Ligand-Gated Ion Channels: New Insights into Neurological Disorders

Review pubs.acs.org/CR

Ligand-Gated Ion Channels: New Insights into Neurological Disorders and Ligand Recognition Damien Lemoine,‡ Ruotian Jiang,‡ Antoine Taly,† Thierry Chataigneau,‡ Alexandre Specht,§ and Thomas Grutter*,‡ ‡

Laboratoire de Biophysicochimie des Récepteurs Canaux, UMR 7199 CNRS, Conception et Application de Molécules Bioactives, Faculté de Pharmacie, Université de Strasbourg, 67400 Illkirch, France § Laboratoire de Chimie Bioorganique, UMR 7199 CNRS, Conception et Application de Molécules Bioactives, Faculté de Pharmacie, Université de Strasbourg, 67400 Illkirch, France † Laboratoire de Biochimie Théorique, UPR 9080 CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France 5.1.2. nAChRs as Targets in PD Treatment 5.1.3. nAChRs as Targets in Neuropathic Pain Treatment 5.1.4. GABAA Receptors as Targets in Epilepsy Treatment 5.1.5. GABAA Receptors as Targets in AD Treatment 5.1.6. GABAA Receptors as Targets in Pain Treatment 5.2. Tetrameric Receptors 5.2.1. AMPA Receptors as Targets in AD Treatment 5.2.2. AMPA Receptors as Targets in PD Treatment 5.2.3. AMPA Receptors as Targets in Epilepsy Treatment 5.2.4. NMDA Receptors as Targets in AD Treatment 5.2.5. NMDA Receptors as Targets in PD Treatment 5.2.6. NMDA Receptors as Targets in Epilepsy Treatment 5.2.7. NMDA Receptors as Targets in Pain Treatment 5.2.8. Kainate Receptors as Targets in Epilepsy Treatment 5.3. Trimeric Receptors 6. Three-Dimensional Structures Illuminate the Mechanisms of Ligand Recognition 6.1. Pentameric Receptors 6.1.1. Orthosteric-Binding Sites 6.1.2. Allosteric-Binding Sites 6.2. Tetrameric Receptors 6.2.1. Orthosteric-Binding Sites 6.2.2. Allosteric-Binding Sites 6.3. Trimeric Receptors 6.3.1. Orthosteric-Binding Sites 6.3.2. Allosteric-Binding Sites 7. Conclusion and Perspective

CONTENTS 1. Introduction 2. A Brief History of the LGICs 3. General Description of the LGIC Superfamily 3.1. Pentameric Receptors 3.2. Tetrameric Receptors 3.3. Trimeric Receptors 4. Contribution of LGIC Dysfunction to Disease 4.1. Alzheimer’s Disease 4.1.1. nAChRs 4.1.2. iGluRs 4.1.3. GABAARs 4.1.4. P2XRs 4.2. Parkinson’s Disease 4.2.1. iGluRs 4.2.2. P2XRs 4.2.3. nAChRs and GABAARs 4.3. Epilepsy 4.3.1. GABAARs 4.3.2. nAChRs 4.3.3. iGluRs 4.3.4. P2XRs 4.4. Hyperekplexia 4.5. Neuropathic Pain 4.5.1. P2XRs 4.5.2. iGluRs 5. LGICs as Attractive Targets for New Therapeutic Agents 5.1. Pentameric Receptors 5.1.1. nAChRs as Targets in AD Treatment © 2012 American Chemical Society

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Special Issue: 2012 Ion Channels and Disease Received: February 24, 2012 Published: September 18, 2012 6285

dx.doi.org/10.1021/cr3000829 | Chem. Rev. 2012, 112, 6285−6318

Chemical Reviews Author Information Corresponding Author Notes Biographies Acknowledgments References Note Added after ASAP Publication

Review

how recent crystal structures have provided new molecular insights into the mechanism of ligand recognition.

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2. A BRIEF HISTORY OF THE LGICS The first evidence showing that a chemical compound can act on the LGICs came from the pioneering work of Claude Bernard (1857) who showed that curare, a substance that South American Indians used to poison arrow heads for hunting, affected the peripheral action of motor nerves on the muscle.1 Inspired by the work of Bernard, John Newport Langley introduced in 1905 the notion of pharmacological receptor.2,3 At the same time, two important features of LGICs were established: first by Elliot who mentioned that a chemical stimulant, today termed neurotransmitter, is released by the nerve ending on each occasion when the impulse arrives at the periphery,4 a hypothesis firmly demonstrated 35 years later by Nachmansohn and co-workers,5 and second by Lillie who proposed that nerve signal would result from a transient change of passive ionic permeability across the cell membrane.6 At that time, of course, receptors remained enigmatic entities, and direct evidence of their existence was made possible by the development of novel concepts and techniques. Two decades after the formalism of ligand binding and channel gating postulated in the 50s by del Castillo and Katz,7 biochemical techniques allowed for the first time to purify a LGIC by Changeux and co-workers using snake venom toxins.8 This was followed later by the key experiment performed by Neher and Sakmann who, by using single-channel recordings, demonstrated that this receptor was indeed an ion channel activated by a ligand (here acetylcholine) where discrete channel openings and closings were resolved.9 Progressively, our basic knowledge about LGICs further increased in the 80s with the first report of the primary sequence of a receptor subunit of a LGIC member following cloning and sequencing. 10−13 However, the three-dimensional structure of these receptors remained unknown until the late 1990s when the first crystal structures of extracellular domains of such receptors or of related homologues were published,14,15 followed several years later by the atomic structures of the nearly full-length receptors.16−18 These structural data provide, more than a century after the concept of receptive substances mentioned by Langley, unprecedented insights into the mechanism that underlies cell signaling at the atomic level.

