic acetylcholine receptors, but similar to that of potassium channels in that a re-entrant loop is present. Residues in this re-entrant second membrane loop control key permeation properties of the ion channel (see below).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Structure of AMPA receptor subunits. The transmembrane topology is shown along with the flip/flop alternatively spliced exon, and the two ligand-binding domains (S1 and S2). Glycosylation sites are shown as trees in the N-terminal region.

Early evidence favored a pentameric structure for glutamate receptors based on the size of chemically cross-linked NMDA receptor protein (Brose et al., 1993) or functional analysis of mixtures of native and mutant subunits with different sensitivity to channel blockers (Ferrer-Monteil and Montal, 1996), although velocity sedimentation analysis was consistent with a smaller protein (Blackstone et al., 1992; Wu and Chang, 1994). Premkumar and Auerbach (1997) inferred a pentameric stoichiometry for NMDA receptors consisting of three NR1 and two NR2 subunits. Their conclusion depended on interpretation of the patterns of single-channel conductances observed in mixtures of native and mutant subunits; the asparagine residue in the M2 segment lining the channel (N616) was changed to a glutamine in the mutant. For example, coexpression in Xenopus oocytes of NR1(N) with a combination of NR2B (N) and NR2B(Q) produced receptors with three patterns of main and subconductance states, corresponding to those seen with 1N/2N, 1N/2Q, and a third pattern that was considered to reflect 1N/2Q/2N receptors. Because only three different channel types were observed, they concluded that there must be two and only two NR2B subunits in a receptor (i.e., Q/Q, Q/N, and N/N). When a mixture of NR1(N) and NR1(Q) were coexpressed with NR2B(Q), six single-channel current patterns were distinguishable, which by similar logic pointed to three NR1 subunits in a functional receptor. An analogous experimental design by Behe et al. (1995) found fewer single-channel patterns, however, and concluded there are only two copies of NR1, not three as Premkumar and Auerbach (1997) found. Behe et al. (1995) concluded that the most parsimonius model involved a tetrameric protein consisting of two NR1 and two NR2 subunits.

Three more recent studies have been interpreted to favor a tetrameric assembly of subunits similar to that of potassium channels (Doyle et al., 1998) or cyclic nucleotide-gated channels (Liu et al., 1998). Laube et al. (1998) found three components in the dose-response curve for activation of mixtures of wild-type and mutant NMDA receptor subunits [NR1(Q387K) and NR2B(E387A) are described further in Table 2] with either of the two coagonists, glutamate or glycine. Binomial fits to the three receptor populations were most consistent with two NR1 and two NR2 agonist-binding subunits per functional receptor. Binomial analysis of the functional effect of incorporating increasing amounts of dominant negative NR1 or NR2 subunits into the receptor also supported a tetrameric assembly. Mano and Teichberg (1998) used a similar strategy to conclude that homomeric GluR1 receptors could be tetrameric complexes. Rosenmund et al. (1998) observed that upon agonist binding, activation of single receptor/channels proceeds through a staircase of openings to three different conductance levels of increasing amplitude. To resolve rapid transitions that occur during normal activation, agonist-binding sites were presaturated with the competitive antagonist, NBQX, before agonist application, so that each agonist-binding site was only made available after an antagonist molecule dissociated from the receptor. The authors proposed a model whereby the dissociation of two antagonist molecules and their replacement by two agonist molecules occurred before the first current step appeared. Current levels that were observed subsequently reflected the binding of single agonist molecules to the receptor, suggesting that each receptor contains four functional antagonist/agonist-binding sites, which is consistent with a tetrameric protein.

Thus, the conclusions of six carefully performed functional evaluations of receptor subunit mixtures (Ferrer-Monteil and Montal, 1996; Behe et al., 1995; Premkuhar and Auerbach, 1997; Laube et al. 1998; Mano and Teichberg, 1998; Rosenmund et al., 1998) are exactly split between a tetramer and a pentamer. In attempting to resolve the difference, several issues should be considered. First, each of the studies described above was designed to identify the number of functional binding sites in a receptor and relied on the assumption that each binding site in a receptor behaves independently of the others. It is well known that the agonist-binding sites of at least one other receptor, the muscle nicotinic acetylcholine receptor, show cooperativity, and negative cooperativity between binding of glutamate and glycine to the NMDA receptor is also well established (reviewed by McBain and Mayer, 1994). Second, the data of Rosenmund et al. (1998) suggest that subconductance states of a channel may be related to the number of agonists bound to the receptor. If this is correct, the interpretation of dose-response curves (Laube et al., 1998) and patterns of main and subconductance states (Behe et al., 1995; Premkuhar and Auerbach, 1997) become more complicated. Finally, post-translational processing could increase subunit complexity and potentially lead to overestimation of the number of functional subunits. Rosenmund et al. (1998) and Laube et al. (1998) argue that although their data favor a tetrameric protein, the possibility of a pentameric structure could not be entirely ruled out. Although the functional results are provocative, an unequivocal determination of the number of subunits in a functional glutamate receptor awaits physical methods that probe the structure of the protein itself.

Table 1 lists at least 14 functional glutamate receptor subunits. What pairing rules determine which subunits coassemble? Is subunit stoichiometry invariant as in the case of muscle nicotinic receptors? In the early 1990s many mixing experiments were carried out to search for instances in which coexpression of subunits from one family (NMDA, AMPA, or kainate) might alter the functional properties of receptors in a different family. These attempts were uniformly unsuccessful, and it is now generally accepted that a subunit will only assemble with others within its own family. It seems that the membrane domains may dominate assembly, because chimeras of AMPA and kainate receptors do assemble if the membrane domains are all from the same subunit (Stern-Bach et al., 1994). Functional homomeric receptors can be formed within the AMPA and kainate subunit families but probably not for NMDA receptors. Functional NMDA receptors can be formed by expression of the NR1 subunit by itself in Xenopus oocytes but not in mammalian cell lines. However, Soloviev and Barnard (1997) showed that a glutamate receptor subunit, XenU1, is endogenously expressed at very low abundance in Xenopus oocytes and can assemble with mammalian NR1 to form functional NMDA receptors. Their finding could explain why expression cloning of NR1 in Xenopus oocytes (Moriyoshi et al., 1991) was originally possible and reinforces the current notion that NR1 must partner with one or more NR2 subunits to form functional receptors.

