Department of Neuroscience and Cell Biology, The University of Texas Medical Branch,
301 University Blvd., Galveston TX, 77555-1069, USA
Abstract Glutamate acts through a variety of receptors to modulate neurotransmission and neuronal excitability. Glutamate plays a critical role in neuroplasticity as well as in nervous system dysfunctions and disorders. Hyperfunction or dysfunction of glutamatergic neuro- transmission also represents a key mechanism of pain-related plastic changes in the central and peripheral nervous system. This chapter will review the classiﬁcation of glutamate receptors and their role in peripheral and central nociceptive processing. Evidence from preclinical pain models and clinical studies for the therapeutic value of certain glutamate receptor ligands will be discussed.
Keywords Ionotropic glutamate receptors · NMDA and non-NMDA receptor antagonists · Metabotropic glutamate receptors · Neurotransmission · Nociception · Plasticity · Analgesia · Preclinical · Clinical
Glutamate is the major excitatory amino acid in the mammalian nervous sys- tem. A highly ﬂexible molecule, glutamate binds to a number of diverse families of receptors and transporters. These include the ionotropic glutamate receptors (iGluRs), which are ligand-gated ion channels, and the metabotropic glutamate receptors (mGluRs), which couple through different G proteins to a variety of signal transduction pathways. Vesicular transporters (vGluT1 and 2) pack- age glutamate into vesicles for synaptic exocytosis whereas plasma membrane glutamate transporters (excitatory amino acid transporters EAAT1–5) remove glutamate from the synaptic space. Glutamate receptor classiﬁcation, struc- ture, and function have been reviewed. This chapter will focus on the role of iGluRs and mGluRs in nociception and pain (for reviews of glutamate re- ceptor classiﬁcation, structure, and function see Michaelis 1998; Anwyl 1999; Dingledine et al. 1999; Schoepp et al. 1999; Lerma et al. 2001; Wollmuth and Sobolevsky 2004; Mayer and Armstrong 2004; Swanson et al. 2005; Mayer 2005).
Ionotropic Glutamate Receptors
The iGluRs allow the ﬂow of cations through the channel in the center of the re- ceptor complex upon binding of an extracellular ligand (glutamate). Typically, this results in the depolarization of the plasma membrane and the generation of an electrical signal, the action potential, that propagates along the axon and triggers the release of transmitter(s) from the synaptic terminal. The iGluRs are tetrameric complexes of subunits transcribed from separate genes (Michaelis
1998; Dingledine et al. 1999; Wollmuth and Sobolevsky 2004; Mayer and Arm- strong 2004; Mayer 2005). Each subunit has four hydrophobic domains but only three transmembrane segments because the second domain forms a pore loop at the inner opening of the ion channel and does not go through the membrane. Thus, different from other ionotropic receptors, iGluRs have an extracellular N-terminal and an intracellular C-terminal domain. The C-terminus contains
sites for phosphorylation and protein–protein interaction and is subject to posttranscriptional processes such as RNA editing and alternative splicing. Phosphorylation of iGluRs by various kinases (including PKA, PKC, CaM ki- nase II, tyrosine kinase) can increase the ion channel function. Interaction with intracellular scaffolding and cytoskeletal proteins may be important for receptor trafﬁcking, anchoring, and signaling.
The iGluRs are subdivided into three major groups based on their phar- macology, structural similarities, and sequence homology (Table 1; Michaelis
1998; Dingledine et al. 1999; Lerma et al. 2001; Wollmuth and Sobolevsky
2004; Mayer and Armstrong 2004; Mayer 2005). Each group includes different subunits that are encoded by different genes: N -methyl-d-aspartate (NMDA) receptors (NR1, NR2A-D, NR3A, and NR3B subunits); α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptors (GluR1–4); and kainate receptors (GluR5–7, KA1 and KA2). Two orphan receptor subtypes (δ1 and δ2) also have been cloned, which share sequence homology with other iGluRs, but their ligand-binding and functional properties remain to be determined.
AMPA and kainate receptors are also referred to as non-NMDA receptors.
