glutamate receptors in the mammalian central nervous sistem

8/7/2019 Glutamate Receptors in the Mammalian Central Nervous Sistem

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GLUTAMATE RECEPTORS IN THE MAMMALIAN

CENTRAL NERVOUS SYSTEM

SEIJI OZAWA*, HARUYUKI KAMIYA and KEISUKE TSUZUKI

Department of Physiology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi,Gunma, Japan 371

(Received 9 October 1997)

AbstractGlutamate receptors (GluRs) mediate most of the excitatory neurotransmission in the mamma-lian central nervous system (CNS). In addition, they are involved in plastic changes in synaptic trans-mission as well as excitotoxic neuronal cell death that occurs in a variety of acute and chronic

neurological disorders. The GluRs are divided into two distinct groups, ionotropic and metabotropicreceptors. The ionotropic receptors (iGluRs) are further subdivided into three groups: a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptorchannels. The metabotropic receptors (mGluRs) are coupled to GTP-binding proteins (G-proteins), andregulate the production of intracellular messengers. The application of molecular cloning technology hasgreatly advanced our understanding of the GluR system. To date, at least 14 cDNAs of subunit proteinsconstituting iGluRs and 8 cDNAs of proteins coznstituting mGluRs have been cloned in the mammalianCNS, and the molecular structure, distribution and developmental change in the CNS, functional andpharmacological properties of each receptor subunit have been elucidated. Furthermore, the obtainedclones have provided valuable tools for conducting studies to clarify the physiological and pathophysiolo-gical signicances of each subunit. For example, the generation of gene knockout mice has disclosed criti-cal roles of some GluR subunits in brain functions. In this article, we review recent progress in theresearch for GluRs with special emphasis on the molecular diversity of the GluR system and its impli-cations for physiology and pathology of the CNS. # 1998 Elsevier Science Ltd

CONTENTS

1. Introduction 5822. Ionotropic receptors 583

2.1. Molecular diversity 5832.1.1. Classication 5832.1.2. Multiplicity of genes, and splicing and editing variants 5832.1.3. Structure (membrane topology, etc.) 583

2.2. AMPA receptors 5842.2.1. Distribution 5852.2.2. Channel properties 586

2.2.2.1. Ion selectivity and rectication properties 5862.2.2.2. Kinetics 5872.2.2.3. Single-channel properties 5872.2.2.4. Relation between functional and molecular properties of native receptors 588

2.2.3. Pharmacology 5882.2.3.1. Agonist binding site 588

2.2.3.2. Competitive antagonists 5902.2.3.3. Drugs that aect desensitization 5902.2.3.4. Channel blockers 590

2.2.4. Physiology 5912.2.4.1. Determinants of EPSC kinetics 5912.2.4.2. Functional signicance of Ca2+ permeability 591

2.2.5. Pathophysiology 5922.3. Kainate receptors 592

2.3.1. Distribution 5932.3.2. Channel properties 593

2.3.2.1. Ion selectivity and rectication properties 5932.3.2.2. Kinetics 5942.3.2.3. Single-channel properties 594

2.3.3. Pharmacology 5942.3.4. Physiology 5942.3.5. Pathophysiology 595

Progress in Neurobiology Vol. 54, pp. 581 to 618, 1998# 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0301-0082/98/$19.00

PII: S0301-0082(97)00085-3

*E-mail: [email protected].

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2.4. NMDA receptors 5962.4.1. Distribution 5962.4.2. Channel properties 597

2.4.2.1. Ca2+ permeability 597

2.4.2.2. Voltage-dependent block by Mg2+ 5972.4.2.3. Molecular determinants of ion permeation 5982.4.2.4. Kinetics 5992.4.2.5. Single-channel properties 5992.4.2.6. Relation between functional and molecular properties of native receptors 600

2.4.3. Pharmacology 6002.4.3.1. Glycine as a co-agonist 6002.4.3.2. Binding sites for agonists and co-agonists 6002.4.3.3. Drugs that aect NMDA receptor function 601

2.4.4. Physiology 6012.4.4.1. Kinetics of EPSC 6012.4.4.2. Targeted disruption of subunit gene 602

2.4.5. Pathophysiology 6022.4.5.1. Neuronal cell death 6022.4.5.2. Psychiatric disturbance 6032.4.5.3. Neuropathic pain 603

3. Metabotropic receptors 6033.1. Molecular diversity 6043.1.1. Multiplicity of genes, and splicing variants 6043.1.2. Structure (membrane topology, etc.) 6043.1.3. Classication 6053.1.4. Pharmacology 6053.1.5. Transduction mechanisms 605

3.2. Physiology 6063.2.1. Regulation of neuronal excitability 6063.2.2. Presynaptic inhibition 6063.2.3. Synaptic plasticity 607

3.3. Pathophysiology 6084. Concluding remarks 608

Acknowledgements 608References 609

1. INTRODUCTION

Glutamate receptors (GluRs) mediate most of theexcitatory neurotransmission in the mammalian cen-tral nervous system (CNS). They also participate inplastic changes in the ecacy of synaptic trans-mission underlying memory and learning, and theformation of neural networks during development(see Mayer and Westbrook, 1987b; Dingledine etal., 1988; Monaghan et al., 1989 for reviews).Ironically, glutamate and related excitatory aminoacids are toxic to central neurons. Excessive acti-

vation of GluRs during stress to the brain, such asischemia, head trauma and epileptic seizures leadsto the death of central neurons. The glutamate neu-rotoxicity may also be involved in the geneses ofvarious neurodegenerative diseases (see Rothmanand Olney, 1987; Choi, 1988; Choi and Rothman,1990; Meldrum and Garthwaite, 1990 for reviews).Thus, the GluRs are intimately involved in both thephysiology and pathology of brain functions.

The GluRs are categorized into two distinctclasses, ionotropic and metabotropic receptors (seeNakanishi, 1992; Seeburg, 1993; Hollmann andHeinemann, 1994 for reviews). The ionotropic recep-tors (iGluRs) contain cation-specic ion channels,

and are further subdivided into three groups:a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA), kainate and N-methyl-D-aspartate

(NMDA) receptor channels. On the other hand, the

metabotropic receptors (mGluRs) are coupled to

GTP-binding proteins (G-proteins) and modulate

the production of intracellular messengers.

The application of molecular cloning technology

has caused dramatic changes in the study of the

GluR system. The rst iGluR was cloned in 1989

with the expression-cloning approach (Hollmann et

al., 1989). The cloning of the rst mGluR was also

accomplished using the same technique in 1991

(Houamed et al., 1991; Masu et al., 1991). To date,

at least 14 cDNAs of iGluRs and 8 cDNAs of

mGluRs have been identied in the mammalian

CNS. In recent years, both physiology and pathol-

ogy of GluR systems have been investigated exten-

sively by using various techniques that manipulate

expressions of GluR genes. The aim of this review is

to summarize recent ndings on GluRs, putting

emphasis on describing the physiological and patho-

logical signicances of the molecular diversity of

GluRs. We will rst describe recent ndings on

iGluRs in terms of their molecular diversity, distri-

bution in the CNS, ion channel properties, pharma-

cology, and physiological as well as

pathophysiological signicances. Then, we willdescribe the molecular diversity, physiology and

pathophysiology of mGluRs.

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2. IONOTROPIC RECEPTORS

2.1. Molecular Diversity

2.1.1. ClassicationTraditionally, iGluRs have been divided into

three major subtypes, AMPA, kainate and NMDAreceptors, on the basis of agonist specicities.However, since neither agonist nor antagonistclearly distinguished between AMPA and kainatereceptors, they were often collectively referred to asnon-NMDA receptors. Cloning studies have demon-strated that they are distinct receptor complexesalthough they can be activated by the same agonists,notably AMPA receptors are activated by kainateand kainate receptors with certain subunit compo-sitions by AMPA. In recent years, several antagon-ists that dierentially block either AMPA or kainatereceptors have been developed.

2.1.2. Multiplicity of Genes, and Splicing and EditingVariants

Molecular cloning and expression studies haverevealed that the diversity of iGluRs is much largerthan expected from electrophysiological and phar-macological studies. Expression cloning in Xenopusoocyte has lead to the identication of two iGluRsubunits, an AMPA receptor subunit (GluR1) andthe principal NMDA receptor subunit (NR1)(Hollmann et al., 1989; Moriyoshi et al., 1991).Additional cDNAs encoding iGluR subunits werecloned by homology cloning and by polymerasechain reaction (PCR)-based strategies. So far, 14

cDNAs, 4 for AMPA receptor subunits (GluR1,GluR2, GluR3 and GluR4), 5 for kainate receptorsubunits (GluR5, GluR6, GluR7, KA1 and KA2),and 5 for NMDA receptor subunits (NR1, NR2A,NR2B, NR2C and NR2D), have been isolated.Phylogenetic trees of these 14 iGluR subunits are il-lustrated in Fig. 1. In addition, two cDNAs for dsubunits (d1 and d2), of which the functions are pre-sently unknown, have been cloned (see Nakanishi,1992; Seeburg, 1993; Hollmann and Heinemann,1994 for reviews). In addition to multiplicity ofgenes, the molecular diversity of iGluRs is furtherincreased by variants due to alternative splicing andRNA editing.

In most cases, the iGluR subunits were cloned

independently and almost simultaneously in severallaboratories, and therefore given dierent names toidentical subunits. In this review, we use gene namesconsistent with those introduced for the rst clonedrepresentative of each subfamily.