1. INTRODUCTION Ligand-gated ion channels (LGICs) mediate intercellular communication by converting the binding of a neurotransmitter that is released from the presynaptic terminal into an ion flux in the postsynaptic membrane. They are integral oligomeric membrane proteins that carry an orthosteric-binding site for the neurotransmitter (the agonist) and an ion channel that spans the membrane. Under resting conditions, the channel is closed, and binding of the agonist triggers a conformational change that opens the gate, a process called gating. This process, which occurs on a millisecond time scale, represents one of the most rapid conformational changes in oligomeric proteins. Once the channel is opened, cations or anions diffuse through the pore at rates approaching tens to hundreds of millions of ions per second. In addition to their well-established role in neurotransmission, it is now recognized that some LGICs are critical in nonexcitable cells, such as endothelial cells, suggestive of a wider functional role of these receptors outside the peripheral and central nervous system. Moreover, modulation of gating can occur by the binding of endogenous or exogeneous modulators at allosteric sites that are topologically distinct from the orthosteric-binding sites. These modulators eventually modify the excitatory/inhibitory balance in the central nervous system. LGICs thus represent attractive targets for new therapeutic agents. In this review, we give an overview of the recent advances in our knowledge of LGICs, focusing on channels that are activated by conventional neurotransmitters (glutamate, acetylcholine, glycine, ATP, serotonin and γ-aminobutyric acid, Figure 1). First, we review

3. GENERAL DESCRIPTION OF THE LGIC SUPERFAMILY Genome and cDNA sequencing analyses revealed that there are three major unrelated vertebrate superfamilies of LGICs, each folded with a unique architecture (Figure 2): first, the so-called Cys-loop receptors, which form pentameric ion channels gated by acetylcholine (ACh), γ-aminobutyric acid (GABA), glycine (Gly), and serotonin or 5-hydroxytryptamine (5-HT); second, the ionotropic glutamate receptors (iGluRs), which are tetrameric nonselective cation channels (Na+, K+, Ca2+) that are activated by glutamate; and finally, the P2X receptors (P2XRs), which are trimeric ion channels gated by ATP.

Figure 1. Chemical structures of conventional neurotransmitters acting on the LGICs.

the involvement of LGICs in neurological disorders and the therapeutic agents that are currently used in clinical treatments. We focus on few selected topics such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) as well as on epilepsy, hyperekplexia, and neuropathic pain, because they are characterized by important LGIC dysfunctions. Then, we describe the chemical tools that have been developed for these receptors to explore structure−function relationships of orthosteric and allosteric binding sites. Finally, we present

3.1. Pentameric Receptors

The first superfamily comprises the excitatory, cation-selective nicotinic acetylcholine receptors (nAChRs), 5-HT3 receptors, and zinc-activated channel (ZAC) and the inhibitory, anionselective GABAA, strychnine-sensitive glycine receptors, and invertebrate glutamate-gated chloride channels (GluCl).19−21 6286


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instead they assemble under strict rules to form a large combinatory display of receptors with different pharmacological and biophysical properties and also with different patterns of expression within the nervous system and other tissues. For instance, nAChRs expressed in the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas (α1)2β1γδ predominates in the embryonic skeletal muscle. This last stoichiometry is remarkably well expressed in the electric organs of the Torpedo and Electrophorus electric fishes where activation of these nAChRs in the electroplax produces electric shocks used to kill prey. Neuronal nAChRs comprise various combinations of α (α2−α10) and β (β2−β4) subunits with variable stoichiometry. A diverse range of neuronal nAChR hetero-oligomers exist in the brain, which creates substantial challenges for targeted drug design.25 For GABAA receptors, there is strong evidence that the subunit composition of brain receptors is mainly 2α, 2β, and 1γ or 1δ subunits.26 There is considerable interest in modulating the activity of human Cys-loop receptors to treat various nervoussystem disorders, such as AD, PD, epilepsy, and neuropathic pain as well as tobacco addiction. In 2005, the prokaryotic homologues of the vertebrate Cys-loop counterparts have been identified, expanding considerably this superfamily.27 Although, they do not carry the canonical cysteine residues, they exhibit conserved residues of the “Cys-loop” and also form pentameric structures.16,28,29

Figure 2. Schematic representation of the membrane topology and subunit assembly of the three families of LGICs: (A) Cys-loop receptors, (B) iGluRs, and (C) P2X receptors. Conserved cysteine residues engaged in disulfide bridges are shown as yellow circles, and N- and C-termini and transmembrane spans are also indicated. Out and in stand for, respectively, outside and inside the cell.

3.2. Tetrameric Receptors

The second superfamily comprises the excitatory NMDA (Nmethyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid), and kainate receptors, named originally according to their preferred agonist (Figures 2B and 14).30 They are tetrameric structures, and mammalian iGluRs are encoded by 18 genes that assemble to form the three main families: AMPA (GluA1−4), kainate (GluK1−5), and NMDA (GluN1, GluN2A−D, GluN3A,B) receptors. A fourth family comprises an “orphan” class of iGluR subunits, GluD1,2, which do not form functional receptors when expressed individually or together in heterologous expression systems.31 However, GluD2 is predominately expressed in Purkinje cells of the central nervous system and a naturally occurring mutant of this receptor found in the Lurcher mouse (which exhibits ataxia and cerebellar dysfunction)32 is sensitive to D-serine.33 Co-assembly of the three main iGluRs within, but not between, the families produces a large number of receptor subtypes in vivo. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand-binding domain (LBD), a transmembrane domain (TMD) composed of three membrane spans (M1, M2, and M3) with a channel lining re-entrant “p-loop” located between M1 and M2, and an intracellular C-terminal domain (CTD). The LBD is formed by the association of two domains, D1 and D2, which are composed mainly of the S1 and S2 peptide segments, respectively (Figure 2B). Unlike the Cysloop receptors, in which the ligand-binding sites are formed at the interface between subunits, in iGluRs the orthostericbinding sites for glutamate are present within the core of each subunit. Note that in NMDA GluN1 subunits, glycine is the agonist (Figure 2B). These receptors have been implicated in neurological diseases such as dementia, traumatic brain injury, and excitotoxicity, a phenomenon leading to neuronal apoptosis in AD and PD. They are also involved in neuropathic pain.