Little is yet known about the exact subunit composition of native glutamate receptors, but immunoprecipitation strategies have shown that NR2A and NR2B subunits can coexist together with NR1 in native NMDA receptors gently solubilized from mammalian brain by sodium deoxycholate at pH 9 (Sheng et al., 1994; Blahos and Wenthold, 1996; Luo et al., 1997; Chazot and Stephenson, 1997); multiple NR1 splice variants can also exist in a receptor assembly (Blahos and Wenthold, 1996). Likewise, NR2D can be immunoprecipitated along with NR1 and either NR2A or NR2B (Dunah et al., 1998). Wenthold et al. (1996), using similar strategies, showed that AMPA receptors immunoprecipitated from the CA1 region of rat hippocampus (primarily pyramidal cells) consisted of two major complexes represented by GluR2 plus either GluR1 or GluR3; very few solubilized receptors appeared to contain both GluR1 and GluR3, but a small fraction of solubilized receptors appeared to be homomeric GluR1. Results from other functional assays appear compatible with heteromultimeric receptors. For example, the glycine dose-response curve of NMDA receptors assembled from NR1-1e, NR2A, and NR2C could not be described as the weighted average of dose-response curves obtained from NR1-1e + NR2A and NR1-1e + NR2C receptors done separately (Wafford et al., 1993). The glycine EC₅₀ for the triple subunit combination was intermediate between those for the heterodimeric combinations. All of these results argue for at least heteroternary NMDA receptors, but do not rule out the presence of some additional binary heteromers consisting of NR1 plus a single type of NR2 subunit.

It is apparent from the studies described above that multiple subtypes exist within the AMPA and NMDA receptor families based on subunit composition. Further complicating matters, it is now clear that multiple AMPA receptor subtypes coexist within the same neuron. Thus, Zhang et al. (1995) showed that AMPA receptor channels with both low and high calcium permeability could be found within the same retinal ganglion neuron. Washburn et al. (1997) showed that polyamine spider toxins, which selectively block GluR2-lacking AMPA receptors (Iino et al., 1996; Washburn and Dingledine, 1996), removed the inwardly rectifying component of AMPA receptor currents in hippocampal interneurons, suggesting coexpression of GluR2-lacking and -containing receptors in the same cell. Likewise, spermine (Ito et al., 1996) or polyamine spider toxins (Tóth and McBain, 1998; F. Laezza, J. Doherty and R. Dingledine, unpublished) eliminate the inwardly rectifying component of evoked synaptic currents in hippocampal interneurons, suggesting that mosaics of GluR2-containing and -lacking receptors are present at individual postsynaptic membranes. Tóth and McBain (1998) have proposed that the expression of calcium-permeable or -impermeable AMPA receptors is determined by the origin of synaptic input onto the dendrites of the target cell. The most direct evidence for multiple AMPA receptor subtypes in a single cell is found in the fusiform cells of the rat dorsal cochlear nucleus. In these cells, postsynaptic AMPA receptors at auditory nerve synapses on basal dendrites contain GluR4 by immunohistochemistry, whereas receptors in parallel fiber synapses on apical dendrites lack GluR4 (Rubio and Wenthold, 1997). This shows directly that subsynaptic receptor targeting can be guided by the GluR4 subunit.

The studies described above indicate that more than one AMPA receptor subtype can coexist within the same neuron. AMPA receptor diversity is even more extreme, however, because it appears that subunit stoichiometry is not fixed for AMPA receptors as it is for muscle nicotinic receptors. Washburn et al. (1997) studied three GluR2-dependent permeation features of recombinant AMPA receptors composed of different subunits in a variety of ratios, and in native receptors expressed by hippocampal interneurons: rectification, Ca²⁺ permeability and sensitivity to external polyamine block. The shape of AMPA receptor current-voltage curves in individual cells could not be described by an algebraic summation of I-V curves from two populations of receptors, those containing and lacking GluR2 in some fixed but unspecified stoichiometry. Moreover, rectification was much less sensitive to the relative abundance of GluR2 than was Ca²⁺ permeability. Both of these results argue strongly that the number of GluR2 subunits in an AMPA receptor is not fixed (see also Geiger et al., 1995). Variable AMPA receptor subunit stoichiometry endows excitatory synapses with a much wider range of responses than previously imagined.