2.1.3. Structure (Membrane Topology, etc.)

iGluR subunits have in common a large extra-cellular N-terminus domain and four hydrophobicmembrane segments (M1M4). From analogy toother ligand-gated ion channels, such as the nic-otinic acetylcholine (ACh) receptor and GABAAreceptor, it was initially proposed that M1M4 aretransmembrane segments (TMITMIV) and the C-terminus is extracellular. However, this conventional

model has been revised in order to accomodate avariety of later ndings. For example, both im-munocytochemical and biochemical studies have

indicated the intracellular location of the C-terminus(Petralia and Wenthold, 1992; Tingley et al., 1993).In an attempt to determine the transmembrane top-ology model of the iGluR subunit, Hollmann et al.(1994) constructed a series of mutants of the AMPAreceptor subunit, GluR1, by introducing N-glycosy-lation consensus sequences at dierent sites alongthe entire length of the protein and analyzed thesemutant receptors for glycosylation, since glycosyla-tion at any given site can be taken as proof of theextracellular localization of the respected site. Basedon these analyses, they have concluded that thereceptor has only three transmembrane domains,which correspond to previously proposed TMI,TMIII and TMIV. According to this three-trans-

membrane domain model, M2 previously assignedfor TMII does not span the membrane, but is con-sidered to either lie in close proximity to the intra-

Fig. 1. Dendrogram of the members of the ionotropic glu-tamate receptor family. Unrooted neighbor-joint tree of 14iGluR subunit proteins of the rat constructed by a clustalw computer program (Thompson et al., 1994). The value,100% minus the sum of the length of the horizontal solidlines between the two subunits, indicates % identity in theamino-acid sequence between them. For example, the %identity in the amino-acid sequence between GluR1 andGluR2 is obtained as follows. The distance of GluR1 andGluR2 to each nearest node is 15% and 14%, respectivelyon the dendrogram, and the distance between these two

nodes is 2%, resulting in a total of 15% + 14% + 2% = 31% distance between GluR1 andGluR2. Therefore, the identity in the amino-acid sequencebetween them is (100% 31%) = 69%. The amino-acididentities among group 1 (GluR1GluR7, KA1, KA2),group 2 (NR1) and group 3 (NR2ANR2D) are low, ap-proximately 20%, and they are combined with dashed lineswhich are not used for estimating the distance. Data usedfor constructing this dendrogram were obtained fromDNA Data Bank of Japan (DDBJ) with accession num-bers, X17184(GluR1), M85035(GluR2), M85036(GluR3),M85037(GluR4), Z11713(GluR5), Z11548(GluR6),M83552(GluR7), X59996(KA1), Z11581(KA2),X63255(NR1), M91561(NR2A), M911562(NR2B),

M91563(NR2C), and D13213(NR2D).

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cellular surface of the plasma membrane or make ahairpin turn within the membrane. Furthermore, theentire region between M3 and M4, previously

believed to be intracellular, is extracellular, and theC-terminus is intracellular. Several lines of evidenceobtained using chimeric proteins and site-directedmutations support the notion that this new model isapplicable not only for AMPA receptor subunits,but also for kainate and NMDA receptor subunits(Kuryatov et al., 1994; Stern-Bach et al., 1994;Bennett and Dingledine, 1995; Wo and Oswald,1995; Hirai et al., 1996; Laube et al., 1997).

A transmembrane topology of the AMPA recep-tor subunit, GluR2, constructed according to thenew model proposed by Hollmann et al. (1994) is

illustated in Fig. 2.

2.2. AMPA Receptors

AMPA receptors mediate fast excitatory neuro-transmission in most of the synapses in the CNS.These receptors were initially named quisqualatereceptors. However, they were renamed AMPAreceptors, since quisqualate was found to act on

Fig. 2. Structure of AMPA receptor subunit GluR2. The 862 amino acids of the mature GluR2 proteinshown in single-letter code are placed according to the three transmembrane domain topology modelproposed by Hollmann et al. (1994). The two RNA editing sites, glutamine(Q)-to-arginine(R) at position586 and arginine(R)-to-glycine(G) at position 743, are indicated by lled squares. The box around

amino acids 744781 indicates the region where alternative splicing variants, ip and op, occur. Thenine amino acids in the ip version indicated by double arrows inside the box are changed to those out-

side of the box in the op version.

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mGluR and AMPA acted more specically onAMPA receptors.

Using the expression cloning technique,

Hollmann et al. (1989) isolated the rst iGluRclone, GluR1, from a rat brain cDNA library. Withsequence information of GluR1, the closely relatedreceptor genes, GluR2, GluR3 and GluR4, werecloned by several groups. The four AMPA receptorsubunits, GluR1GluR4, are of similar size (H900amino acids), and share 6873% amino acidsequence identity (Boulter et al., 1990; Keina nen etal., 1990; see Seeburg, 1993; Hollmann andHeinemann, 1994; Bettler and Mulle, 1995; forreviews). Each of the GluR1GluR4 subunits existsin two dierent forms, ``ip'' and ``op'', created byalternative splicing of a 115-base pair (bp) regionimmediately preceding M4 (see Fig. 2, columndrawn with thick lines) (Sommer et al., 1990). RNA

editings further increase the diversity of receptorsubunits. A glutamine residue (Q; CGA) in M2 isencoded in the genes for GluR1GluR4. However,GluR2 cDNA clones from adult animals contain anarginine (R; CGG) at this position termed the Q/Rsite (Fig. 2, a lled square in the M2 segment ofGluR2; also see Fig. 4) (Sommer et al., 1991). Thiscodon change due to the adenosine (A)-to-guanosine(G) alteration is generated by site-directed nuclearRNA editing, and only low levels of unedited RNAare present in fetal brain (Burnashev et al., 1992b;Higuchi et al., 1993). RNA editing at the second siteoccurs in positions termed R(arginine; AGA)/G(glycine; GGA) sites immediately preceding thealternative spliced modules, `` ip'' and `` op'' of

GluR2, GluR3 and GluR4 (Fig. 2, the other lledsquare) (Lomeli et al., 1994).

AMPA receptors are either homomeric or hetero-meric oligomers composed of these multiple subu-nits. Remarkable dierences in functional propertiesof native AMPA receptors are consequences ofdierent assemblies of these subunits.

2.2.1. Distribution

AMPA receptors are distributed ubiquitouslythroughout the CNS, although regional dierencesin the distribution are conspicuous. The regionaldierence in densities of AMPA-binding receptorswas previously examined with [3H]AMPA binding

studies (Monaghan et al., 1984; Olsen et al., 1987;Insel et al., 1990). Brain regions that are rich in highanity [3H]AMPA binding include the hippo-campus, which has slightly higher densities in CA1than CA3, and higher densities in the pyramidal celllayer than stratum radiatum and stratum oriens.Levels of binding in the molecular layer of the den-tate gyrus and the supercial layer of the cerebralcortex are also very high. Intermediate levels ofbinding are found in deeper layer cortex and in thecaudate-putamen. Lower levels are found in thediencephalon, midbrain and brainstem. In the cer-ebellum, binding levels are low as a whole, but morebinding is found in the molecular layer than in thegranule layer (Monaghan et al., 1984; Olsen et al.,

1987).After the molecular identication of receptor sub-

units, distributions of AMPA receptors were exam-

ined in more detail using in situ hybridizationhistochemistry. Systematic surveys have revealed re-gional expression patterns of GluR1GluR4 in

adult rat brains (Keina nen et al., 1990; Sommer etal., 1990; Monyer et al., 1991). In the hippocampus,the GluR1, GluR2 and GluR3 mRNAs are abun-dantly expressed in the pyramidal cell layer and den-tate gyrus with no apparent gradient of expressionbetween CA1 and CA3. Expression of GluR4mRNA is much less abundant than that of GluR1GluR3 mRNAs, and is relatively higher in CA1 anddentate gyrus than in CA3CA4. In the cerebralcortex, the expression patterns of GluR1, GluR3and GluR4 mRNAs dier among layers, whileGluR2 mRNA is uniformly found in all layers. Onlylow levels of GluR1 and GluR3 occur in layers IIIand IV, whereas GluR4 expression appears to bestrong in this region. In the cerebellum, GluR1

mRNA is expressed in Purkinje cells, but not ingranule cells. GluR2 mRNA is abundant inPurkinje cells and granule cells. GluR3 mRNA isexpressed in Purkinje cells, stellate-basket cells, withno detectable expression in granule cells. GluR4mRNA is expressed at high level in granule cells.Both GluR1 and GluR4, but not GluR2 andGluR3, are expressed in Bergmann glial cells(Keina nen et al., 1990).

Developmental and regional dierences in ex-pressions of two alternative splice variants, ip andop, have been studied using in situ hybridizationhistochemistry (Sommer et al., 1990; Monyer et al.,1991). AMPA receptor subunits are expressed pre-dominantly in the ip form in embryonic brains.

The op forms are expressed at low levels prior topostnatal day 8, and gradually increase throughoutthe brain, reaching adult levels by postnatal day 14.Thus, excitatory neurotransmission in the adultbrain appears to be mediated mainly by AMPAreceptors carrying the op module. However, it hasbeen noted that certain neuronal populations, exem-plied by hippocampal CA3 pyramidal cells, expressonly ip modules even in the adult stage (Monyer etal., 1991).

The GluR2 RNA editing to replace the gene-encoded Q to R is developmentally regulated(Sommer et al., 1991). At the early developmentalstage (embryonic day 14), a small percentage of theGluR2 does not undergo editing, and therefore the

unedited form coexists with the edited form. In post-natal stages, however, virtually all GluR2 exists inthe edited form (Sommer et al., 1991; Burnashev etal., 1992b). The editing at the R/G site in GluR2GluR4 is also developmentally regulated. The extentof R/G editing in the embryonic brain is generallysmall, but increases markedly during developmentup to 55% toH100% in the adult brain (Lomeli etal., 1994).

A comprehensive study of AMPA receptor immu-nohistochemistry was performed on sections of ratbrain, which were immunolabeled with antibodiesmade against peptides corresponding to the C-term-inal portions of GluR1, GluR2/3 and GluR4(Petralia and Wenthold, 1992). Regional distribution

patterns of AMPA receptor subunits in the brainwere generally consistent with those revealed by insitu hybridization studies. The subcellular distri-



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butions of AMPA receptors were examined usinganti-GluR1, anti-GluR2/3 and anti-GluR4 in thecerebral cortex and hippocampus of rat brain

(Petralia and Wenthold, 1992). Immunolabelingwith these antibodies is detected in the cytoplasmand on the plasma membrane of the cell body anddendrites. The labeling in the cytoplasm has a spottydistribution with accumulation on the outer mem-brane of mitochondria and nucleus and on microtu-bules. It is possible that at least a part of labelingsrepresents receptors being transported to and fromthe plasma membrane. The receptors on the plasmamembrane are localized predominantly at the post-synaptic densities. These postsynaptic labelings seemto be conned to the intracellular side of the mem-brane (Petralia and Wenthold, 1992). This obser-vation suggests an intracellular location for theantigenic site (C-terminal portion), being consistent

with the three transmembrane topology model foriGluR subunit proteins (Hollmann et al., 1994).Developmental changes in the subcellular localiz-

ation of GluR1 and GluR2/3 were examined in cul-tured rat hippocampal neurons byimmunohistochemistry (Craig et al., 1993). GluR1and GluR2/3 are relatively uniformly distributed insomata, axons and minor processes at the earlystage in cultured neurons obtained from embryonicday 18 rat. When the dendrites begin to develop, thereceptor subunits become polarized to the dendrites.In matured pyramidal cells (obtained from embryo-nic day 18 and 24 weeks in culture), clusters ofGluR1 and GluR2/3 are restricted to a subset ofpostsynaptic sites in the dendritic spines. The target-

ing of these receptor subunits appears to occur intwo stages that develop sequentially: rst, exclusionfrom the axon; second, enrichment at postsynapticsites (Craig et al., 1993).