They are pentameric structures formed by the assembly of five identical or related subunits and are frequently referred to as Cys-loop receptors due to the presence in the extracellular domain of a loop of approximately 13 residues flanked by two canonical cysteine residues connected by an intrasubunit disulfide bridge (Figure 2A). All subunits of the Cys-loop superfamily are homologous and thus have evolved from a common ancestral gene.22 As a consequence, the biochemical and subsequent site-directed mutagenesis experiments gathered for two decades on the nAChR conferred a leading position on this receptor over other members of this superfamily. Indeed, it was established that the N-terminal domain of ∼200 amino acids is extracellular and contains the orthosteric-binding site, which lies at the interface of two adjacent subunits (Figure 2A).23 There are many allosteric-binding sites in the Cys-loop receptors, including the famous benzodiazepine and general anesthetic binding sites. Four transmembrane segments that span the membrane follow the extracellular N-terminal domain, and consequently the C-terminus is located extracellularly. The second segment, M2, lines the channel pore, and consequently, the channel is formed by the association of five M2 segments. The second intracellular loop is of variable size and amino acid sequence. Concerning the genetic input, at least 17 genes code for human nAChR subunits termed α1−α10, β1−β4, γ, δ, and ε; 19 genes code for GABAA subunits termed α1−α6, β1−β3, γ1−γ3, δ, ε, θ, π, and ρ1−ρ3 (these ρ subunits have sometimes been called GABAC receptors),24 while only few genes code for 5-HT3 (5-HT3A−E), glycine (α1−α4, β) and ZAC subunits (note that only one subunit has been identified so far for ZAC). All these subunits do not associate randomly to form receptors; 6287


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early forms of AD.50 These nAChRs, especially the homomeric α7- and heteromeric α4β2-containing nAChRs, are normally well expressed in different brain regions including the hippocampus.51 With use of several experimental approaches such as patch-clamp electrophysiology, nAChR immunolabeling, and in situ hybridization, the ultrastructural distribution of these receptors has been confirmed at dendritic, somatic, and axonal parts of neurons.52−56 Therefore, according to this distribution, these nAChRs play a major role in the control of neurotransmitter release and memory process, including the induction of hippocampal long-term potentiation (LTP), a form of synaptic plasticity.42,44,51 A decreased density of nAChRs has been consistently described in autopsy brain samples from patients with AD, by radioligand binding experiments.57−60 Analysis of mRNA expression for nAChR subunits from the hippocampus of AD patients indicated that the messenger expression was unmodified for α3 and α4 subunits, whereas that of α7 subunits was increased.61,62 Western blot-based methods and immunohistochemistry identified differential nAChR subunit expression in brain samples from AD patients in comparison to age-matched controls. It has been shown that in the temporal cortex, protein levels of α4 subunit were decreased.63−66 In contrast, those of α3 subunit either decreased64 or were unaltered.63 Similarly, protein levels of α7 subunit were either reduced65,66 or unaltered,63,64 whereas those of β2 subunits have been shown to be unaltered.64 On the other hand, the level of α3, α4, and α7 subunits decreased in the hippocampus, but not those for β2.64 Therefore, a large reduction of α4 subunit-containing and α7 nAChRs was observed in the cerebral cortex and hippocampus of AD patients. In vitro experiments indicate that long-term exposure (7 days) of PC12 cells to Aβ1−40 at concentrations in the nanomolar range (i.e., similar to the concentrations in the plasma and cerebrospinal fluid of AD patients), induced a decrease in mRNA, nAChR binding sites, and protein levels of α3 and α7 subunits.67 For β2 subunits, the mRNA level was unchanged but the protein level was reduced.67 An increase in lipid peroxidation has also been observed in response to short exposure (48 h) to Aβ1−40 at micromolar concentrations, and to Aβ1−42 at nanomolar concentrations.68,69 This effect was accompanied by an antioxidant-sensitive decrease in both nAChR binding sites, α3 and α7 subunits, in response to higher concentrations (in the micromolar range).68,69 Therefore, it was proposed that free radical-mediated cellular membrane damage induced a reduction of membrane insertion of the corresponding receptors.68,69 Altogether, these studies rather suggest that alteration of nAChR expression in the brain of AD patients has a broad range of causes ranging from Aβ chronic exposure to lipid peroxidation. A recent work highlighted a neuroprotective effect of α7 nAChRs during the early stages of the disease, which are associated with early cognitive decline, possibly by decreasing Aβ accumulation.70 However, another study has shown that deletion of the gene encoding for α7 subunit (α7 nAChR knockout) in a transgenic mouse model of AD not only improved cognitive deficits but also protected the brain from synaptic dysfunction,71 suggesting strongly a deleterious role of α7 nAChR in AD. These results revealed the complexity of the relationships between nAChRs and AD. In addition, a recent study identified autoantibodies against α7 nAChR in blood plasma of AD patients capable of binding to the fragment α7(1−208) and of decreasing nAChR brain levels.72,73 These autoantibodies may thus represent another important risk