Functional NMDA receptors are obligate heteromeric assemblies of NR1 sub- units with NR2A-D or, less commonly, with NR3A,B subunits (Michaelis
1998; Dingledine et al. 1999; Wollmuth and Sobolevsky 2004; Mayer and
Armstrong 2004; Mayer 2005). NMDA receptors have binding sites for glu- tamate (formed by the NR2 subunits) and the co-agonist glycine (strychnine- insensitive glycineB site formed by NR1) as well as binding sites for polyamines (NR2B) and phencyclidine (PCP site inside the ion channel), which can modu- late NMDA receptor function. Ion currents through NR2B-containing NMDA receptor heteromers have a much slower decay time (i.e., longer duration) compared to NR2A-containing receptors. NMDA receptors are unique in that
occupation of the glycine binding site and removal of the Mg2+ blockade of the ion channel are required for full activation. Furthermore, NMDA receptors
are tonically inhibited by protons, and Zn2+ (NR2A>NR2B) enhances whereas polyamines (NR2B) relieve proton inhibition. Compared to non-NMDA re-
ceptors, NMDA receptors respond more slowly to glutamate due to the tonic and voltage-dependent inhibition by Mg2+; they participate in slow synaptic transmission, desensitize only weakly, and have a high permeability to Ca2+ (tenfold greater than to Na+).
NMDA is the diagnostic ligand for these receptors. d-AP5 (d-2-amino-5- phosphonopentanoic acid) is the most commonly used competitive antagonist at the glutamate binding site. Noncompetitive antagonists that bind to the PCP site in the NMDA receptor channel include the high-afﬁnity compound MK-
801 (dizocilpine; 10,11-dihydro-5-methyldibenzocyclohepten-5,10-imine) and
AC, adenylylcyclase; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AP5, d-2-amino-
5-phosphonopentanoic acid; APDC, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate; CHPG, (RS)-2- chloro-5-hydroxyphenylglycine; CNQX, cyano-7-nitroquinoxaline-2,3-dione; CP-101606 (traxoprodil; (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol); CPCCOEt, 7-(hydroxy- imino) cyclopropa[b]chromen-1a-carboxylate ethyl ester; DHPG, (S)-3,5-dihydroxyphenylglycine; DXM, dextromethorphan; EGLU, (2S)-alpha-ethylglutamic acid; GIRK, G protein-activated inwardly rectifying potassium channels; LAP4, L-(+)-2-amino-4-phosphonobutyric acid; LSOP, l-serine-O- phosphate; LY293558, (3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl] decahydroisoquinoline-3- carboxylic acid; LY341495, 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid; LY354740, [1S, 2S,5R,6S]-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid; LY367385, (S)-
(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid; LY379268, (−)-2-oxa-4-aminobicylco hexane-
4,6-dicarboxylic acid; LY382884, (3S, 4aR, 6S, 8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a- decahydroisoquinoline-3-carboxylic acid; MPEP, 2-methyl-6-(phenylethynyl)pyridine; NBQX, 2,3- dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f ]quinoxaline-7-sulphonamide; NMDA, N-methyl-d-aspartic
acid; Ro 25–6981, (R-R*,S*)-alpha-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine
propanol; UBP1112, α-methyl-3-methyl-4-phosphonophenylglycine; UBP302, (S)-1-(2-amino-2- carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione; VGCC, voltage-gated calcium channel Numbers in parentheses refer to receptor subtype/subunit selectivity *Denotes drugs tested/used
clinically +Also binds to other classes of receptors (α1, 5HT1α, and σ)
clinically used agents such as ketamine, memantine, and dextromethorphan with its main metabolite dextrorphan. Noncompetitive antagonists selective for NR2B subunit-containing NMDA receptors include CP-101606 [traxoprodil; (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol)
and Ro 25–6981 (R-R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-
1-piperidine propanol] and their parent compound ifenprodil, which also
binds to other receptors besides glutamate receptors (Table 1; Michaelis 1998; Dingledine et al. 1999; Parsons 2001; Wollmuth and Sobolevsky 2004; Mayer
AMPA and kainate receptors can be homo- or heteromeric tetramers. AMPA
receptors (Michaelis 1998; Dingledine et al. 1999; Wollmuth and Sobolevsky
2004; Mayer 2005) are composed of subunits GluR1–4, which are encoded by separate genes. All AMPA receptor subunits exist as two splice variants, ﬂip and ﬂop, which yield altered kinetics such that the ﬂop forms show more rapid desensitization. Each subunit has a ligand binding site made up from the N-terminal region; the C-terminus plays a role in receptor trafﬁcking. Different from NMDA receptors, native AMPA receptors mediate fast synaptic transmission, have fast gating kinetics, desensitize rapidly, and show a lower
permeability to Ca2+. Ca2+ permeability of AMPA receptors is determined by the GluR2 subunit, which in its native form is impermeable to Ca2+ due to posttranscriptional RNA editing that changes a single amino acid in the pore-
forming second membrane domain from glutamine (Q) to arginine (R). Most neurons express GluR2 in combination with one or more of the GluR1, 3, and
4 subunits, yielding AMPA receptors with low Ca2+ permeability.