2.2.2. Channel Properties

2.2.2.1. Ion selectivity and rectication properties

AMPA receptor channels had been considered tobe permeable only to Na+ and K+, and almostimpermeable to Ca2+ in central neurons. However,it was found that AMPA receptors displayed a sub-stantial permeability to Ca2+ (PCa/Palkali-metalI2.3according to the GoldmanHodgkinKatz (GHK)equation) and a strong inward rectication in a

small population of cultured rat hippocampal neur-ons (type II neurons) (Iino et al., 1990; Ozawa andIino, 1993). In contrast, AMPA receptors displayeda slight outward rectication and little permeabilityto Ca2+ (PCa/Palkali-metal < 0.18) in most neurons(type I neurons). The permeability sequence for theAMPA receptor in type II neurons to ve divalentcations (Ca2+, Sr2+, Ba2+, Mg2+, and Mn2+) wasBa2+ (1.3) > Ca2+ (1.0) > Sr2+ (0.9) > Mg2+

(0.8) > Mn2+ (0.7). The corresponding sequence forthe NMDA receptor was Ba2+ (1.2) > Ca2+

(1.0) > Sr2+ (0.8) > Mn2+ (0.3)bbMg2+ (


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fore unedited Ca2+-permeable AMPA receptorscould exist during brain development.

2.2.2.2. Kinetics

AMPA, glutamate and kainate are representativeagonists of AMPA receptors. Except kainate, theseagonists evoke rapidly and profoundly desensitizingresponses in AMPA receptors (Kiskin et al., 1986;Trussell et al., 1988; Tang et al., 1989). Kinetics ofdesensitization of AMPA receptors were investi-gated in outsideout membrane patches includingeither native or recombinant receptors by rapid ap-plication of agonists via a piezo-driven solutionexchange device (Colquhoun et al., 1992;Mosbacher et al., 1994; Geiger et al., 1995). Timeconstants of desensitization of current responses to110 mM glutamate ranged from 1 to 16 ms in out-

sideout patches derived from a variety of centralneurons (Tang et al., 1989; Trussell and Fischbach,1989; Colquhoun et al., 1992; Hestrin, 1992; Trussellet al., 1993; Barbour et al., 1994; Geiger et al., 1995;Ha usser and Roth, 1997). This large dierence inthe desensitization kinetics seems to be due to thesubunit composition of AMPA receptors containedin each membrane patch. Mosbacher et al. (1994)measured desensitization of dierent recombinanthomo- and heteromeric AMPA receptors. Amonghomomeric GluR1, GluR3 and GluR4 receptors,the GluR4 op channel showed the fastest desensiti-zation time constant, 0.9 ms, and GluR3 ip theslowest one, 4.8 ms when 1 mM glutamate wasapplied rapidly. In heteromeric receptors assembled

with either the ip or op forms of GluR2 andGluR4, GluR2 op/GluR4 op channel showed thefastest time constant, 0.8 ms, whereas GluR2 ip/GluR4 ip channel the slowest one, 6.1 ms. Exceptfor GluR1 channel, the channels assembled withop variants showed a faster desensitization timeconstant than those with ip variants. It is thuslikely that alternative splicing of AMPA receptorsubunits regulates channel kinetics which aect theshape of synaptic currents (Mosbacher et al., 1994).

Desensitization kinetics of AMPA receptors arealso regulated by RNA editing at the R/G site.Receptors assembled with the edited forms showeda slower desensitization rate than those with theunedited forms (Lomeli et al., 1994).

The rate of deactivation for both native andrecombinant AMPA receptors was estimated bymeasuring decay time constants of responses pro-duced by 1 ms pulses of 1 mM glutamate. Thesevalues ranged from 0.9 to 3.3 ms in native AMPAreceptors (Geiger et al., 1995). In general, there wasa positive correlation between desensitization anddeactivation time constants in native AMPA recep-tors. For example, both desensitization and deacti-vation time constants in hippocampal CA3pyramidal cells (15.2 ms and 3.0 ms, respectively)are much slower than the corresponding values inrelay neurons of the medial nucleus of the trapezoidbody (MNTB, 1.7 ms and 0.9 ms, respectively)(Geiger et al., 1995). However, in recombinant

GluR4 op receptors, the desensitization time con-stant is as fast as the deactivation time constant(0.60.9 ms). Thus, the time course of current decay

is determined almost exclusively by desensitizationkinetics in this case, not depending on the durationof agonist application (Mosbacher et al., 1994).

2.2.2.3. Single-Channel properties

Single-channel properties of the non-NMDAreceptors, both AMPA and kainate receptors, areless well characterized than those of the NMDAreceptor due to a combination of three diculties.(1) single-channel conductances of non-NMDAreceptors are of smaller amplitude than those ofNMDA receptors; (2) the open time of non-NMDAchannels is of briefer duration than that of NMDAchannels and (3) non-NMDA receptors adopt moremultiple conducting states than NMDA receptors.The issue is further complicated by the fact thatboth AMPA and kainate lack specicity for activat-

ing AMPA and kainate receptors, respectively.Previous experiments on a variety of central neur-ons have indicated that non-NMDA receptors arelinked to channels with a wide range of conduc-tances; some channel events are too small to beresolved as discrete channel openings (low conduc-tance responses), whereas others have clearly resol-vable multiple conductance levels (high conductanceresponses) (Cull-Candy and Usowicz, 1987, 1989a,b;Jahr and Stevens, 1987; Ascher and Nowak, 1988a;Cull-Candy et al., 1988; Ozawa et al., 1991b; Wyllieet al., 1993). Low conductance responses aredetected as noise increases, and the single channelconductance values are estimated to be as low as1 pS (much lower in some cases,H140 fS) with the

use of noise analysis (Cull-Candy et al., 1988;Ozawa et al., 1991b; Wyllie et al., 1993).

The low conductance responses are activatedmainly by kainate. Wyllie et al. (1993) have shownthat AMPA produces discrete openings with twoconductance levels of 6 and 10 pS in the outsideoutpatch where kainate produces only low conductanceresponses in rat cerebellar granule cells. A simple ex-planation for this result is that the low conductanceresponses are due to activation of kainate receptors.However, the possibility that a single GluR channelmay open to dierent conductance levels, dependingon the agonist, cannot be ruled out. It is also poss-ible that although kainate activates 6/10 pS events,their open times are too short to be detected as full

openings. In patches where kainate produces highconductance responses withH10, H20 and H30 pSopenings, AMPA and glutamte also activate re-sponses with the same conductances (Wyllie et al.,1993). Since the relative proportion of these conduc-tance levels is constant among dierent patches forall three agonists, it seems likely that three multipleconductances originate from a single AMPA recep-tor channel activated by AMPA, kainate and gluta-mate (Wyllie et al., 1993). Kinetic analysis of singlechannel events in high conductance responses hasindicated that the mean open time is as brief as0.5 ms for all agonists, and that few bursts of open-ings occur, in other words, most channel activationsare composed of single openings (Wyllie et al.,

1993).It is reasonable to assume that the single-channel

properties of AMPA receptors depend on the subu-



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nit composition. Swanson et al. (1997) have investi-gated the eects of RNA editing at the Q/R site,and splice variation of ip and op cassette on the

single-channel properties of recombinant AMPAreceptors formed from GluR2 and GluR4 subunits.Native AMPA receptors expressed in rat cerebellargranule cells are known to be composed of both ipand op forms of GluR2 and GluR4, with the opisoform increasing with age (Monyer et al., 1991;Mosbacher et al., 1994). In these recombinantAMPA receptors, the single-channel conductance ishighest for the Ca2+-permeable heteromeric chan-nels [GluR2(Q) (unedited form of GluR2) op/GluR4 ip], lowest for the Ca2+-impermeablehomomeric channels formed entirely of editedGluR2 ip or op, and intermediate for the Ca2+-impermeable heteromeric channels formed of bothedited and unedited subunits (GluR2 ip/GluR4 ip

and GluR2 op/GluR4 ip). For the rst group thesingle-channel conductance levels activated by gluta-mate are 8, 15 and 24 pS, whereas they are


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Fig. 3. Molecular analysis of native AMPA receptors at the single cell level using patch-clamp reversetranscription (RT)-polymerase chain reaction (PCR) method. (A) Experimental procedures of patch-clamp RTPCR method. Electrophysiological properties of AMPA receptor were rst analyzed inwhole-cell patch-clamp congurations. Then, the cell contents which include mRNAs of AMPA receptorsubunits were aspirated into the patch pipette. The harvested cell contents were expelled to a test tubefor reverse transcription (RT) reaction. The cDNA products were then subjected to PCR amplication.The cDNAs were amplied 1071011 at the rst 40 PCR cycles. The second PCR was performed toobtain enough amplied product (>100 ng/ml) for restriction analysis. For the PCR, a common primerpair for GluR1GluR4, which amplies GluR1GluR4 without changing the initial proportion, wasused. The sizes of the nal PCR products were approximately 750 bps. Four restriction endonucleases,Bgl 1, Bsp 1286I, Eco 47III, and Eco RI that selectively digest GluR1, GluR2, GluR3, and GluR4 PCRproducts, respectively, were chosen, and the restriction reaction was analyzed by agarose gel electrophor-esis. Vertical bars in the left, lower column indicate the positions of restriction sites on the GluR1GluR4 fragments. For more details see Lambolez et al. (1992) and Bochet et al. (1994). (B), (C)Rectication properties and subunit composition of AMPA receptors in cultured rat hippocampal neur-ons. (B) Current-voltage (IV) relations of responses of AMPA receptors in type I and type II neurons.The IV relation in the type I neuron displays a slight outward rectication, whereas that in the type IIneuron shows a strong inward rectication. Inset: Whole-cell currents evoked by ionophoretic appli-cations of kainate at holding potentials of60, 40, 20, 0, +20, +40 and +60 mV in type I and typeII neurons. Kainate almost exclusively activated AMPA receptors in these experimental conditions. (C)

Restriction analysis of AMPA receptor subunits in four type I (left) and four type II (right) neurons.Note the absence of digestion product with the enzyme specic for GluR2 fragment in the four type IIneurons and presence of these fragments in the four type I neurons. The Hae III digests of FX174 were

used as molecular weight markers (from Bochet et al., 1994).



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the agonist-binding sites, S1 and S2, should belocated extracellularly.