3.3. Trimeric Receptors

The third superfamily of LGICs is composed by the excitatory ATP-gated P2X receptors (Figure 2C).34 They are mostly selective to cations, except for one subtype (P2X5), which also conducts chloride ions. They are trimeric structures formed by the assembly of three identical or related subunits. Each subunit possesses intracellular N- and C-termini and two transmembrane segments (M1 and M2), joined by a cysteine-rich ectodomain. There are seven genes (P2RX1−7) encoding P2X receptor subunits (P2X1−7) found in diverse organisms ranging from amoeba35 and parasitic worms36 to fish37 and mammals,38 indicating that ATP signaling including P2X receptors is a general mechanism that extends far beyond that underlying cell signaling in neurobiology. However, to date no evidence supports the existence of P2X receptors in prokaryotes. Similarly to the Cys-loop receptors, the orthosteric-binding sites are located at the interface between subunits (Figure 2C). After ∼20 years of struggle for the acceptance of purinergic neurotransmission,39 P2X receptors are now wellrecognized therapeutic targets. They have been implicated in many disorders including thromboembolism, bladder instability, pain, and inflammation.39,40

4. CONTRIBUTION OF LGIC DYSFUNCTION TO DISEASE LGICs have been shown to be involved in several diseases including neurodegenerative disorders. Among those, this review mostly deals with AD and PD, as well as with epilepsy, hyperekplexia, and neuropathic pain because they are characterized by major LGIC dysfunctions. In the following sections, receptor families are classified according to their importance in the disease. 4.1. Alzheimer’s Disease

4.1.1. nAChRs. AD is a neurodegenerative disorder that is characterized by progressive cognitive decline, accompanied by the loss of neurons and synapses, especially the cholinergic synapses, in the basal forebrain, cerebral cortex, and hippocampus. AD is the major cause of dementia in elderly people,41 and may affect around 25 million patients worldwide.42 The two major biochemical mechanisms associated with AD are the extracellular accumulation of senile plaques in cerebral cortical regions of the patients and the formation of neurofibrillary tangles composed of hyperphosphorylated tau protein in the cytoplasm of particular cortical pyramidal neurons.41,42 Senile plaques (or amyloid plaques) are formed following aggregation of oligomerized amyloid-β peptides (Aβ).43−45 The sequential proteolytic cleavage of the amyloid precursor protein (APP), a transmembrane glycoprotein, by β- and γ-secretases, produces monomeric Aβ.46,47 The two major isoforms of Aβ are composed of 40 and 42 amino acids, termed, respectively, Aβ1−40 and Aβ1−42, and may represent the most neurotoxic species in AD.42 This Aβ neurotoxicity is due in part to complex interactions with the cholinergic system and more precisely with the nAChRs. Early studies with enzymatic cholinergic markers such as choline acetyltransferase and acetylcholinesterase (AChE) provided evidence that cholinergic neurotransmission was reduced in AD.48,49 This cholinergic deficit is supposed to underlie the impairment of memory in the disease and takes place in the cerebral cortex and hippocampus of patients.48 Besides the loss of cholinergic neurons at advanced stages of late-onset AD, there is a marked dysfunction of nAChRs in the 6288


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of the peptides, as well as on the location and composition of nAChRs, and also on the experimental conditions. Further evidence for a possible direct interaction between Aβ and α7-nAChRs was provided by more detailed molecular studies.83 A computer-simulated docking analysis proposed a model in which agonist-binding loop C of the human α7 nAChR was implicated in the binding of Aβ1−42,84 involving the segment V12−K28 from Aβ1−42, in agreement with previous work.78 These data were recently confirmed by a site-directed mutagenesis study, which revealed the critical role of the AChbinding site residue Y188 from loop C of the α7 nAChRs in the agonist-like action of Aβ.85 Hence, these data argue in favor of a direct binding of Aβ peptide and α7 nAChR, but more direct evidence using, for instance, engineered site-directed affinity labeling or crystallographic studies is needed to definitively locate the Aβ-binding site in nAChR. Other nAChR subtypes have been investigated as significant targets for Aβ, in particular the α4β2 nAChRs.42 However, the affinity of Aβ1−42 to α4β2 nAChRs is 5000-fold lower than that to α7 nAChRs.74 In addition, conflicting results arose from the fact that nanomolar Aβ1−42 concentration is able to either inhibit α4β2 nAChRs86 or potentiate its activation (Figure 3).87 Nevertheless, the most promising results came from the recent discovery in rodents of a novel, functional, and naturally occurring nAChR subtype in basal forebrain cholinergic neurons.88 This presumed heteromeric α7β2 nAChR was functionally inhibited by nanomolar concentrations of Aβ oligomers51,88 and could thus represent an attractive target for the development of AD treatment. Altogether, these studies clearly reveal the major role of the interactions between Aβ and nAChRs in the pathophysiology of AD and help define nAChRs as potentially important therapeutic targets in the disease. The design of molecules or strategies able to disrupt these complex interactions might become a valuable approach for the treatment of AD patients. Several ligands of the nAChRs have already been developed to hopefully provide future therapeutic applications in AD (for review, see ref 89). 4.1.2. iGluRs. Even if the mechanism underlying AD has been abundantly investigated through the deficit of the cholinergic neurotransmission, alternatives involving the alteration of glutamatergic neurotransmission have also been examined, including the dysfunction of NMDA and AMPA receptors.42,60 Under physiological conditions, glutamatergic neurotransmission, and predominantly NMDA receptors, is the main component underlying LTP in synaptic transmission.90 Aβ has been shown to inhibit NMDA-mediated LTP in hippocampus.91 A series of investigations have been undertaken to further explore the mechanisms leading to the alteration of the glutamatergic system in AD. First, Aβ has been shown to induce a decrease in levels of both NMDA and AMPA receptors by mechanisms such as increased endocytosis of synaptic receptors.92−94 Second, Aβ induces a calcium influx leading to an abnormal elevation of intracellular Ca 2+ concentrations and oxidative stress in a phenomenon called excitotoxicity through a mechanism requiring an excessive activation of NMDA receptors (Figure 3).95,96 The formation of reactive oxygen species depends upon the binding of Aβ to, or in close proximity to, NMDA receptors in hippocampal neurons.96 Excitotoxicity has been demonstrated to take place in cholinergic neuronal apoptosis in response to Aβ.95,97 Finally, in experiments with heterologous expression of GluA1, GluA2, and GluA3 subunits of AMPA receptors in Xenopus oocytes, nanomolar concentrations of Aβ1−40 significantly