A high-resolution crystal structure (approximately 1.9 Å) has recently been obtained for the ligand-binding domain of GluR2 complexed with the agonist kainate (Armstrong et al., 1998). This achievement followed and built upon much effort devoted to model the structure of glutamate receptor subunits, which in turn was made possible by the realization (Nakanishi et al., 1990) that glutamate receptors share weak sequence homology with a large family of bacterial amino acid-binding proteins whose structures had been solved to high resolution. A conserved amino acid-binding pocket (Oh et al., 1993, 1994; Sun et al., 1998) is proposed to exist in all glutamate receptors. This pocket would be formed from two globular domains (S1 and S2) drawn from the sequence adjacent to the M1 domain and the M3-M4 loop, respectively (Fig. 2A). In the bacterial proteins, the two lobes of the binding pocket are in a dynamic equilibrium of open and closed states; binding of ligand stabilizes the closed form of the clamshell structure. Four studies support the idea that the agonist-binding site of glutamate receptors is also a bilobular structure. First, swapping of S1 and S2 domains between GluR3 and GluR6 subunits caused the expected change in agonist pharmacology (Stern-Bach et al., 1994). Second, a soluble (nonmembrane bound) "minireceptor" consisting of these two domains from GluR4 or GluR2 joined by a hydrophilic spacer peptide was able to bind AMPA, glutamate, kainate, quisqualate, and CNQX with the expected affinities (Kuusinen et al., 1995; Arvola and Keinänen, 1996). Similarly, a soluble glycine-binding site with correct pharmacology was preserved in a fusion protein consisting of the S1 and S2 lobes of the NR1 subunit connected by a linker (Ivanovic et al., 1998). Third, inserting the S1 domain from GluR6 into GluR2 decreased the affinity for AMPA and increased kainate affinity (Tygesen et al., 1995). Finally, deletion of the N-terminal 400 amino acids, and the C-terminal 90 amino acids, from GluR6 left a membrane-bound core homomeric receptor that displayed normal [³H]kainate-binding properties (Keinänen et al., 1998). These findings support the concept that individual glutamate receptor subunits, like many other proteins, are constructed in a modular fashion: a pore-forming domain similar to that of potassium channels plus two separate domains that form a ligand-binding site similar to those of the bacterial periplasmic binding proteins (Wo and Oswald, 1995; Paas, 1998). The N-terminal 400 amino acids appear to play no significant role in ligand binding but may be the locus of many modulatory functions in some (e.g., NMDA) receptors. The crystal structure of the ligand-binding domain of GluR2 confirmed the bilobular structure (Fig. 2, A and B) and revealed additional details about the ligand-binding site.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Crystal structure of the GluR2 subunit and identification of agonist-binding residues. A, schematic of a glutamate receptor subunit with the two domains that contain agonist-binding residues colored in orange (S1) and turquoise (S2). The flip/flop region is indicated in violet. B, space-filled representation of the kainate-bound S1 and S2 domains joined by an 11-residue linker peptide, with coloration the same as in A. The flop domain is helical and located on a solvent-exposed face of the protein. The position of a single-kainate agonist molecule (black) within a deep gorge of the protein is indicated; the two disulfide-bonded cysteines (C718 and C773) are shown in yellow. Red asterisks mark the positions of S662 and S680 (lower left), which in GluR6 are important for PKA, and N721 (adjacent to the yellow C722), which in GluR5 and GluR6 controls agonist sensitivity. C, backbone representation of the subunit, with kainate (black) docked into its binding site. The kainate-binding residues are shown as stick figures in magenta, the two cysteines in yellow, and the flop helix structure in violet. The two green residues (E402 and T686) do not directly bind to kainate but instead interact with each other, helping to hold the clamshell in the closed conformation. D, close-up view of the ligand-binding pocket. The binding residues are in space-filled representation, with atoms colored conventionally (gray = carbon, light blue = nitrogen, red = oxygen). These images were created in rasmol from the pdb file graciously provided by E. Gouaux (Armstrong et al., 1998).

Figure 2B shows a spacefill model of the GluR2 structure, with color-coded S1 and S2 lobes folding around the kainate molecule (shown partly buried in black). An immediate conclusion from inspection is that the ligand-binding pocket appears to be entirely contained within a single subunit rather than being at the interface between two subunits. The flip/flop domain (violet) is an -helical structure on the side opposite the ligand-binding gorge. The subunit backbone is shown in Fig. 2C, where the residues that make contact with the kainate molecule are shown in magenta and kainate itself in black. The two residues in green (E402 and T686) do not participate directly in kainate binding but instead interact with one another to help hold the S1 and S2 lobes together. The two conserved cysteines that play a role in redox modulation of NMDA receptors are disulfide-bonded and shown in yellow. Figure 2D shows an enlarged view of the ligand-binding pocket, with electrostatic or polar interactions indicated by dotted lines. It is visually clear that kainate, binding deep within the S1 to S2 cleft, stabilizes the closed form of the clamshell structure by simultaneously bonding with residues in both S1 and S2 lobes.

How does ligand binding lead to channel opening? One can speculate that closure of the S1 and S2 lobes places a torque on the receptor that is transmitted to the channel region. The resulting mechanical force could increase the likelihood that the channel structure itself undergoes a conformational change to the open state. Whether desensitization is caused by a time-dependent relaxation of the "molecular spring" that connects the S1 and S2 lobes to the membrane, to partial unbinding of the agonist molecule that releases one of the lobes, to further closure of the cleft as residues in the outer reaches of the two lobes interact when the nearly closed clamshell breathes, or to some other event, remains for future work.

The ability of different agonists to either bind or activate glutamate receptors has now been assessed in well over 100 mutants in an effort to identify residues important for agonist binding. Residues that have been shown to influence agonist potency by more than a fewfold in kainate, AMPA, and NMDA receptors are identified in Table 2. These residues have been mapped onto the sequence of the GluR2 subunit so their positions can be compared. It is immediately apparent that several amino acids appear in homologous positions in the different subunits. An example is R485, the guanidinium group of which interacts with a carboxyl group of kainate in GluR2 (Fig. 2D). This arginine is also present in NR2B and cKBP and, when mutated to a lysine or serine, abolishes agonist responses (Table 2). Other examples are found by perusing Table 2. It is remarkable that, with one exception, the correspondence is excellent between the predictions made by functional evaluation of point mutations and the direct structural identification of residues in or near the binding pocket. That exception is Pro478. In GluR2, the carbonyl group of P478 is positioned near T480 and appears to bind to the nitrogen of kainate (not shown in Fig. 2D), but mutation of the homologous proline in chick KBP had no effect on agonist binding (Table 2).

Certain residues do not participate directly in agonist recognition but instead serve allosteric roles. For example, E402 in GluR2 has its homologs in the NR1, NR2B, GluR1, and chick KBP; in all of these subunits mutation of the E402 homolog has mild-to-substantial effects on the potency of the respective agonists (Table 2). E402 does not directly contact the kainate molecule in GluR2 but instead interacts with T686 of the opposite lobe to shape the binding pocket (Fig. 2C). Other examples of important modulatory or allosteric residues are N721, which lies adjacent to the S2 lobe cysteine and regulates agonist selectivity in GluR5 and GluR6 (Swanson et al., 1998), and S662 and S680, which in GluR6 are involved in phosphorylation by protein kinase A (PKA; Raymond et al., 1993; Wang et al., 1993; Basiry et al., 1999). The positions of these residues are marked with red asterisks in Fig. 2C.