2.2.3.2. Competitive antagonistsQuinoxalinediones such as 6-cyano-7-nitroqui-

noxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxa-line-2,3-dione (DNQX) are potent competitiveantagonists at non-NMDA receptors (Honore et al.,1988). Both CNQX and DNQX potently displace[3H]AMPA binding (IC50=H0.30.5 mM), but theyalso signicantly displace [3H]kainate (IC50=H1.52.0 mM) (Sheardown et al., 1990). NBQX is a moreselective competitive antagonist for AMPA recep-tors, which has approximately 30-fold selectivity forAMPA over kainate receptors (IC50=H0.15 vsH4.8 mM) (Sheardown et al., 1990). Recently,another selective competitive antagonist of AMPAreceptors, YM90K has been synthesized by displace-

ment of the cyano group of CNQX with a 1-imida-zolyl group (Ohmori et al., 1994).

2.2.3.3. Drugs that aect desensitization

Two categories of drugs potentiate responses ofAMPA receptors by slowing the rate of desensitiza-tion. The rst are the pyrrolidinones aniracetam,piracetam, and the related compound 1-(1,3-benzo-dioxol-5-ylcarbonyl)-piperidine (1-BCP) (Ito et al.,1990; Isaacson and Nicoll, 1991; Ozawa et al.,1991a; Tang et al., 1991; Vyklicky et al., 1991;Gouliaev and Senning, 1994; Staubli et al., 1994;Desai et al., 1995). The second are the benzothiadia-zines CTZ, diazoxide, and 7-chloro-3-methyl-3-4-

dihydro-2H-1,2,4 benzothiadiazine (IDRA21)(Yamada and Rothman, 1992; Patneau et al., 1993;Zivkovic et al., 1995). Very recently, a novel sulfo-nylamino compound, 4-[2-(phenyl-sulfonylami-no)ethylthio]-2,6-diuoro-phenoxyacetamide(PEPA), which is structurally distinct from pyrroli-dinone or benzothiadiazine compounds, has beenfound to potentiate responses of AMPA receptorsby either abolishing or markedly slowing the rate ofdesensitization (Sekiguchi et al., 1997). Since mostof these drugs positively modulate glutamatergicsynaptic transmission, it is expected that they havethe potential for therapeutic use as memory- andcognition-enhancing drugs (Isaacson and Nicoll,1991; Ozawa et al., 1991a; Tang et al., 1991;

Yamada and Tang, 1993).With regard to the action site of CTZ, Partin et

al. (1994) have found that the ip and op splicevariants of homomeric GluR1 AMPA receptorsshow strikingly dierent sensitivity to CTZ; recep-tors composed of op forms are much less aectedby CTZ than those of ip forms. They have furthershown that although the ip and op forms ofGluR1 subunits have dierences in amino acidsequence at three separate areas, the large dierencein the sensitivity to CTZ is fully explained by asingle amino acid at position 750, namely, the dier-ence is reversed completely by exchange of serine-750 (ip) and glutamine-750 (op) (Partin et al.,1995). These results suggest that a part of the bind-

ing site for CTZ includes residue 750 in GluR1.The roles of a single residue at position 750 of

GluR1 in controlling sensitivity to CTZ, and also

aniracetam were further investigated by Partin et al.(1996). The kinetic analyses of responses of GluR1AMPA receptors with various mutations at position

750 have suggested that both modulators bind at ornear this critical site within the ip/op domain, butthat they act by distinct mechanisms: aniracetamslows desensitization by decreasing the closing rateconstant for ion channel gating, whereas CTZ eitherabolishes or reduces desensitization by stabilizing anondesensitized agonist-bound closed state (Partinet al., 1996).

Atypical 2,3-benzodiazepines such as 1-(4-amino-phenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzo-diazepine (GYKI 52466) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-(3N-methylcarba-mate)-2,3-benzodiazepine (GYKI 53655) are highlyselective noncompetitive antagonists of AMPAreceptors (Tarnawa et al., 1989; Donevan and

Rogawski, 1993; Palmer and Lodge, 1993; Zorumskiet al., 1993). It has been suggested that they counter-act the eect of CTZ by increasing the rate of desen-sitization of AMPA receptors by binding to theCTZ binding site or a neighbouring site (Palmer andLodge, 1993; Zorumski et al., 1993; Desai et al.,1995). However, a more recent study indicates thattheir action sites are not identical to the CTZ site,since the serine (S) to glutamine (Q) mutated GluR1ip, which is not aected by CTZ, is similarly antag-onized by these drugs (Partin and Mayer, 1996).

2.2.3.4. Channel blockers

A variety of spider and wasp toxins such as

JSTX, argiotoxin and philanthotoxin are known toblock glutamatergic synaptic transmission (Kawai etal., 1982; Abe et al., 1983; Jackson and Usherwood,1988; Washburn and Dingledine, 1996). JSTX, thetoxin from Nephila clavata, has a polyamine moietyconnected to a phenyl ring, and carries 23 positivecharges at physiological pH (Aramaki et al., 1987).It has been shown that JSTX specically blocksinwardly rectifying and Ca2+-permeable AMPAreceptors lacking the edited GluR2 subunit(Blaschke et al., 1993; Iino et al., 1996). At low con-centrations (


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meable AMPA receptors in a highly voltage-depen-dent manner (Herlitze et al., 1993; Washburn andDingledine, 1996; Koike et al., 1997).

2.2.4. Physiology

2.2.4.1. Determinants of EPSC kinetics

At most central synapses, both AMPA andNMDA receptors are activated during synaptictransmission. AMPA receptors mediate fast neuro-transmission. Their rapid kinetics are suitable forthis purpose. In contrast, neurotransmissionmediated by NMDA receptors occurs more slowlyand lasts for a much longer period, since their kin-etics are much slower than those of AMPA recep-tors.

The time course of EPSCs mediated by AMPAreceptors depends on two factors, that is, the gluta-

mate concentration transient at the synapse and theproperties of the postsynaptic receptors. Theamount of glutamate released from the presynapticterminal and the rate at which it is removed by dif-fusion and/or uptake, determine the transmitter con-centration in the synaptic cleft. The anity of thereceptors for glutamate and their deactivation anddesensitization kinetics control the time course ofsynaptic currents produced by the transmitter avail-able in the synaptic cleft. It seems likely thatremoval of transmitter by diusion occurs veryrapidly at central synapses, as is the case in the frogneuromuscular junction. An experimental estimationhas indicated that the peak glutamate concentrationat central glutamtergic synapses reaches 1.1 mM and

decays with a time constant of 1.2 ms (Clements etal., 1992). This estimation is supported by the obser-vations that in outsideout patches of both hippo-campal and visual cortical neurons a brief (1 ms)application of 1 mM glutamate produces a responsethat mimics the time course of EPSCs (Colquhounet al., 1992; Hestrin, 1992). It seems likely that thesojourn of the transmitter in the synaptic cleft isvery brief in most synapses, and that deactivationrather than desensitization plays a major role indetermining the decay rate of EPSCs since the timeconstant for the decay of EPSCs is faster than thatfor desensitization in most cases tested. Forexample, Hestrin (1992, 1993) has shown that thedecay time constants areH2 ms for both deactiva-

tion and EPSCs, but 8 ms for desensitization invisual cortical neurons. At some synapses, however,there is evidence that desensitization plays a role indetermining the time course of EPSCs, as describedbelow. First, CTZ, which markedly reduces desensi-tization but has little eect on deactivation, pro-longs some EPSCs (Yamada and Rothman, 1992;Yamada and Tang, 1993). A striking eect of thisdrug has been reported at the chick calycealsynapses of the magnocellullaris (Trussell et al.,1993). It has also been reported that EPSCs evokedin two types of cerebellar neurons, Purkinje cellsand interneurons (stellate and basket cells), bystimulation of parallel bers have markedly dierentkinetics: the Purkinje cell EPSCs decay slowly

(t =H7.3 ms), whereas the interneuron EPSCshave much faster kinetics (t =H1.5 ms). Sincethere are no dierences in the kinetics of both deac-

tivation and desensitization between patches takenfrom these two types of neurons, it has beensuggested that the complex geometry of synapses

between parallel bers and spines of Purkinje cellsdisturbs the diusion of transmitter, therebyprolonging EPSCs (Barbour et al., 1994). In suchcases, the time course of the EPSCs are inuencedby desensitization kinetics.

As described in Section 2.2.2.2, kinetics for desen-sitization and deactivation of AMPA receptorsdepend on which subunit gene is expressed. Thus,the time course of fast EPSCs is nely regulated indierent synapses by assembling dierent subunitsinto the receptors.

2.2.4.2. Functional signicance of Ca2+ permeability

Native AMPA receptors are likely to be hetero-

mers composed of GluR1GluR4 subunits. SinceGluR2, which determines the Ca2+ permeability inrecombinant AMPA receptors, is ubiquitouslyexpressed in the CNS, native AMPA receptors inmost central neurons exhibit low permeability toCa2+. However, native AMPA receptors with highCa2+ permeability have been reported in a varietyof cells in the CNS. Among these cells, the Ca2+

permeability is highest in type II cultured rat hippo-campal neurons and cerebellar Bergmann glia, fol-lowed by hippocampal interneurons, MNTB relayneurons and neocortical interneurons in rat brainslices (Iino et al., 1990; Burnashev et al., 1992a;Jonas et al., 1994; Geiger et al., 1995; Isa et al.,1996; Itazawa et al., 1997). It has also been noted

that even in similar types of neuron, such as hippo-campal and neocortical interneurons, the Ca2+ per-meability varies from cell to cell. The patch-clampRTPCR experiments indicate that the relativeabundance of GluR2 expressed in each neurondetermines the degree of Ca2+ permeability (Bochetet al., 1994; Jonas et al., 1994; Geiger et al., 1995).Most brain neurons with the Ca2+-permeableAMPA receptors are GABAergic and express aCa2+ binding protein, parvalbumin (Bochet et al.,1994; Jonas et al., 1994; Leranth et al., 1996; Kondoet al., 1997).

The Ca2+-permeable AMPA receptors areinvolved in the excitatory synaptic transmission in apopulation of hippocampal and neocortical nonpyr-

amidal, and spinal dorsal horn neurons (McBainand Dingledine, 1993; Gu et al., 1996; Isa et al.,1996; Itazawa et al., 1997). It is expected that thesereceptors provide a synaptically activated route forCa2+ entry, and play a role in modulating a long-term synaptic function. This notion is supported bythe observation that the amplitude of miniatureEPSCs is enhanced for a prolonged period afterCa2+ entry through Ca2+-permeable AMPA recep-tors in spinal dorsal horn neurons (Gu et al., 1996).