Kainate receptors (Michaelis 1998; Dingledine et al. 1999; Chittajallu et al.
1999; Lerma 2003; Wollmuth and Sobolevsky 2004; Mayer 2005) are tetrameric assemblies of low-afﬁnity GluR5–7 subunits and high-afﬁnity KA1 and 2 sub- units. Homomeric expression of KA1 and KA2 does not form functional ion channels, but KA1 and KA2 contribute to heteromeric receptor complexes when expressed with the other subunits. GluR5–7 can form homomeric and heteromeric kainate receptors. Like the other iGluRs, kainate receptor sub- units undergo alternate splicing and RNA editing, yielding a number of phar- macologically and functionally distinct receptors. Similar to AMPA receptors, kainate receptors with Q to R editing in the GluR5 or GluR6 subunits are im-
permeable to Ca2+; receptors with unedited subunits are calcium permeable. Interestingly, some actions of kainate may involve the interaction of ionotropic kainate receptors with a G protein. Kainate receptors contribute to postsynaptic excitatory responses and plasticity; presynaptic kainate receptors can increase
or decrease the release of glutamate and inhibit the release of the inhibitory transmitter γ-aminobutyric acid (GABA) (resulting in disinhibition).
The pharmacological differentiation of AMPA and kainate receptors has been difﬁcult (see Table 1; Michaelis 1998; Dingledine et al. 1999; Chittajallu et al. 1999; Wollmuth and Sobolevsky 2004). Naturally, AMPA and kainate show some selectivity for AMPA- and kainate-preferring receptors, respec- tively. ATPA [(RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl) propionic acid] is a more selective kainate receptor agonist, particularly for GluR5. The most commonly used non-NMDA receptor antagonists are CNQX (cyano-7- nitroquinoxaline-2,3-dione) and NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro- benzo[f ]quinoxaline-7-sulfonamide), which have limited selectivity for AMPA over kainate receptor subunits. GYKI53655, a 2,3-benzodiazepine, is cur- rently the most selective AMPA receptor antagonist (non-competitive allosteric modulator). The competitive antagonist LY293558 [(3S,4aR,6R, 8aR)-6-[2- (1(2)H-tetrazole-5-yl)ethyl] decahydroisoquinoline-3-carboxylic acid], which has been tested in phase II clinical trials, shows higher selectivity for AMPA re- ceptors over GluR6 and GluR7 kainate receptors, but it is also active at the GluR5 subunit (Sang et al. 2004). Antagonists that distinguish kainate from AMPA re- ceptors have only recently become available. LY382884 [(3S, 4aR, 6S, 8aR)-6-(4- carboxyphenyl)methyl-1, 2, 3, 4, 4a, 5, 6, 7, 8, 8a-decahydroisoquinoline-3-carb- oxylic acid] and UBP302 [(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxyben- zyl)pyrimidine-2,4-dione] are GluR5-selective antagonists at concentrations that do not affect AMPA receptors (Bortolotto et al. 2003; More et al. 2004).