The comparison of mutagenesis and molecular modeling in Fig. 2 and Table 2 provides support for the idea that all glutamate receptor subunits have a similar folding pattern, with ligand specificities probably accounted for by differences in amino acids at key positions as argued by Paas (1998). Analysis of mutants of the NR1 and NR2 subunits led to the proposal that the glycine coagonist docking site of NMDA receptors is exclusively on the NR1 subunit, whereas glutamate binds to the NR2 subunit (Kuryatov et al., 1994; Hirai et al., 1996; Laube et al., 1997; Anson et al., 1998; see Table 2).

The combination of structure determination and functional evaluation of mutants is thus producing internally consistent views of glutamate receptor subunit structures. Not surprisingly, before the GluR2 structure had been solved, numerous investigators had constructed molecular models by aligning the S1 and S2 sequences from glutamate receptors onto the structure of the amino acid-binding proteins, and then adjusting the new structure based on energy minimization in concert with functional information provided by mutants (Stern-Bach et al., 1994; Paas et al., 1996; Sutcliffe et al., 1996; Laube et al., 1997; Swanson et al., 1997a). The general low-resolution picture provided by homology modeling is remarkably congruent across several glutamate- and glycine-binding sites of various glutamate receptor subunits. All models incorporated two hinged lobes that close upon an agonist molecule within a cleft, and this general scheme is confirmed by the GluR2 structure shown in Fig. 2. However, it is well known that identification of residues contacting ligands from electrophysiological evaluation of mutants is not straightforward, because the measured agonist EC₅₀ is influenced not only by binding affinity but also by the ease with which the agonist-bound structure undergoes the conformational change leading to channel opening (e.g., Colquhoun and Sakmann, 1998). As caveats to the uncritical interpretation of mutagenesis experiments, one only needs to recall the once widely held but mistaken views that the acetylcholine-binding pocket of acetylcholinesterase consists of acidic and basic residues, and that the selectivity filter of potassium channels is derived from -orbitals of aromatic residues. Both of these views were overturned when crystal structures became available (Harel et al., 1993; Doyle et al., 1998). Further understanding of the structure of the ligand-binding sites of glutamate receptors will require solving the structures of other subunits liganded to a variety of agonist and antagonists. This work is important to direct and interpret a myriad of mutagenesis studies and, in the long run, to facilitate the design of new drugs. The structure of the pore inferred from functional measurements is discussed below.

	IV. RNA Modifications That Promote Molecular Diversity

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As described elsewhere in this review, functional diversity in glutamate receptors is determined in large part by which genes are expressed in a given neuron. In addition, ionotropic glutamate receptor subunits are subject to post-transcriptional alterationsalternative splicing and RNA editingboth of which give rise to a high structural and functional diversity.

All four AMPA receptor subunits occur in two alternatively spliced versions, flip and flop, that are encoded by exons 14 and 15 (in GluR2) positioned just before the M4 domain (Figs. 1-3) (Sommer et al., 1990; Monyer et al., 1991). Flip variants predominate before birth and continue to be expressed in adult rats, whereas flop variants are in low abundance before the eighth postnatal day and are up-regulated to about the same level as the flip forms in adult animals. The flip forms of most subunits desensitize more slowly and less profoundly than the flop forms (Table 4). Desensitization in the flip forms is more potently attenuated by cyclothiazide, whereas PEPA (4-[2(phenylsulfonylamino)ethylthio]2,6-difluoro-phenoxyacetamide) preferentially reduces the desensitization of the flop forms (see below).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Alternative splicing and editing of AMPA receptor subunits. The flip/flop and C-terminal splice variants of the AMPA receptor subunits are depicted schematically. The regions buried in the membrane are shown as boxes M1 to M4. The Q/R- and R/G-editing sites are indicated, as well as the defined phosphorylation sites in GluR1, Ser845 for PKA and Ser831 for PKC and CAMKII. Homologous C termini contain the same pattern.

C-terminal splice variants are found in GluR2, GluR4, and the kainate receptor subunits GluR5 to 7 (Figs. 3 and 4). A small percentage of GluR2 protein exhibits a long C terminus (Köhler et al., 1994). The cerebellum expresses GluR4c (Gallo et al., 1992), which has a C terminus that is shorter than that of GluR4 and is homologous to the tail of GluR2short. GluR5 cDNAs display four different C-tails and, additionally, an exon encoding 15 amino acids in the N terminus occurs in some transcripts (Sommer et al., 1992; Gregor et al., 1993). GluR6 and GluR7 each have two splice variants that differ in their C termini (Gregor et al., 1993; Schiffer et al., 1997a). When expressed as homomeric receptors in HEK 293 cells, GluR7a receptors gave rise to 5- to 10-fold larger currents than GluR7b (Schiffer et al., 1997a). Additional functional differences among the different splice variants have not been reported, but the different C termini may bind to different intracellular proteins and thus influence receptor targeting. For example, association of glutamate receptors with recently identified proteins containing PDZ domains is dependent on the C-terminal amino acids [e.g., GluR2 binding to glutamate receptor-interacting protein (GRIP), see below].

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Alternative splicing and editing of kainate receptor subunits. The diagram shows the basic structures of rat (prefix r) and human (prefix h) kainate receptor subunits, including the membrane-buried domains M1 to M4, the alternatively spliced cassettes, and editing sites in both M1 and M2 membrane domains.