Very recently, mice lacking the GluR2 subunithave been produced by gene targeting (Jia et al.,1996). Surprisingly, most of the GluR2-lacking micesurvived and the adult mutants appeared healthyand fully capable of caring for themselves. In these

mice, the Ca2+

permeability of AMPA receptors inhippocampal CA1 pyramidal cells increased 9-fold.The long-term potentiation (LTP) in the CA1



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synapse was markedly enhanced and became non-saturable. Furthermore, the LTP could be inducedin the presence of blockers of both NMDA recep-

tors and high-voltage activated Ca

2+

channels (Jiaet al., 1996). These results suggest that Ca2+ entrythrough Ca2+-permeable AMPA receptors can par-ticipate in the regulation of synaptic plasticity in cer-tain conditions.

2.2.5. Pathophysiology

There is overwhelming evidence that the gluta-mate-induced excessive Ca2+ entry under pathologi-cal conditions leads to neuronal cell death (see Choi,1988; Choi and Rothman, 1990 for reviews).Neurological disorders in which this mechanismmay be involved include global and focal ischemia,hypoglycemia, physical trauma, drug abuse, meta-

bolic poisoning, certain food toxicities, epilepsy,AIDS-related dementia, Parkinson disease, motorneuron diseases (including amyotrophic lateral scler-osis), Huntington disease, and Alzheimer disease(see Lee, 1996 for review). The glutamate-inducedCa2+ entry may occur through any of three routes:NMDA receptor channels, Ca2+-permeable AMPAreceptor channels and voltage-dependent Ca2+

channels. Initially, the NMDA receptor, which ishighly permeable to Ca2+, was thought to playmajor roles in causing neuronal cell death in variouspathological conditions. The excessive activation ofthe NMDA receptor can cause severe neuronalinjury, and specic antagonists of this receptorpowerfully protect against NMDA neurotoxicity

(Rothman and Olney, 1987). However, since potentAMPA receptor antagonists such as NBQX,YM90K and GYKI 52466 have become available,evidence has been accumulating that AMPA recep-tor antagonists are eective in preventing neuronalcell death caused by brain ischemia (Sheardown etal., 1990; Le-Peillet et al., 1992; Yatsugi et al.,1996). This suggests that AMPA receptors play arole in the pathogenesis of ischemic neuronal celldeath.

Transient but severe global or forebrain ischemiacauses injury in specic populations of neurons, es-pecially in the hippocampal CA1 pyramidal cells(Kirino, 1982). Specic blockers of the NMDAreceptor are protective in moderate but not severe

ischemia (Buchan and Pulsinelli, 1991), whereasNBQX is eective in preventing delayed CA1 celldeath following severe ischemia (Sheardown et al.,1990; Buchan et al., 1991). It has been reported thatfollowing severe transient forebrain ischemia,GluR2 gene expression is preferentially reduced inCA1 hippocampal neurons at a time point that pre-ceded their degeneration (Pellegrini-Giampietro etal., 1992, 1994). Since the Ca2+ permeability of theAMPA receptor is increased by a reduced expressionof GluR2, the result suggests that the increasedCa2+ entry caused by a switch in AMPA receptorsubunit gene expression is a mechanism underlyingdelayed death of CA1 neurons. This notion is sup-ported by the nding that after ischemia the EPSCs

in CA1 neurons in the hippocampus of gerbil aremediated by Ca2+-permeable AMPA receptors(Tsubokawa et al., 1995).

Mechanisms for reduced ischemic damage byAMPA receptor antagonists would be as follows.Firstly, blockade of the AMPA receptor prevents

membrane depolarization, which in turn attenuatesthe activations of the NMDA receptor and voltage-activated Ca2+ channel, thereby reducing Ca2+

entry. Secondly, blockade of the AMPA receptordirectly reduces Ca2+ entry through the AMPAreceptor lacking GluR2. It should also be taken intoaccount that the AMPA receptor assembled withGluR2 is slightly permeable to Ca2+ (PCa/Palkali-metal (0.1) (Iino et al., 1990; Jonas and Burnashev,1995). Prolonged periods of activation of generallypoorly Ca2+-permeable AMPA receptors could pro-vide neurons with sustained load of Ca2+, whichwould eventually cause severe cell injury. This mech-anism may underly the vulnerability of cerebellarPurkinje cells (Brorson et al., 1995).

To elucidate functional signicances of the Q/Rsite editing in the GluR2 subunit, Brusa et al. (1995)generated heterozygous mice to harbor an editing-incompetent GluR2 allele by targeting the editingsite complementary sequence (ECS) in intron 11. Adouble-stranded RNA structure found in the pre-mRNA between the editing site in exon 11 and theECS is indispensable for the nuclear process of theQ/R site editing (Higuchi et al., 1993). In thesemutant mice, the abundance of GluR2 mRNA was70% of that in their wild-type littermates andH25%of the transcripts were unedited at the Q/R site. TheCa2+ permeability of AMPA receptors in principalneurons such as hippocampal CA1 pyramidal cells,neocortical pyramidal cells and cerebellar Purkinje

cells in these mice wasH57 times higher than inthat in the wild-type mice. These mutant mice devel-oped severe seizures and died by postnatal day 20(Brusa et al., 1995). This result contrasts with thereport by Jia et al. (1996) that the GluR2-ablatedmice showed no signs of seizure and adult mutantsappeared healthy. Thus, the overexpression of une-dited GluR2 diers from the disruption of GluR2 inits pathophysiological signicances, although bothmanipulations increase the Ca2+ permeability ofAMPA receptors in principal neurons in the brain.

2.3. Kainate Receptors

Although kainate is a potent agonist of the

AMPA receptor, it also activates a distinct class ofiGluRs, i.e. kainate-preferring receptors (kainatereceptors). A family of kainate receptors has beencloned by using low-stringency hybridization screen-ing with AMPA receptor subunit probes, and vesubunits, termed GluR5, GluR6, GluR7, KA1, andKA2, have been identied (see Seeburg, 1993;Hollmann and Heinemann, 1994; Bettler and Mulle,1995 for reviews). GluR5GluR7 may represent thelow anity kainate-binding site with KD ofH50 nM, whereas KA1KA2 correspond to thehigh anity kainate-binding site (KD=H5 nM) inthe neuronal membranes revealed by earlier radioli-gand binding studies. GluR5GluR7 are of similarsize (H900 amino acids) and share 7580% amino

acid sequence identity with each other, and H40%with AMPA receptor subunits GluR1GluR4.KA1KA2 are somewhat larger than GluR5GluR7

S. Ozawa et al.592


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(H970 amino acids), and share 70% amino acidsequence identity with each other, andH40% witheither GluR1GluR4 or GluR5GluR7. As in the

case of GluR2, RNA editing occurs at the Q/R sitein the M2 segment of GluR5GluR6, but not inGluR7. In contrast to GluR2, the Q/R site editing isincomplete during development, and signicantamounts of both edited and unedited versions coex-ist in adult brain. Editing at two additional sites inM1, i.e. I(isoleucine)/V(valine) and Y(tyrosine)/C(cysteine) sites, has also been found in the GluR6subunit. Alternative splicing of GluR5 further addsto receptor diversity (see Seeburg, 1993 for review).

Despite their widespread distribution throughoutthe CNS, physiological signicances of kainatereceptors largely remain unknown, since the nonde-sensitizing kainate-induced response at AMPAreceptors precludes detection of the smaller and

rapidly desensitizing response of kainate receptors.Recently, however, the kainate receptor-mediatedresponses have been studied using novel pharmaco-logical tools which selectively interact with eitherAMPA or kainate receptors.

2.3.1. Distribution

Radioligand binding studies using [3H]kainatehave revealed specic kainate binding sites withboth low (KD=H50 nM) and high (KD=H5 nM)anities. These binding sites are abundant through-out the entire CNS, although some brain areas suchas the hippocampal CA3 region and the granularlayer of the cerebellum show intense labeling (Foster

et al., 1981; Monaghan and Cotman, 1982; Represaet al., 1987; Miller et al., 1990).

More recently, kainate receptor subunits havebeen localized at the mRNA level using in situ hy-bridization histochemistry (Wisden and Seeburg,1993; Bahn et al., 1994), and at the protein levelusing immunohistochemistry (Huntley et al., 1993;Vickers et al., 1993; Roche and Huganir, 1995;Siegel et al., 1995). Each subunit, which forms kai-nate receptors, is dierentially distributed in theCNS. The KA1 mRNA occurs mainly in the CA3region and dentate gyrus of the hippocampus. TheKA2 transcript is almost universally expressed. TheGluR5 mRNA expression is found in the cingulateand piriform cortex, the subiculum, various septal

nuclei, and cerebellar Purkinje cells. The GluR6mRNA is most abundant in cerebellar granule cells,with lower levels in caudate-putamen and hippo-campus. The GluR7 transcripts are present in thedeep cerebral cortex, cingulate cortex, subiculum,caudate-putamen, reticular thalamus, and stellate/basket cells in the cerebellum. The combined ex-pression pattern of these ve subunits approximatesthe autoradiographic pattern of [3H]kainate,suggesting that kainate binding sites represent thedistribution of kainate-preferring receptors. Thisidea is also supported by the ndings that kainateand AMPA receptor subunits can coexist in thesame neurons (Mackler and Eberwine, 1993), butthey do not seem to coassemble with each other

(Wenthold et al., 1994).Immunohistochemical studies using nonselective

monoclonal antibodies against GluR5GluR7

suggested their presence in dendrites (Siegel et al.,1995). Using somewhat more specic antibodiesagainst GluR6GluR7 and KA2, immunolabeling

was also detected in some postsynaptic densities(Petralia et al., 1994a). Presynaptic localization ofthe receptors has been suggested by the stainingwith the antibody against GluR6GluR7 in pre-sumptive unmyelinated axons in the CA3 region,possibly mossy bers. This is in agreement with thending obtained by autoradiographic studies thathigh-anity kainate binding in stratum lucidum ofthe CA3 region is markedly reduced following theselective lesion of the dentate granule cells (Represaet al., 1987).