factor for the development of the disease in relation to nAChRs. Among the main mechanisms proposed to explain alterations of the nAChR function in AD, a direct molecular interaction between Aβ and nAChRs has been frequently proposed. Aβ1−42 appeared to display very high affinity (in the picomolar range) binding to α7 nAChRs in synaptic membranes prepared from cerebral cortex74 and hippocampus,75,76 although this remains controversial.77 In post-mortem human brain samples from AD patients, a close association between Aβ1−42 and α7 nAChRs has been proposed, precisely in senile plaques of AD hippocampal sections, resulting in the formation of a stable protein complex.78 Functional assays revealed that depending on the concentration, Aβ either activates or inhibits α7 nAChRs (Figure 3). It has been shown that picomolar Aβ positively

Figure 3. Interplay between Aβ and LGICs on mechanisms of synaptic plasticity. Interactions of Aβ with nAChRs can result in receptor activation or inhibition, depending on Aβ concentration and receptor subtype. Aβ also impairs plasticity through NMDA receptors by aberrant Ca2+ influx and excessive generation of reactive oxygen species (ROS). Reprinted with permission from ref 42. Copyright 2010 Springer.

modulated synaptic plasticity in the hippocampus via presynaptic α7 nAChRs, as measured by an increase in LTP,76 and activated α7 nAChR heterologously expressed in oocyte, whereas higher concentrations (in the nanomolar range) promoted desensitization, a phenomena usually observed following continuous agonist exposure.79 In agreement, nanomolar Aβ concentration blocks ACh-induced nicotinic postsynaptic currents when tested on α7 nAChRs in primary cultures of hippocampal neurons,80 and hippocampal slices,81 suggesting an inhibitory effect at higher Aβ doses (Figure 3). In addition, a recent study suggested that the oligomeric state of Aβ also displays different functional effects. Fibrillar Aβ exerts neurotoxic effects in part by blockade of α7 nAChRs, whereas oligomeric Aβ may act as a ligand activating α7 nAChRs, thereby stimulating downstream signaling pathways.82 It thus appeared that Aβ displays distinct regulatory roles depending on the state of aggregation and concentrations 6289


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potentiated ionic currents, whereas Aβ1−42 had no effect.98 Altogether, these mechanisms are likely to contribute to the early memory deficit and cognitive alterations of AD. 4.1.3. GABAARs. In the brain of mammals, the excitatory/ inhibitory balance is controlled by both the GABAergic system and glutamatergic neurotransmission. GABAergic neurons represent the majority of the estimated 10% of inhibitory neurons in hippocampus, and GABAA receptors mediate most of the fast inhibitory neurotransmission in the vertebrate brain.99 The GABAergic system is assumed to be, on the whole, unaltered in AD.99 Concentrations of GABA have been shown to be either decreased100 or unaltered101 in the cerebrospinal fluid of patients with AD. In contrast, a consistent reduction of GABA concentrations has been observed in post-mortem samples from the temporal cortex, occipital cortex, and cerebellum of AD patients,102 confirming previous observations.103,104 In addition, only subtle modifications at the level of some subunits of GABAA receptors have been identified.99 Indeed, immunohistochemical studies have indicated a decrease of α1 subunit of GABAA receptors in the hippocampus from patients with severe AD, whereas β2/β3 subunits were rather well preserved.105,106 From radioligand binding studies, it seemed that α5 subunit-containing GABAA receptors were also unaffected in the hippocampus except in the CA1 area as well as in entorhinal and perirhinal cortex.107 Western blot investigations have revealed a moderate but significant reduction of the α5 subunit level in the CA1/CA2 and CA3 subregions from patients with severe AD.108 It has been shown by experiments based on hippocampal administration of Aβ that the peptide specifically induced a loss of rat hippocampo− septal GABAergic neurons, which normally play an essential role for synchronization in the hippocampal network and contribute to memory processing.109 Overall, these modest region-dependent alterations of GABAA receptor subunits in AD patients and the loss of rat hippocampo−septal GABAergic neurons are assumed to be at the basis of an increased incidence of seizures in AD and could contribute to the memory deficit.99,109 Recently, two studies reported that in knock-in mice that carried alleles for human apolipoprotein E4 (apoE4), the major known genetic risk factor for AD, the survival of GABAergic interneurons in the hilus of the dentate gyrus was decreased in an age- and tau-dependent manner.110,111 Consequently, reducing tau and enhancing GABA signaling are potential strategies to treat or prevent apoE4-related AD. 4.1.4. P2XRs. Finally, it has been shown that ATP release from cells during neuronal excitation or injury might contribute to the chronic inflammation seen in AD. In particular, it is known that P2X7 receptors are upregulated in the brain of patients with AD and in animal models.112,113 Stimulation of these receptors enhanced the degenerative lesions mediated by Aβ,114−116 whereas their blockade not only induced neuroprotection in an animal model of AD117 but also reduced the formation of amyloid plaques in a mouse model of familial AD.118 However, a recent report suggested that P2X7 receptors have beneficial effects through the alternative processing of APP into sAPPα, which has been reported to exert neuroprotection.119 Regardless of these apparently inconsistent data, it appears therefore that P2X7 receptors may represent a new and important therapeutic target in relation to AD. More recently, another P2X receptor subtype, P2X4, has been shown to be involved in Aβ-induced neuronal death through an enhanced toxic effect of Aβ1−42.120