The NR1 subunit contains three alternatively spliced exons: exon 5 in the N terminus (also called the N1 cassette) and exons 21 and 22 in the C terminus (also called C1 and C2 cassettes). Exon 22 (C2) contains an alternate acceptor splice site that, when used, splices out part of exon 22 including the stop codon and engages a new reading frame that encodes an alternative cassette C2' before a stop codon is reached. Several nomenclatures are used for the eight NR1 splice variants, some related to structure and others referring to the chronological appearance of the clones (see Hollmann et al., 1993; Zukin and Bennett, 1995). According to Hollmann's nomenclature, NR1-1 is the full-length clone containing both C-terminal exons, NR1-2 lacks exon 21, NR1-3 lacks exon 22, and NR1-4 lacks both (Fig. 5). The lower case letters a and b indicate the presence (a) or absence (b) of exon 5. These splice variants vary considerably in their properties and are differentially localized in the adult and developing animal (e.g., Laurie and Seeburg, 1994; Laurie et al., 1995; Nash et al., 1997; Paupard et al., 1997; Weiss et al., 1998). For example, recombinant NR1 receptors lacking exon 5 (N1 cassette) have a higher affinity for NMDA, are potentiated by Zn²⁺ when expressed without NR2 subunits in Xenopus oocytes but are more sensitive to block by Zn²⁺ and protons when expressed with NR2 subunits, and show stronger potentiation by polyamines through relief of proton inhibition (see below; Durand et al., 1993; Hollmann et al., 1993; Traynelis et al., 1995, 1998). Receptors containing N1 without C1 and C2 are more strongly enhanced by phorbol esters than those with C1 or C2 (Durand et al., 1993). Interestingly, after optic nerve crush, retinal ganglion cells down-regulated NR1 subunits and expressed preferentially receptors lacking exon 5 before cell death (Kreutz et al., 1998). However, the remaining NR1-b subunits seem to be crucial for survival because experimental reduction of NR1-b by treatment with antisense oligonucleotides increased retinal ganglion cell death after nerve crush.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Alternative splicing of NMDA receptor subunits. The different NR1 subunit splice variants arise from alternative splicing of the exons 5, 21, and 22, giving rise to the cassettes N1, C1, C2, and C2'.

The C1 cassette found in NR1-1 and NR1-3 is involved in receptor clustering, i.e., it binds to neurofilaments and the intracellular protein yotioa (Ehlers et al., 1998; Lin et al., 1998). Furthermore, the C1 cassette contains protein kinase C (PKC) phosphorylation sites and binds to calmodulin. Clustering and interaction with these regulators can be inhibited by PKC phosphorylation in the C1 cassette (Ehlers et al., 1995). After kindling a transient reduction of C1-containing splice variants was found in rats (Kraus et al., 1996; Vezzani et al., 1995), but whether the expected functional receptor alterations contribute to the kindled state is unknown. NR1 variants with the C2''cassette (NR1-3 and -4) interact with postsynaptic density (PSD)-95 proteins (see below).

Some glutamate receptor RNAs are post-transcriptionally modified by RNA editing, which leads to single-amino acid exchanges (reviewed by Seeburg, 1996). In this process, selected adenosines are deaminated to inosines by dsRNA adenosine desaminases (Rueter et al., 1995; see below). Inosines base pair like guanosines, which changes the amino acid codon. To date, editing has not been demonstrated for any NMDA receptor RNA, but AMPA and kainate receptor RNAs are edited at multiple positions.

In the primary transcript of GluR2, GluR5, and GluR6, a glutamine codon in the M2 domain (CAG) can be edited to an arginine (CIG) at the Q/R site (Figs. 1, 3, and 4). The arginine in edited versions of GluR2 causes low calcium permeability (Hume et al., 1991), low single-channel conductance (Swanson et al., 1996), and an approximately linear current-voltage relation even in heteromeric receptors (Verdoorn et al., 1991; Hume et al., 1991; Egebjerg and Heinemann, 1993; Washburn et al., 1997). Editing at the Q/R site in GluR6 also controls anion permeability (Burnashev et al., 1996). Furthermore, some of the AMPA receptor subunits, GluR2-4, are edited at the R/G site, which is located just before the flip/flop exons (Fig. 3). The glycine codon (IGA) replacing the genomically encoded arginine (AGA) in GluR3 and GluR4 reduces and speeds up recovery from desensitization (Lomeli et al., 1994). Finally, the kainate receptor subunits GluR5 and GluR6 can be edited in M1 at the I/V and Y/C sites (Köhler et al., 1993). Nutt et al. (1994) identified two potential editing sites in human GluR7 that are not the result of adenosine deaminations, based on sequencing reverse transcription-polymerase chain reaction (RT-PCR) products of fetal and adult human brain, which revealed different codons in the amino-terminal region of GluR7: Ser310 (TCC) or Ala (GCC) and Arg352 (CGG) or Gln (CAG). Whether these changes are due to editing as originally proposed or to polymorphisms (Schiffer et al., 1997b) awaits resolution. Since in GluR6 the edited amino acids in M1 can influence the ion permeability in receptors that have a Q at the M2 Q/R site, it has been argued that M1 may influence the structure of the open channel (Köhler et al., 1993; Burnashev et al., 1995, 1996). Changing the homologous amino acids in M1 of recombinant GluR4 had no effect on ion permeability, implying subtle differences in the channel structure between AMPA and kainate receptors. In single-cultured hippocampal neurons, editing of GluR5 and GluR6 occurred at different levels at all three sites, producing eight different species of mRNA (Ruano et al., 1995).

The enzymology of editing has received a lot of attention, with the ultimate aim of manipulating the editing process itself. Editing of glutamate receptors was initially found to depend on intronic sequences containing an editing complementary sequence, which base pairs with the exonic sequences (Higuchi et al., 1993; Egebjerg et al., 1994; Herb et al., 1996). The dsRNA structure is recognized by one of two editing enzymes, the first called DRADA or dsRAD (recently renamed ADAR1) and the second called RED1 or DRADA2 (now ADAR2) (Bass et al., 1997). Recombinant RED1 edits the GluR2 Q/R site (Melcher et al., 1996; Lai et al., 1997a; O'Connell et al., 1997), whereas the Q/R site of GluR6 can be edited by recombinant DRADA (Herb et al., 1996). Both recombinant enzymes can edit the R/G site. However, editing might not depend on the editing enzymes DRADA or RED1 alone, since some cells clearly express the mRNA for these enzymes but have no editing activity (Lai et al., 1997b). Moreover, the site selectivity and the efficiency of recombinant DRADA is changed by incubation with nuclear extracts (Dabiri et al., 1996), suggesting the contribution of additional proteins in the editing process. In addition to these deaminations that result in changes of the amino acid sequence, other adenosines are edited in introns and exons of GluR2 and GluR6, but do not change the coding sequence.