Since kainate elicits large nondesensitizing currentresponses at AMPA receptors, the native kainate

receptor-mediated response was dicult to detect inisolation until very recently. Therefore, the channelproperties have mostly been examined in recombi-nant kainate receptors. In expression studies, kai-nate-evoked currents are observed in homomericreceptors of GluR5 or GluR6 subunit (Egebjerg etal., 1991; Sommer et al., 1992), while functionalchannels are not formed by GluR7, KA1 or KA2expression alone (Werner et al., 1991; Bettler et al.,1992; Herb et al., 1992; Lomeli et al., 1992).Homomeric GluR5 or GluR6 channels show rapiddesensitization to the continuous application of kai-nate. When KA2 subunit is combined with eitherGluR5 or GluR6, the time course of desensitizationand current-voltage (IV) relation changes from

those of homomeric GluR5 or GluR6 channels(Herb et al., 1992). Specically, heteromeric chan-nels of KA2 and the unedited version of GluR5(Q)at the Q/R site show more rapid desensitization anda less inwardly rectifying IV relation than thehomomeric GluR5(Q) channels. Furthermore,GluR6/KA2 channels can be activated by AMPA,which does not activate homomeric GluR6 recep-tors. Since in situ hybridization studies (Wisden andSeeburg, 1993; Bahn et al., 1994) suggest that non-functioning subunits (GluR7 or KA1KA2) co-loca-lize with functioning subunits (GluR5GluR6) inthe same populations of neurons, the native kainatereceptors may be heteromers composed of dierentcombinations and ratios of these subunits.

2.3.2.1. Ion selectivity and rectication properties

In contrast to AMPA receptors, homomericGluR6 receptors show substantial Ca2+ per-meability. RNA editing at the Q/R site in the M2region plays a role in determining the Ca2+ per-meablity as well as the rectication properties(Ko hler et al., 1993). Unlike AMPA receptors, bothedited and unedited versions of GluR6 at the Q/Rsites expressed channels with relatively high Ca2+

permeability. Although one group reported that thePCa/Palkali-metal value was greater in the edited formthan in the unedited form (Ko hler et al., 1993),another group reported the opposite (Egebjerg and

Heinemann, 1993). As for the rectication proper-ties, the edited GluR5GluR6 homomers (Egebjerget al., 1991) or heteromers coexpressed with KA2



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(Herb et al., 1992) form channels with either a linearor slightly outwardly rectifying IV relation,whereas the unedited GluR5GluR6 homomers or

the heteromers with KA2 showed a strong inwardrectication (Herb et al., 1992; Sommer et al., 1992).

2.3.2.2. Kinetics

One striking dierence in channel propertiesbetween AMPA and kainate receptors exists in thedesensitization proles of responses to continuousapplications of kainate. Until recently, it has beenbelieved that kainate produces nondesensitizing cur-rents at AMPA receptors and rapid and completedesensitizing currents at kainate receptors, allowinga clear-cut functional distinction between thesereceptors. However, studies using the fast drug ap-plication device have revealed the presence of asmall, and rapidly desensitizing component in kai-

nate-activated current at AMPA receptors (Patneauet al., 1993). Furthermore, kainate receptor-mediated currents pharmacologically isolated byusing a noncompetitive AMPA receptor antagonistGYKI 53655 contain substantial steady-state re-sponses (H30% of peak) in cultured hippocampalneurons (Wilding and Huettner, 1997). Thus, thedesensitization prole does not necessarily give anabsolute distinction between the AMPA and kainatereceptors. As a whole, however, it is still true thatkainate-activated currents at the kainate receptorare desensitized more markedly than those at theAMPA receptor.


Single-channel properties of kainate receptorswere studied in the expression system using HEK293 cells transfected with kainate receptor subunitsGluR5, GluR6, and KA2 (Howe, 1996; Swanson etal., 1996). It was concluded that the single-channelconductance is signicantly reduced by Q/R siteediting. Homomeric unedited GluR6(Q) receptorsexhibited directly resolvable single-channel conduc-tance of 8, 15, 25 pS and the noise analysis gave amean conductance of 5.4 pS, whereas the edited ver-sion GluR6(R) showed an extremely low conduc-tance of 225 fS (Swanson et al., 1996). A similar lowconductance value for the GluR6(R) receptor(H230260 fS) was reported by another author

(Howe, 1996). GluR5(Q) exhibited resolvable con-ductance of 5, 9, 14 pS, while GluR5(R) had anextremely low (


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receptors. The functional native kainate receptorswere rst characterized in rat dorsal root ganglion(DRG) neurons (Huettner, 1990), where high levels

of GluR5 as well as KA2 mRNA have beenobserved (Bettler et al., 1990; Herb et al., 1992).Since there is a close correspondence in pharmaco-logical and electrophysiological characteristicsbetween recombinant GluR5 receptors and nativekainate receptors found in DRG neurons, GluR5may be a major component of the native receptorsof these neurons. Except for the case of DRG neur-ons, the characterization of kainate receptor-mediated currents, despite their widespread distri-bution, has been hampered by the larger AMPAreceptor-mediated currents. The diculty in record-ing kainate receptor-mediated currents could be alsodue to their desensitizing properties as well as theirpossible localization on distal dendrites or axon

terminals (Represa et al., 1987; Huntley et al., 1993;Petralia et al., 1994a). Although apical dendrites ofhippocampal pyramidal cells represent strong immu-nolabeling of kainate receptor subunits (Good et al.,1993; Petralia et al., 1994a), only AMPA receptor-mediated responses were detected in outsideoutpatches derived from the dendrites of these neuronsin slice preparations (Jonas and Sakmann, 1992;Spruston et al., 1995).

In the presence of GYKI 52466, that selectivelyblocks AMPA receptor-mediated responses, Lermaand colleagues have detected the functional kainatereceptor-mediated responses in cultured embryonichippocampal neurons (Lerma et al., 1993; Paternainet al., 1995). In the majority of cells, the electro-

physiological properties were similar to those ofhomomeric GluR6(Q) receptors. The kainate recep-tor-mediated current rapidly and almost completelydesensitized in response to continuous application ofkainate, and the IV relation displayed a stronginward rectication. A more recent study using thepatch-clamp RTPCR has shown that most of thecultured neurons express GluR6 mRNA and a fewcells express GluR5, but GluR7 and KA1KA2 sub-units are not detected (Ruano et al., 1995).Furthermore, analysis of the editing state of the Q/R site of the GluR6 subunit has revealed that theunedited variant is predominantly expressed,although in some cases both unedited and editedvariants coexist in the same cell. In addition, this

study has revealed that the rectication properties ofkainate responses are directly correlated with therelative abundance of edited vs unedited versions ofGluR6 in a single neuron (Ruano et al., 1995).

In contrast to the above results, native kainatereceptor-mediated currents isolated by using GYKI53655 in cultured hippocampal neurons from 2- to5-day-old postnatal rats show incomplete desensiti-zation to kainate (leaving H30% steady-state cur-rents of the peak currents). Furthermore, the IVrelation of kainate receptors from neonatal neuronshas been shown to be almost linear (Wilding andHuettner, 1997). The reason for these dierencesbetween embryonic and neonatal origins of the cul-tured neurons are currently unknown.

The presence of the functional kainate receptorsin hippocampal neurons suggests the involvement ofthe native kainate receptor in the fast glutamatergic

transmission. It has been reported that autaptic re-sponses in primary cultures as well as the populationsynaptic responses at CA1 and dentate gyrus

synapses in slice preparations are completelyblocked by the AMPA receptor-selective antagonistGYKI 53655 at its saturating concentration(100 mM), suggesting that fast synaptic transmissiondoes not involve the kainate receptor activation (seeLerma et al., 1997 for review). However, recent stu-dies have demonstrated that high-frequency stimu-lation of the hippocampal mossy bres generatesslow excitatory postsynaptic currents mediated bykainate receptors in hippocampal CA3 neuronswhich express many of the kainate receptor subunits(Castillo et al., 1997; Vignes and Collingridge,1997).

In addition to postsynaptic contributions, severallines of evidence suggest presynaptic roles for kai-

nate receptors. Earlier studies by Represa et al.(1987) suggested that kainate binding sites arehighly localized on hippocampal mossy ber term-inals in the CA3 region. Since selective destructionof the mossy bers reduces the convulsive eect ofkainate on CA3 neurons (Debonnel et al., 1989;Gaiarsa et al., 1994), it has been proposed that kai-nate acts at presynaptic kainate receptors on mossyber terminals and generates epileptiform activity.This idea is supported by the nding that unmyeli-nated presumptive mossy bers are labeled by theGluR6GluR7 antibody (Petralia et al., 1994a).With regard to functional roles of presynaptic kai-nate receptors, Chittajallu et al. (1996) have recentlyshown that kainate suppresses KCl-stimulated

[3

H]glutamate release from hippocampal synapto-somes and also suppresses the NMDA receptor-mediated synaptic responses measured in the CA1region of the slice preparations in the presence ofAMPA receptor antagonist GYKI 52466. Based onthese observations, it was suggested that kainatereceptors on the presynaptic terminals at hippocam-pal CA1 synapses negatively regulate the synapticrelease of glutamate. On the other hand, severaldierent groups have reported that kainate enhancestransmitter release from synaptosomes preparedfrom the hippocampal CA3 region (Gannon andTerrian, 1991; Malva et al., 1995, 1996). Therefore,modulating actions of presynaptic kainate receptorsmay vary from synapse to synapse (i.e. suppression

vs potentiation on CA1 and CA3 synapses, respect-ively). Presynaptic localization of the functional kai-nate receptors is also described in C-bers of theprimary aerents (Agrawal and Evans, 1986).

2.3.5. Pathophysiology

Kainate has a potent convulsive action whenapplied in vivo. Intracerebral or parenteral adminis-tration of kainate in rats results in limbic seizuresand a pattern of brain damage which resemblesthose of human temporal lobe epilepsy patients (seeNadler, 1981; Ben-Ari, 1985 for reviews). Domoateintoxication following ingestion of contaminated

mussels has been reported to cause a clinical syn-drome which includes seizures as well as anterogradememory decits (Teitelbaum et al., 1990). Since



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some neuronal populations, i.e. CA3 hippocampalpyramidal cells or thalamic reticular neurons, arehighly sensitive to kainate-induced damage (Nadler

et al., 1978) and their distribution approximatesthose of kainate binding sites, involvement of kai-nate receptors in the epileptic seizure has been pro-posed. Actually, several non-NMDA antagonistsprotect against certain forms of epileptic seizures(Chapman et al., 1991). Since selective lesion of hip-pocampal mossy bers reduces not only the convul-sive eect of kainate on CA3 neurons (Debonnel etal., 1989; Gaiarsa et al., 1994), but also the high a-nity kainate binding sites localized presumably atpresynaptic mossy ber terminals (Represa et al.,1987), it has been proposed that the kainate-inducedconvulsion is due to the presynaptic action onmossy ber terminals which leads to a massive glu-tamate release (Gaiarsa et al., 1994). The obser-

vation that kainate enhances glutamate release fromsynaptosomes prepared from the hippocampal CA3region (Gannon and Terrian, 1991; Malva et al.,1995, 1996) is consistent with this notion. In ad-dition, a loss of CA3 and hilar neurons was inducedby over-expression of GluR6 using a viral vector inorganotypic slice cultures (Bergold et al., 1993).