4.2. Parkinson’s Disease

4.2.1. iGluRs. PD is a neurodegenerative disease characterized by a selective and progressive alteration of the nigrostriatal dopaminergic pathway associated with depletion of dopamine. In fact, it is the second most common neurodegenerative disease after AD.121 The exact etiology of PD is still uncertain and seems to be dependent upon genetic and environmental factors.122 It must be noticed that the pathogenic relationship between LGICs and PD does not seem to be as clear as that seen in AD. A complex relationship exists between the dopaminergic system and NMDA receptors.123 It has been shown that D1 receptor activation potentiated NMDA receptor-mediated currents124,125 and led to the rapid trafficking of NMDA receptor subunits to the cell surface of striatal neurons.126 In addition, the administration of L-3,4-dihydroxyphenylalanine (LDOPA) is one of the major therapeutic strategies to relieve the symptoms of PD. However, adverse effects often occur in response to the chronic treatment with L-DOPA such as dyskinesias, abnormal motor responses that involve NMDA receptors, further evidencing the interplay between NMDA receptors and the dopaminergic system.127 It has been shown that in animal models of PD, NMDA antagonists increased the survival of neurons in the substantia nigra pars compacta128 and reduced dyskinesias induced by L-DOPA.129,130 4.2.2. P2XRs. Microglia-dependent inflammatory processes in substantia nigra and striatum seem to play a major role in the neurodegeneration associated with PD.121 Microglial cells are the resident macrophages of the central nervous system and represent, with astrocytes, the major source of proinflammatory molecules in response to brain injury. It has been known that P2X7 receptors are involved in microglia-induced inflammatory processes,131 and a recent study showed that blockade of these receptors located in microglia, but also in astroglia, partially prevented depletion of striatal dopamine stores, suggesting that P2X7 may be involved in PD.132 However, it has been shown from a more recent study that in toxin-induced animal models of PD, mRNA expression of P2X7 increased in the striatum, but protein expression remained unchanged.133 This study further showed that genetic deletion or pharmacological inhibition of P2X7 receptors did not change survival rate or depletion of striatal endogenous dopamine content, suggesting that P2X7 receptor deficiency or inhibition did not support the survival of dopaminergic neurons in models of PD.133 These apparent conflicting data thus warrant further investigations to identify the exact implication of P2X7 receptors in PD. 4.2.3. nAChRs and GABAARs. As observed in AD, reduced nAChR expression has been shown to occur in the cerebral cortex of PD patients134 and in animal models of PD,135 including receptors containing α4,122,135 α6,122,135 and α7 subunits.134 nAChRs could play a protective role against neurotoxicity in the striatum and relieve L-DOPA-induced dyskinesias as observed in animal models of PD.122,136 It is estimated that drugs targeting α4β2- and α6β2-nAChRs could represent an additional strategy in the treatment of PD.122 Finally, an inverse correlation has been found between dopamine and GABA concentrations in the striatum,137 a brain region that expressed high levels of extra-synaptic α4βδ GABAA receptors. These alterations could thus be related to sleep disorders, which are among the nonmotor symptoms of PD.138 6290


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4.3. Epilepsy

of GABAA receptors have also been found in association with generalized epilepsy with febrile seizures139,150−154 or childhood absence epilepsy with febrile seizures.155,156 They are located in the extracellular loop linking M2 to M3 (γ2K289M),150 in the intracellular loop linking M3 to M4 (γ2Q351X),151 in M3 (α1A332D),147 or in the extracellular Nterminal domain of the γ2 or α1 subunits (Figure 4).139 These mutations were associated either with a decreased sensitivity to GABA or benzodiazepines150,152,155 or with a default in cellsurface targeting of the immature protein.151,154 For one of these mutations (P83S), no apparent effect on receptor surface expression and functionality was reported, but additional experiments are needed to identify the phenotype of this mutant.139 Mutations of genes encoding other subunits of GABAA receptors have also been described in relation with epilepsy. For instance, mutations of GABRA6 (coding for the α6 subunit) have been reported in association with idiopathic generalized epilepsy,157 whereas mutations of GABRB3 (coding for the β3 subunit), which are located in the extracellular Nterminal domain of the receptor, causing hyperglycosylation and reduced GABA currents, were found to be associated with childhood absence epilepsy.158 Finally, two variants in GABRD encoding the δ-subunit were proposed as susceptibility alleles for generalized epilepsy.159 These mutations (E177A and R220H), which are located in the extracellular domain of the receptor, altered channel gating.160 The fact that GABAA receptor channelopathies are associated with epilepsy is in agreement with the imbalance theory in which it is assumed that loss-of-function in inhibitory transmission may underlie the neuronal hyperexcitability and contributes to epilepsy.161,162 However, this view might be too simple to solely explain the mechanism of seizures. In certain forms of epilepsy, such as temporal lobe epilepsy, potential compensatory mechanisms leading to an increase in GABAergic neurotransmission have been observed because in basal conditions GABAergic neurotransmission can also be excitatory.163−165 Mutations in the δ subunit gene of extrasynaptic GABAA receptors have been shown to be associated with genetic forms of human epilepsy.138 Because these particular receptors are responsible for tonic inhibition, a change of their activity during the ovarian cycle due to their high sensitivity to neurosteroids such as progesterone participates in catamenial epilepsy, a form of epilepsy in women characterized by variable seizures depending on the phases of the menstrual cycle.166 However, triggering tonic inhibition is not a universal strategy to fight all epilepsies. Indeed, it has been shown that membrane hyperpolarization that occurs following enhanced tonic conductance in thalamic relay neurons elicited absence seizures.138 4.3.2. nAChRs. Besides GABAA receptors, several nAChR mutations have also been described in relation to epilepsy and more precisely to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE),140 which is associated with violent motor seizures at night.167 The analysis of genomic DNA from patients has highlighted a complex relationship between the mutations of the gene coding for α4 subunits of nAChRs (CHRNA4) on chromosome 20 and the ADNFLE symptoms in humans.140,167−170 The first mutation of CHRNA4, originally identified in one large Australian pedigree167 and later in a Spanish family,171 corresponded to a missense mutation leading to a substitution of serine with phenylalanine in position 6′ (we use the prime numbering system that aligns M2 residues of all