During development, editing occurs at different levels for all editing sites. The GluR2 Q/R site is the most vigorously edited site and this is even essential for survival. After E14 in rats >99% of GluR2 mRNA has arginine at the Q/R site. Removal of the editing complementary sequence in one GRIA2 allele in mice, which reduced the efficiency of Q/R site editing by about 25%, resulted in epilepsy and early death, attesting to the importance of efficient GluR2 editing (Brusa et al., 1995). The physiological role, if any, of unedited GluR2(Q) is unclear, however, as mice engineered to contain a genomic arginine codon in the GluR2 Q/R site were phenotypically similar to normal mice (Kask et al., 1998). Also, in other species, GluR2 is nearly fully edited, with exceptions found in tissue from older humans (Nutt and Kamboj, 1994) and in fish, where the Q/R site arginine in GluR2 is genomically encoded (Kung et al., 1996). A lower extent of editing is found at the other editing sites, which gives rise to a high number of functional variants. During development, editing at the other sites increases gradually and, in the adult, only about 50% of GluR5 and 80% of GluR6 are edited at the Q/R site (reviewed by Seeburg, 1996). About 80 to 90% of GluR2-4 are edited at the R/G site, except for GluR4flip (50%) (Lomeli et al., 1994). Kindling did not change editing of the Q/R site in GluR2, 5, or 6 (Kamphuis and Lopes da Silva, 1995), whereas transient ischemia in rats increased Q/R editing for GluR5 but reduced Q/R editing for GluR6 (Paschen et al., 1996). These results indicate that the editing processes are themselves tightly regulated.

	V. Post-translational Modifications

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Phosphorylation of ion channels is an important regulatory mechanism that may underly synaptic plasticity. The location of phosphorylation sites on glutamate receptor subunits and the functional consequences of phosphorylation have received much attention in the past 5 years. The responses of ionotropic glutamate receptors to agonists is usually potentiated after phosphorylation, but phosphorylation of NR1 can also disrupt channel clustering in transfected cells. The localization of the phosphorylation sites in glutamate receptor subunits was hotly debated for several years due partly to early uncertainties regarding receptor topology. Most of the controversies have recently been resolved and most experimentally veriified phosphorylation sites seem to be located intracellularly.

Like many other proteins, glutamate receptors are under tight control by various phosphokinases (reviewed by Roche et al., 1994; Soderling et al., 1994; Smart, 1997). Neuronal AMPA receptor activation can be potentiated by PKA (Knapp et al., 1990; Greengard et al., 1991; Wang et al., 1991; Blackstone et al., 1994), PKC (Wang et al., 1994a), calcium/calmodulin kinase II (CAMKII; McClade-McCulloh et al., 1993; Tan et al., 1994), and other unspecified kinases (e.g., Nakazawa et al., 1995). The potentiation by PKA of native AMPA receptors in cultured neurons appears to be due to an increase in channel open probability (Knapp et al., 1990) or open time (Greengard et al., 1991). Ser845 of GluR1 is a probable PKA target, because the C-terminus of the GluR1(S845A) mutant could not be phosphorylated after incubation with PKA (Roche et al., 1996). Moreover, the Western blot signal of hippocampal slices with antibodies directed againstphosphorylated Ser845 increased after forskolin treatment (Mammen et al., 1997). PKA activation appears to increase the open probability in recombinant GluR1 receptors but not in GluR1 (S845A) receptors (Banke and Traynelis, 1998). Changes in the phosphorylation state of Ser845 is specifically associated with synaptic plasticity. During chemically induced long-term depression, a decrease of phosphorylated Ser845, but not phosphorylated Ser831, was found (Lee et al., 1998). On the other hand, 15 min after a kindling stimulus phosphorylation of Ser845, but not Ser831, was specifically increased by about 25% (Wang et al., 1998). This suggests a role for the GluR1 PKA phosphorylation site in synaptic depression and enhancement. In Purkinje cells, according to immunocytochemistry with antibodies directed against Ser696 of GluR2, phosphorylation is suggested to occur between the M3 and M4 segments, which is a proposed extracellular domain (Nakazawa et al., 1995). Exposure to AMPA increased immunostaining, but the kinases responsible have not been identified yet. However, several early studies of recombinant or native receptors did not find AMPA receptor phosphorylation by PKA in this region (Tan et al., 1994, Moss et al., 1993), leading to the suggestion that the association of PKA with A kinase-associated proteins (AKAPs) may in some cases be required for potentiation of AMPA receptors (Rosenmund et al., 1994).

GluR1 and GluR2/3 in the postsynaptic densities could be phosphorylated by endogenous kinases in the presence of calcium and calmodulin (Hayashi et al., 1997). Phosphorylation of AMPA receptors by CAMKII and possibly PKC was produced by electrical stimulation patterns that induced long-term potentiation (LTP). Phosphorylation by CAMKII correlated temporally with the increased AMPA receptor-mediated responses during LTP (Barria et al., 1997a). The idea that phosphorylation of AMPA receptors by CAMKII mightcontribute to synaptic plasticity is supported by the finding that mice engineered to lack the  subunit of CAMKII are devoid of LTP and short-term potentiation as well as long-term depression of synaptic transmission (reviewed by Soderling, 1996). Benke et al. (1998) used nonstationary noise analysis to find evidence for an increased single-channel conductance of synaptic AMPA receptors after LTP, but it is not known whether receptor phosphorylation is involved.