Interestingly, kindling, an experimental model ofepilepsy, causes sprouting of aberrant mossy bersin hippocampal CA3 region (see Ben-Ari andRepresa, 1990 for review). The involvement of kai-nate receptors in such a morphological plasticity hasto be tested in future studies using more specicpharmacological tools as well as gene targeting tech-nology.

2.4. NMDA Receptors

NMDA receptors mediate excitatory neurotrans-mission in the CNS in dierent ways from AMPAreceptors. They are characterized by voltage-depen-dent block by Mg2+ (Mayer et al., 1984; Nowak etal., 1984), a high permeability to Ca2+

(MacDermott et al., 1986; Mayer and Westbrook,1987a), and slow gating kinetics (Lester et al., 1990).These unique properties have attracted the interestof neuroscientists, since they provide the NMDAreceptor with a molecular basis for synaptic plas-ticity.

Using the expression cloning technique,

Moriyoshi et al. (1991) have cloned the fundamentalNMDA receptor subunit, NMDA1R (NR1), from arat brain cDNA library. Upon expression inXenopus oocytes, this subunit was capable of form-ing homomeric receptor channels that displayedcharacteristic NMDA properties. However, theamplitude of current responses obtained with thehomomeric NR1 receptors in oocytes was muchsmaller than that obtained with brain mRNA,suggesting the existence of additional subunits toform heteromeric receptors. Additional four recep-tor subunits, NMDAR2A (NR2A)-NMDAR2D(NR2D) (also termed e1e4 by Mishina and col-leagues), were cloned in the laboratories of Seeburg,Mishina and Nakanishi by PCR and cross hybridiz-

ation techniques (Ikeda et al., 1992; Kutsuwada etal., 1992; Meguro et al., 1992; Monyer et al., 1992;Ishii et al., 1993). Although NR2 subunits do not

form functional NMDA receptor channels by them-selves, when one of them is coexpressed with NR1,current responses of the heteromeric receptors

increase by several orders. Since recombinant het-eromeric NMDA receptors display dierent proper-ties depending on which of the four NR2 subunitsare assembled with NR1, the NR2 subunits can beregarded as modulatory subunits, whereas NR1serves as a fundamental subunit to form heteromericNMDA receptors (see Nakanishi, 1992; Seeburg,1993; Mori and Mishina, 1995 for reviews).

NR1 and NR2ANR2D subunits are composedof 938 (main isoform among splice variants, seebelow), 1464, 1482, 1250 and 1323 amino acids, re-spectively. NR1 shows low but signicant homology(2528% amino acid sequence identity) with otheriGluR subunits and shares a similar hydrophobicityprole to them. Amino acid sequence identities

between the NR1 and NR2 subfamilies are as low asH18%, and those among the NR2 families are ashigh as H40% to H50% (see Nakanishi, 1992;Hollmann and Heinemann, 1994; Mori andMishina, 1995 for reviews). In spite of a substantialdivergence in the amino acid sequences of NR2 sub-units from NR1, they possess structural character-istics similar to those of NR1 as will be elaboratedin Sections 2.4.2 and 2.4.3.

For the NR1 subunit, the existence of eightalternative splice variants, that is, the main isoform,four variants with N-terminal insertion and threevariants with C-terminal deletion, have beenreported. The relative abundance of these variantsare H67, H15 and H18%, respectively (Durand et

al., 1993; Sugihara et al., 1992; Yamazaki et al.,1992; Hollmann et al., 1993; see Mori and Mishina,1995 for review).

2.4.1. Distribution

To determine NMDA receptor distribution,ligand binding studies were conducted by using anumber of dierent radioligands, which specicallybind to NMDA receptors such as [3H]glutamate,[3H]1-(1-(2-thienyl)-cyclohexyl)piperidine ([3H]TCP),[3H]3-((2)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid ([3H]CPP) and [3H]5-methyl-10,11-dihy-dro-5H-dibenzo[a,d]cyclohepten-5,10-imine([3H]MK-801) (Monaghan et al., 1983; Maragos et

al., 1988; Subramaniam and McGonigle, 1991; seeMonaghan et al., 1989 for review). Results of thesestudies have shown that NMDA receptors are foundthroughout the brain but predominantly within theforebrain. The highest levels in the entire brain arefound in the CA1 region of the hippocampus.Furthermore, quantitative comparisons of the distri-bution of NMDA receptors determined by bindingsites of the dierent ligands have suggested the pre-sence of multiple pharmacologically distinct types ofNMDA receptors (see Monaghan et al., 1989 forreview).

Following the molecular cloning of NMDA recep-tor subunits, the distribution of each subunit wasexamined using in situ hybridization histochemistry

(Moriyoshi et al., 1991; Kutsuwada et al., 1992;Monyer et al., 1992, 1994; Watanabe et al., 1992;see review Mori and Mishina, 1995). In the adult

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rodent, the NR1 mRNA is distributed ubiquitouslythroughout the brain. In contrast, the four NR2transcripts display distinct regional patterns. The

NR2A mRNA is distributed widely in the brain, butmore so in the cerebral cortex, hippocampus, andcerebellum. The NR2B transcript is selectively pre-sent in the forebrain with high level expression inthe cerebral cortex, hippocampus, septum, caudate-putamen and olfactory bulb. The NR2C mRNA isexpressed predominantly in the granule cell layer ofthe cerebellum, with weak expression in the olfac-tory bulb and thalamus. Low levels of the NR2Dtranscript are detected in the thalamus, brain stemand olfactory bulb. The NR2C and NR2D tran-scripts are found in a subset of hippocampal neur-ons, most likely interneurons (Monyer et al., 1994).

Expression patterns of the NR2 subunits are alsoregulated developmentally in rodent brains

(Watanabe et al., 1992; Monyer et al., 1994). NR2Band NR2D mRNAs occur prenatally, whereasNR2A and NR2C mRNAs are rst detected aroundbirth. The most prominent change is the switchfrom NR2B to NR2C expression which occurs inthe cerebellar granule cells. NR2B mRNAs, whichare abundantly expressed in the cerebellar granulecells on embryonic day 7, almost disappear, and arereplaced by NR2C mRNAs. Since the functionalproperties of NMDA receptor channels such as thedegree of voltage-dependent Mg2+ block and deacti-vation kinetics depend on which of the four NR2 isassembled, it is conceivable that dierent spatial andtemporal patterns of expressions of the NR2 genesare designed for ne tuning of NMDA receptor

functions in both embryonic and adult brains.An immunohistochemical study of the distri-

bution of the NR1 subunit was performed on sec-tions of adult rat brain, which were immunolabeledwith a selective antibody recognizing 30 C-terminalamino acid residues (Petralia et al., 1994b). The re-gional pattern of the expression obtained by thisstudy agrees with the results of in situ hybridizationstudies in that most neurons throughout the brainexpress NR1 transcripts. In this study, the ultra-structural localization of immunostaining was alsoexamined in the hippocampus, cerebral cortex, andcerebellar cortex. Major stainings were localized inpostsynaptic densities apposed by unstained presyn-aptic terminals with mainly round vesicles, and in

associated dendrites.


2.4.2.1. Ca2+ permeability

The NMDA receptor channel has characteristicion permeation properties. The alkali-metal cations,Na+, K+ and Cs+ ions, permeate through thechannel with a low selectivity, but major dierencesfrom non-NMDA receptor channels exist in the per-meation properties for Ca2+ and Mg2+. Ca2+ ishighly permeant, whereas Mg2+ is a potent blockerof the NMDA channel (Mayer et al., 1984; Nowaket al., 1984; MacDermott et al., 1986; Mayer andWestbrook, 1987a; Ascher and Nowak, 1988b).

The relative Ca2+

to the alkali-metal cation per-meability in the NMDA receptor has been mostcommonly estimated by using the GHK equation.

According to this equation, the ratio of the per-meability coecients of Ca2+ and alkali-metal (Cs+

or Na+), PCa/Palkali-metal was estimated to be 4.0

(Mayer and Westbrook, 1987a) or 6.2 (Iino et al.,1990) in cultured central neurons when ionic con-centrations were used for the calculation. If ionic ac-tivities were used instead of concentrations, thevalue became 10.6 or 14.3. To predict the actualCa2+ inow through the NMDA channel in physio-logical conditions using this information, however,the following two points should be kept in mind.Firstly, the GHK equation is based on the assump-tion that there is no interaction among ions per-meating through the channel. This is obviously notthe case for the NMDA channel. The single-channelconductance of the NMDA channel decreases as theCa2+ concentration in external saline is increased,despite that the Ca2+ permeability of the NMDA

channel is much higher than that to the alkali-metalcations (Ascher and Nowak, 1988b; Gibb andColquhoun, 1992; Jahr and Stevens, 1993; Tsuzukiet al., 1994; Premkumar and Auerbach, 1996; Iinoet al., 1997). It is likely that permeation through theNMDA channel involves binding of permeant cat-ions, and Ca2+ binds to the site within the channelmore tightly and stays there longer than the alkali-metal cation, therefore reducing the amplitude ofthe single-channel current. This inhibitory eect ofCa2+ on the single-channel conductance can be wellexplained by assuming a model for one-ion channelbased on the EyringLa uger theory (La uger, 1973;Iino et al., 1997). Secondly, the relative Ca2+ per-meability of the NMDA channel is usually measured

in external solutions containing unphysiologicallyhigh concentrations of Ca2+ (>10 mM). To esti-mate the proportion of whole-cell current carried byCa2+ (fractional Ca2+ current) through nonspeciccationic channels, Neher and colleagues have devel-oped a technique combining whole-cell patch-clamprecording with uorescence measurement using fura-2 (for review see Neher, 1995). With the use of thistechnique, Burnashev et al. (1995) measured frac-tional Ca2+ current through recombinant NMDAchannels expressed in HEK 293 cells. When theHEK cells were bathed in physiological external sol-ution containing 135 mM Na+, 5.4 mM K+ and1.8 mM Ca2+, fractional Ca2+ currents throughNR1/NR2A and NR1/NR2C NMDARs were 11

and 8.2%, respectively. The corresponding valuespredicted by the relative Ca2+ permeabilitiesobtained according to the GHK equation were 14and 10%.

2.4.2.2. Voltage-Dependent block by Mg2+

In external medium containing physiological con-centrations of Mg2+ (H1 mM), the NMDA recep-tor-mediated current is maximal between 20 and30 mV, and is reduced at more hyperpolarized po-tentials despite the increased electrical driving force.The inward current is negligible at 80 mV, and theIV relation of the NMDA response thus exhibits aclear negative slope conductance between 30 and

80 mV. The negative slope conductance is elimi-nated by removing Mg2+ from the external solution(Mayer et al., 1984; Nowak et al., 1984).