Epilepsy is a heterogeneous neurological disorder characterized by sudden recurrent episodes termed seizures that affects approximately 50 million people worldwide.139 While multiple origins and aspects depending upon the affected brain areas can cause epilepsy, there is a general agreement that epilepsy is caused by a hypersynchrony of brain neuron firing.140 There are several subtypes of epilepsy, but when the etiology of the disease is considered, the classification includes idiopathic and symptomatic epilepsies.141 Symptomatic epilepsies include birth accidents, abnormal neurodevelopment, injection, vascular diseases, and head trauma, as well as brain tumors.142 In idiopathic epilepsy, genetic predisposition plays a significant role and is directly related to mutations of genes encoding for ion channels, both voltage-gated and ligand-gated.142−144 It is estimated that more than 100 different heterozygous mutations of genes encoding ion channels are associated with ca. 10 different forms of epilepsy,142 making idiopathic epilepsy a prototypical case of channelopathies. 4.3.1. GABAARs. Many studies have consistently shown the importance of the genetic alteration of GABAA receptors in epilepsy (Figure 4).142,145,146 In this context, several mutations

Figure 4. Location in a consensus sequence of a Cys-loop receptor of pathological mutations (indicated by colored spheres for each family of receptors) involved in neurological disorders: (A) general view; (B) enlarged view of M1 to M3.

of the gene (GABRA1) encoding the α1 subunit of GABAA receptors have already been described in association with either autosomal dominant juvenile myoclonic epilepsy,147,148 sporadic cases of childhood absence seizures,149 or idiopathic generalized epilepsy.139 These mutations induced a loss-offunction of corresponding GABAA receptors with decreased sensitivity to GABA147 or reduced total cell surface expression, possibly by increased retention of the receptor in the endoplasmic reticulum.139,148,149 Mutations of GABRG2 and GABRA1 genes coding for the γ2 and α1 subunits, respectively, 6291


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have been found to be associated with seizures.184 These antibodies were found in the anti-NMDA receptor encephalitis, which has been assumed to account for some unexplained newonset epilepsies in young women.184 The antibodies have been shown to induce a decrease in density of NMDA receptors at neuronal cell surface in hippocampus.185,186 They interact selectively with an epitope located in the extracellular domain of GluN1 subunits.187 It is assumed that this antibody-induced decrease of NMDA receptor density predominantly inactivates GABAergic neurons leading to the disinhibition of central excitatory pathways and finally to the frontostriatal syndrome, a characteristic of this form of encephalopathy.185 In patients with temporal lobe epilepsy, there is evidence that NMDA receptors are increased in dentate granule cells and that NMDA receptors are strongly involved in excitatory postsynaptic potentials.188 In fact, it appeared from several studies that the exact nature of the alteration of GluN1 and GluN2 subunits seemed to be clearly dependent upon the type of epilepsy and was also cell-specific.189 Further studies are required to determine conditions in which the targeting of NMDA receptors could prove to be beneficial and safe in the treatment of epilepsy.189 4.3.4. P2XRs. In more recent years, the role of P2X receptors in epileptic seizures has also been explored. It has been shown that P2X7 receptors were upregulated in the brain of rats and mice, in which status epilepticus was induced by injection of chemical compounds.190−192 Following status epilepticus, microglia cells are activated through P2X7 receptor activation,191 and interestingly, blockade of these receptors by the antagonists pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and suramin (see Figure 19 for chemical structures) markedly decreased, but not completely, microglial activation.193 This led to the conclusion that ATP-mediated microglial activation may be an important pathway in epileptogenesis. Finally, it has been reported that P2X4 receptor expression has been altered in seizure-sensitive gerbil hippocampus,194 a genetic model of epilepsy, and in the chronic phase of temporal lobe epilepsy induced by pilocarpine,192 suggesting that these receptors may also be involved in epilepsy.