The CAMKII and most PKC phosphorylation sites were initially proposed to be located between M3 and M4, which would place them on the extracellular side of the membrane. However, later studies showed that Ser627, which was believed to be involved in the potentiation by CAMKII, is not phosphorylated (Yakel et al., 1995; Roche et al., 1996). Ser831 in the C terminus of GluR1 was recently identified as the CAMKII phosphorylation acceptor site using mutagenesis and antibodies directed againstphosphorylated peptides (Roche et al., 1996; Barria et al., 1997b; Mammen et al., 1997). This serine is also the target of PKC and is unique in GluR1, not being found in GluR2-4. In hippocampal slices, Ser831 and also the other PKA target Ser845, were found to be phosphorylated under basal conditions by immunoblotting (Mammen et al., 1997). Barria et al. (1997b) noted that Ser831 is not a consensus site for either PKC or CAMKII and is only poorly phosphorylated by CAMKII after LTP induction. The fact that Ser831 is not a good substrate might be a physiological checkpoint ensuring that only strong synaptic input would lead to phosphorylation and potentiation of postsynaptic currents.

PKA has also been shown to phosphorylate recombinant GluR6 homomeric receptors (Raymond et al., 1993), and this phosphorylation of the GluR6 protein has been suggested to underlie an enhancement of whole-cell current responses (Raymond et al., 1993; Wang et al., 1993) similar to that observed with GluR1. These two studies have utilized site-directed mutagenesis to identify extracellularly localized serine residues (S684A and S666A) that are important for control of the PKA potentiation of GluR6, but it seems unlikely that these presumably extracellular serines themselves are phosphorylated (Basiry et al., 1999). Serines in the homologous position of GluR2 (S662 and S680) are located distant to the binding pocket in the crystal structure (Armstrong et al., 1998; red asterisks in Fig. 2C). The mechanism underlying the PKA-induced potentiation of GluR6, like that of GluR1, appears to be an increase in the open probability without any apparent change in response time course. Interestingly, calcineurin, a serine/threonine phosphatase that is colocalized with PKA, has the opposite effect, decreasing open probability (Traynelis and Wahl, 1997).

The effects of tyrosine kinases on non-NMDA glutamate receptors have received little study. Cotransfection of GluR1 and v-src resulted in phosphorylation of GluR1 (Moss et al., 1993), but this experiment could not distinguish between a direct or indirect effect of v-src. In synaptic membranes no phosphorylated tyrosines could be identified on GluR1 to GluR4, GluR6, GluR7, or KA2 (Lau and Huganir, 1995).

NMDA receptors can be phosphorylated by PKA, PKC, and CAMKII, and the Ca²⁺/calmodulin-dependent phosphatase calcineurin inhibits NMDA receptor function (Lieberman and Mody, 1994). In the brain, between 10 and 70% of NR1 and NR2 subunits seem to be phosphorylated at one or more sites by PKA or PKC. This variable proportion of phosphorylated subunits should substantially increase molecular and functional heterogeneity in the NMDA receptor family (Leonard and Hell, 1997).

PKC activation has been shown to enhance NMDA receptor function in different neuronal preparations. Activation of µ opioid receptors (Chen and Huang, 1992), the protease-activated receptor PAR1 (Gingrich et al., 1997), phosphoinositol-coupled metabotropic glutamate receptors (Aniksztejn et al., 1992), and muscarinic acetylcholine receptors (Markram and Segal, 1990; Dildy-Mayfield and Harris, 1994) all potentiated neuronal or recombinant NMDA receptors presumably via activation of PKC. Phosphorylation by PKC increases the opening probability and decreases the affinity for extracellular Mg²⁺ (Chen and Huang, 1992), but the mechanisms underlying these effects are unknown. Activation of PAR1 in hippocampal neurons by thrombin or an agonist peptide can also potentiate NMDA receptor responses in a Mg²⁺- and voltage-dependent manner that is reminiscent of PKC-induced relief of external Mg²⁺ blockade (Gingrich and Traynelis, 1998). Tingley et al. (1997) identified Ser890, Ser896, and Thr879 in the C1 cassette of the NR1 subunit as PKC targets by using antibodies directed against phosphorylated peptides. However, the C1 cassette does not seem to be responsible for PKC-induced potentiation, because recombinant homomeric NR1 receptors lacking the C1 cassette (NR1-2a) show even higher potentiation by PKC than receptors containing C1 (Durand et al., 1993). Some phosphorylation sites responsible for the Mg²⁺-independent PKC-induced potentiation in NR1 subunits lacking C1 (Durand et al., 1993; Wagner and Leonard, 1996) might lie within NR1 but outside of C1, or might instead reside in the Xenopus XenU1 subunit (Soloviev and Barnard, 1997). However, this Mg²⁺-independent potentiation also requires the carboxyl terminus of the NR2 subunit, suggesting it may be a property of the NMDA receptor channel region. Thus, receptors containing NR2C and NR2D are insensitive to phorbol ester-induced enhancement of function in Xenopus oocytes, whereas receptors containing NR2A and NR2B are potentiated (Mori et al., 1992). Alternatively, phosphorylation of sites in C1 may actually cause inhibition and the site(s) for potentiation may be on the NR2 subunit. Receptors without C1 would then be potentiated to a larger degree than receptors with C1. Additional work is needed to resolve these possibilities.

The large potentiation by PKC in C1-lacking homomeric NR1 receptors might also be explained partially by the observation that without C1 there is no receptor clustering. Indeed, PKC activation and phosphorylation of NR1 at Ser890 within the C1 cassette can inhibit clustering of NR1 (Ehlers et al., 1995; Tingley et al., 1997). This might also explain why in some studies PKC reduced NMDA receptor-mediated currents (e.g., Markram and Segal, 1992). Of great interest is the observation that calcium influx through NMDA receptors appears to amplify the potentiation by PKC, as shown by Zheng and Sigworth (1997) who compared wild-type NMDA receptors with receptors carrying NR1 mutations that reduce calcium permeability.