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Single-channel studies have shown that theNMDA receptor has a conductance of 4050 pS insalines containing no Mg2+ (Nowak et al., 1984;

Ascher and Nowak, 1988b). In the presence ofMg2+, the single-channel current occurs in bursts ofshort-lasting openings separated by brief closures,which reect a fast transition between blocking andunblocking of the channel by Mg2+ (Nowak et al.,1984; Ascher and Nowak, 1988b). The block byMg2+ may be explained by assuming that the poreof the channel has a wide mouth located near theextracellular space in which hydrated cations entereasily, and a narrow constriction located deep in themembrane through which only dehydrated Mg2+

ions can pass. The apparent electrical distance ofthe site of Mg2+ block from the outside of the mem-brane was calculated to be between 0.64 and 1.0(Ascher and Nowak, 1988b; Jahr and Stevens, 1990;

Premkumar and Auerbach, 1996). Since the speed ofthe replacement of water molecules immediately sur-rounding the ion is much slower for Mg2+ than forother physiological ions (Na+, K+ and Ca2+), thepermeation of Mg2+ through the channel would behindered more markedly than that of the permeantions. This notion is supported by the fact that Ni2+

and Co2+, around which water molecules arereplaced as slowly as Mg2+, mimic the eect ofMg2+, but not Cd2+, Sr2+ and Ba2+ around whichthe rate of water exchange is 1000 times faster thanMg2+ (Diebler et al., 1969; Mayer and Westbrook,1985; Ascher and Nowak, 1988b; see Mayer andWestbrook, 1987b for review). More membranehyperpolarization would increase the probability

that Mg2+

occupies the entrance of the constrictionregion, thereby increasing the degree of the Mg2+

block.

2.4.2.3. Molecular determinants of ion permeation

Inspection of the primary structures of theNMDA receptor subunits, NR1 and NR2ANR2D,has revealed that the positions corresponding to theQ/R site in the M2 segment of the AMPA receptorsubunits are occupied by asparagine (N) in theNMDA receptor subunits (Fig. 4) (see Nakanishi,1992; Seeburg, 1993; Hollmann and Heinemann,1994; Mori and Mishina, 1995 for reviews). This Nsite governs both Ca2+ permeability and Mg2+

block in the NMDA channel (Burnashev et al.,1992c; Mori et al., 1992; Sakurada et al., 1993).Replacing the asparagine in this site of the NR1subunit with glutamine by site-directed mutagenesisleads to a reduction of Ca2+ permeability and a les-ser degree of Mg2+ block. The importance of theasparagine is more convincingly supported by theresult that replacing this amino acid with argininealmost completely abolishes both Ca2+ permeabilityand Mg2+ block (Burnashev et al., 1992c; Sakuradaet al., 1993). Asparagine in the corresponding site ofthe NR2 subunits (NR2A and NR2B) also plays animportant role in the permeability of the divalentcations. Replacing the asparagine in this site ofNR2 with glutamine markedly reduces the Mg2+

block and causes a permeability to Mg2+

(Burnashev et al., 1992c; Mori et al., 1992). Thus,asparagine in the N site of the NMDA receptor sub-

units guarantees the hallmark of the NMDA recep-tor that the channel is highly permeable to Ca2+

with no permeability to Mg

2+

. As described inSection 2.2.2.1, the selectivity of the Ca2+-per-meable AMPA receptors for divalent cations is verylow, and the receptors display a substantial per-meability to Mg2+. In the mutants of AMPA recep-tors containing an asparagine in the Q/R site, therelative permeability ratio between Ca2+ and Mg2+,PCa/PMg, becomes higher than in those containingglutamine (Burnashev et al., 1992b).

Although the amino-acid residues in the N siteplay crucial roles in determining the Mg2+ block inthe NMDA channel, they are not the sole factordetermining the degree of the Mg2+ block. Thisproperty diers depending on the particular NR2subunit coexpressed with NR1, that is, the NR1

NR2A and NR1NR2B channels are more sensitiveto Mg2+ than the NR1NR2C and NR1NR2Dchannels (Kutsuwada et al., 1992; Monyer et al.,1992, 1994). In external solution containing 1 mMMg2+, the current response was largest at 25 mVin the former two receptor channels, whereas it wasaround 45 mV in the latter two. Furthermore, theblock by Mg2+ on the inward current was muchstronger in the range between 25 and 80 mV forthe former than the latter (Monyer et al., 1994).

The profound eects of the amino-acid residues inthe N site on the permeation of divalent cationsstrongly suggest that structural elements includingthis site constrict the permeation pathway of theNMDA channel, serving as an ion selectivity lter.

The cross-sectional diameter of the constriction inthe wild-type NR1NR2A receptor was estimatedto be 0.55 nm by measuring the relative permeability

Fig. 4. Amino acid sequences of the M2 segments of

AMPA and NMDA receptor subunits. Amino-acidsequences of the M2 segments of AMPA receptor subunits(GluR1GluR4) and NMDA receptor subunits (NR1 andNR2ANR2D). Boxes indicate the Q(glutamine)/R(arginine) site of the AMPA receptor subunits and theN(asparagine) site of the NMDA receptor subunits. Theamino-acid residues in these sites determine permeations ofdivalent cations both in AMPA and NMDA receptorchannels. N + 1 site, one position downstream to the C-terminus from the N-site, is occupied by asparagine inNR2ANR2D, and also contributes to the formation ofion selectivity lter of the NMDA channel. For more

detail, see Wollmuth et al. (1996).

S. Ozawa et al.598


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of dierently sized organic cations (Villarroel et al.,1995; Wollmuth et al., 1996). The eects of mutatingthe amino-acid residues at the N sites and the adja-

cent two sites of both NR1 and NR2A subunits onthe pore size were further investigated (Fig. 4).When the NR1 N-site arginine was replaced by thesmaller glycine residue (G), the pore size of themutated NR1(N598G)NR2A channel wasincreased to 0.75 nm. In the NR2 subunit, both Nsite and N + 1 site, one position downstream to theC-terminus from the N site, are occupied by aspara-gine (Fig. 4). When the asparagine was replacedwith glycine at these two sites, the eect of theN + 1 site mutation (NR2A(N596G)) was muchmore prominent than that of the N site mutation(NR2A(N595G)), increasing the pore size from0.55 nm to 0.67 nm. The double mutation,NR1(N598G)NR2A(N596G), increased the pore

size to 0.87 nm, the sum of the increase produced bythe individual mutations (Wollmuth et al., 1996).The pore size of the wild NR1NR2A channel is

similar to that of the native NMDA receptor esti-mated in cultured rat hippocampal neurons(H0.450.57 nm) (Zarei and Dani, 1995). This sizeis smaller than that of the nicotinic acetylcholinereceptor (H0.700.74 nm, Dwyer et al., 1980).

2.4.2.4. Kinetics

It was reported that desensitization of the NMDAchannel in whole-cell recordings of cultured centralneurons was reduced by high concentrations of gly-cine and low levels of Ca2+. In the external Mg2+-

free solution containing 10 mM glycine and 0.2 mMCa2+, very little desensitization was observed in re-sponse to continuous application of either NMDAor glutamate (Vyklicky et al., 1990). In outsideoutpatches, however, NMDA responses exhibited cleardesensitization irrespective of concentrations of gly-cine and/or Ca2+. Even in the solution containig10 mM glycine and 0.01 mM Ca2+, the steady-stateresponse declined to about 20% of the initial peakamplitude with a time constant ofH210 ms (Satheret al., 1990). Although the reason for this dierencebetween the two conditions is unknown, it shouldbe noted that even in the outsideout conguration,the rate of desensitization in the native NMDAreceptor is much slower than in the AMPA recep-

tor.Both activation and deactivation kinetics are also

much slower in NMDA receptors than AMPAreceptors. When NMDA currents were evoked by 5-ms glutamate pulses in outsideout patches of cul-tured hippocampal neurons of neonatal rats, the1090% rise-time wasH10 ms, and the decay couldbe tted by a sum of two exponentials (tfastI90 msand tslowI260600 ms) (Lester et al., 1990). Duringthe long decaying period, bursts of channel openingspersisted which indicates that the agonist remainsbound to the receptor for this period (Lester et al.,1990).

Dierences in the decay time constants amongrecombinant NR1NR2 NMDA channels were esti-

mated following 300-ms glutamate pulses in Mg2+

-free solution containing 10 mM glycine. The timeconstants diered markedly depending on which

subunit of NR2 was assembled with NR1. Thevalues were H120 ms, H400 ms, H380 ms andH4800 ms, for NR2ANR2D, respectively (Monyer

et al., 1994).


The native NMDA receptor hasH50 pS openingsand H40 pS sublevels in various central neurons(Nowak et al., 1984; Jahr and Stevens, 1987; Ascheret al., 1988). As described in Section 2.4.2.1, how-ever, the single-channel conductance decreases asthe extracellular Ca2+ concentration is increased.For example, in rat CA1 pyramidal cells, the con-ductance of the main state is 51 pS in 1 mM Ca2+

and 148 mM Na+ solution, and is reduced to 42 pSwhen the Ca2+ concentration is raised to 2.5 mM(Gibb and Colquhoun, 1992). In addition to this 50/

40 pS state, a lower conductance state (H38/18 pS)has been reported in cultured cerebellar neurons(Cull-Candy and Usowicz, 1987). Farrant et al.(1994) have shown that the single-channel propertiesof the NMDA receptor in cerebellar granule cellsmarkedly changes during early development. At anearly stage (before postnatal day 13), most openingswere to the 50/40 pS state. In contrast, the majorityof channel openings (65%) were to the lower con-ductance state (H33/20 pS) at postnatal days 1923.Furthermore, the mean open time of the 50 pS state,3.4 ms, was reduced to 0.85 ms for the 33 pS con-ductance state (Farrant et al., 1994).

Expression studies have shown that both NR1NR2A and NR1NR2B NMDA receptors have

H50/40 pS openings (Stern et al., 1992; Tsuzuki etal., 1994), whereas NR1NR2C and NR1NR2Dreceptor openings have lower conductances (H35/20 pS) (Stern et al., 1992; Wyllie et al., 1996). Theseresults strongly suggest that the changes in the singlechannel properties of the NMDA receptor in cer-ebellar granule cells during the early development isdue to developmental changes of expressions of theNR2 subunits. This notion is further s

glutamate receptors in the mammalian central nervous sistem

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