Little is known about regulation of NMDA receptors by CAMKII or PKA. The substrate for CAMKII phosphorylation appears to be Ser1303 in NR2B and perhaps the homologous serine in NR2A. This site was found to be phosphorylated in hippocampal neurons, but its function is so far unknown (Omkumar et al., 1996). A PKA phosphorylation site resides in the C1 cassette of NR1 (Ser879; Tingley et al., 1997), but the physiological changes in NMDA receptor function observed after PKA activation seem to be indirect. In hippocampal neurons, PKA activation by  adrenergic receptors potentiated NMDA receptor activity apparently by inhibiting the phosphatase calcineurin (Raman et al., 1996). In contrast, the phosphorylation of NR1 by PKA or PKC can antagonize its interaction with spectrin in vitro and might have direct functional consequences (Wechsler and Teichberg, 1998).

The activation of NMDA receptors can be inhibited by the serine and threonine phosphatases 1, 2A, or 2B (calcineurin). In acutely dissociated dentate gyrus granule cells, calcineurin can be activated by calcium entry through NMDA receptors and shorten the open time of NMDA receptors (Lieberman and Mody, 1994). Phosphatases 1 and 2A reduced the opening probability of NMDA receptors in cultured hippocampal neurons (Wang et al., 1994b). By second-to-second adjustment of the activity of kinases and phosphatases, the responsiveness of NMDA receptors to stimuli can thus be fine-tuned.

Several studies showed that activation of tyrosine kinases increases NMDA receptor-mediated responses in neurons (reviewed by Wang and Salter, 1994; Köhr and Seeburg, 1996; Gurd, 1997; Lu et al., 1998; Zheng et al., 1998). Glutamate-activated currents in HEK 293 cells transfected with NR1/NR2A could be potentiated by including Src or Fyn kinases in the patch pipette (Köhr and Seeburg, 1996). Src and Fyn kinases were unable to potentiate a receptor consisting of NR1 plus a C-terminal truncation mutant of NR2A, however, suggesting that the tyrosine phosphorylation sites might lie within the C-terminal domain of NR2A (Köhr and Seeburg, 1996). Src appears to be an endogenous kinase that regulates NMDA receptors, because 1) an anti-Src antibody applied to the cytoplamic surface of spinal dorsal horn neurons reduced the open probability of NMDA receptors; 2) application to the cytoplasmic surface of a high-affinity peptide that activates Src [EPQ(pY)EEIPIA] increased channel activity in inside-out patches whereas the unphosphorylated peptide, which does not activate Src, was ineffective; and 3) anti-Src antibodies coimmunoprecipitated NR1 from synaptic membranes (Yu et al., 1997). These results taken together suggest that Src may be a regulatory component of the subsynaptic protein complex that contains NMDA receptors.

How does Src kinase potentiate NMDA receptor activation? Zheng et al. (1998) found that Src potentiation of NR1/NR2A receptors could be prevented by a trace concentration (10 µM) of EDTA or other divalent chelators, and also showed that intracellular application of Src altered the Zn²⁺ sensitivity of the receptor, thereby reducing tonic inhibition by ambient Zn²⁺ present as a contaminant in the solution. Three C-terminal tyrosines (Y1105, Y1267, and Y1387) that are found in NR2A were required for this effect of Src. Only Y1267 is unique for NR2A; both Y1105 and Y1387 are also found in NR2B and 2C. The effect of Src was traced to a reduction in Zn²⁺ potency for high-affinity block (IC₅₀ = 90 nM) of NR1/NR2A receptors. With the likely assumption that ambient Zn²⁺ concentrations in the brain are in the range of several hundred nanomolar, Zheng et al. (1998) proposed that one of the synaptic functions of Src may be to regulate the degree of tonic inhibition of NR2Acontaining NMDA receptors by extracellular Zn²⁺. Interestingly, NR1/NR2B receptors could also be potentiated by Src, but only in the presence of higher concentrations of Zn²⁺ that approximate the low potency (IC₅₀ = 3-10 µM) block of these receptors by Zn²⁺. The low potency for Zn²⁺ inhibition of NR2B, NR2C, and NR2D (see below) may explain the failure of Köhr and Seeburg (1996) to observe potentiation of NR1 coexpressed with NR2B, NR2C, or NR2D. In oocytes with a nominally Zn²⁺ -free bath solution, not only NR2A, but also NR2B and even NR2D-heteromeric and NR1-homomeric receptors were potentiated by Src and by an endogenous kinase that was activated by insulin (Chen and Leonhard, 1996). The potentiation by Src of NR1/NR2B and NR1/NR2D was much less than that of NR1/NR2A receptors, and the potentiation by insulin may be partially due to a PKC-dependent mechanism because insulin receptors are also linked to phospholipase C.

Which NMDA receptor subunits can be phosphorylated on tyrosine residues? Moon et al. (1994) reported that the major tyrosine-phosphorylated protein in the PSD is NR2B. Tyrosines in 2 to 4% of NR2A and NR2B subunits, but not the NR1 subunit, were found to be phosphorylated in synaptic plasma membranes (Lau and Huganir, 1995). This low proportion of tyrosine-phosphorylated subunits contrasts to the high degree of NR2 subunit phosphorylation by PKA and PKC (up to 70%). The lower level of tyrosine phosphorylation of NR2A may allow for large increases in response to environmental stimuli. Indeed, an endogenous tyrosine kinase was found to increase phosphorylation of NR2A but not NR2B or NR1 in synaptic membranes by about 7-fold (Lau and Huganir, 1995). The endogenous kinases responsible for tyrosine phosphorylation of NR2B in synaptic membranes have not been fully identified, although exogenous Fyn could phosphorylate NR2A and NR2B in the PSD fraction (Suzuki and Okumara-Noji, 1995) and Src and NMDA receptors coimmunoprecipitate (Yu et al., 1997). The phosphorylation of NR2B by Fyn kinase antagonized the interaction of NR2B with spectrin in vitro (Wechsler and Teichberg, 1998), so one function for Fyn kinase may be to target NMDA receptors to the subsynaptic membrane.

Tyrosine kinase-enhanced synaptic currents through NMDA receptor channels have been proposed to play a role in LTP induction. O'Dell et al. (1991) blocked LTP induction by tyrosine kinase inhibitors in the CA1 region, and fyn knockout mice are impaired in LTP and spatial learning (Grant et al., 1992