Cys-loop receptor subunits) of M2 lining the ion channel (Figure 4). Interestingly, this critical position was the first to be originally identified in Torpedo nAChR by photoaffinity labeling using chlorpromazine (see section 6.1.2 below).172 Heterologous expression of the corresponding human mutant α4(S252F)β2 nAChR (note that this mutation was initially numbered S248F because it is homologous to S248 of Torpedo α1 subunit)167 in Xenopus oocytes indicated that the mutation is associated with faster desensitization, less inward rectification and the absence of Ca2+ permeability in comparison to wildtype α4β2 nAChR.169 Another mutation of CHRNA4 gene associated with ADNFLE has been detected in a Norwegian family.168 This mutation inserted a leucine residue near the extracellular end of M2 after position 17′ (776ins3 in CHRNA4).168 Heterologous expression in Xenopus oocytes of the corresponding mutant α4(263insL)β2 nAChR indicated an increase in the apparent affinity for ACh but lower Ca2+ permeability, which at the cellular level may correspond overall to a loss-of-function.168 Two other mutations of α4 nAChR, all located in M2, S256L(10′)173 and T265I(19′),174 have been associated with ADNFLE (Figure 4), and analyses of functional properties of these mutants indicated an increased ACh sensitivity.174,175 Mutations of the gene encoding for β2 subunits (CHRNB2) of nAChR have also been identified in association with ADNFLE.176−178 Heterologous expression of the mutant α4β2(V287L) nAChRs in human embryonic kidney (HEK)293 cells indicated that the mutation slowed channel desensitization, which in turn left the ion pore open for a longer time.176 Another mutation (V287M) at the same position (22′), corresponding to the extracellular end of M2 (Figure 4), has also been detected, and the functional consequence was a marked increase of the ACh sensitivity (gain-of-function) as investigated in Xenopus oocytes.177 An additional mutation of CHRNB2 encoding for the mutant I312M in the β2 subunit, which was the first M3 domain mutation, has also been shown to drastically increase the sensitivity of the receptor to acetylcholine.178 Overall, these mutations, affecting both α4 and β2 subunits, led in many cases to a net gain-of-function of α4β2 nAChR, which in turn could induce seizures by increasing the α4β2 nAChR-stimulated glutamate release in ADNFLE.144,179 This strongly supports the etiological role of CHRNA4 and CHRNB2 in ADNFLE. Cases of nonfamilial nocturnal frontal lobe epilepsy (NFLE) are more common than the familial type ADNFLE and also involve nAChR mutations, as recently reported with mutations found in CHRNB2180 and CHRNA4 genes,181 which encoded respectively, for β2V337G and α4R308H mutants. These mutations are both located at the intracellular loop linking M3 to M4 (Figure 4). Finally, a mutation of the gene CHRNA2 encoding for the α2 subunit has been found to be associated with a form of sleep-related epilepsy looking like ADNFLE.182 Heterologous expression of the mutated subunit α2(I279N) associated with either β4 or β2 subunits showed that this mutation, located at M1, resulted in a gain-of-function receptor and caused a reduced inhibition by carbamazepine,182,183 an antiepileptic drug frequently used for the treatment of frontal lobe seizures, indicating that the compound may not be useful for treating patients with this mutation.183 Altogether, these studies revealed a clear pathogenic role for several nAChR subunits in various forms of epilepsy. 4.3.3. iGluRs. As observed for AD with antibodies against nAChRs, autoantibodies directed against NMDA receptors

4.4. Hyperekplexia

Hyperekplexia is a rare, nonepileptic, paroxysmal neurogenetic disorder.195 It is a human startle syndrome so-named because it is characterized by abnormal responses to startling events of acoustic or tactile origin. The first presentation of the disease is a neonatal hypertonia.195,196 It is associated with alterations in glycinergic neurotransmission including mutations of the genes GLRA1 and GLRB encoding α1 and β subunits of glycine receptors, respectively. These receptors are responsible for postsynaptic hyperpolarization and synaptic inhibition in the brainstem and spinal cord.197 Mutations of GLRA1 on chromosome 5q33-35 account for about 80% of hereditary hyperekplexia.198 Familial hyperekplexia has been shown to be caused by many mutations of GLRA1 (Figure 4).195,197,199 These mutations include missense,200−214 nonsense,215,216 and frameshift mutations,215 and for most of them, they are situated in the transmembrane-spanning domain (Figure 4). It is noteworthy that mutations of GLRA1 to be initially described (R271Q/L) were the first defined channelopathy affecting LGICs in association with a disease.211 Apart from protein truncation (Y202X, Y279X, S296X, and R316X),200,201,215,216 or alteration of trafficking/assembly of the receptor (R218Q, 6292


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S231R, I244N, R252H, and R392H),203,205,206,217,218 mutations R218Q, P250T, V260M, Q266H, S267N, R271Q/L, K276E, and Y279C disrupt the allosteric signal transduction pathway linking glycine binding to chloride channel gating, resulting either in a decrease of agonist sensitivity or in loss-offunction.207,214,217−219 For some mutations (M147V and G342S), no functional effect was detected.215 Recently, a novel mutation of GLRA1 has been identified.213 This missense mutation in GLRA1 induced an amino acid substitution (W170S) in the extracellular ligand-binding domain of the corresponding glycine receptor α1 subunit, and in this case, hyperekplexia is associated with mild mental retardation.213 Mutations linked to hyperekplexia were also found in the GLRB gene. It has been found that a missense mutation (G229D) reduced sensitivity to agonist-mediated activation in α1β(G229D) glycine receptor, and the splice site mutation (IVS5 + 5G→A) resulted in the exclusion of exon 5 from GLRB transcripts.220 Finally, a second mutation has recently been found in the GLRB gene, but the functional consequence of the mutation M117R at the level of the receptor is so far unknown.221

4.5.1. P2XRs. Among various neurotransmitters and endogenous ligands, ATP plays a significant role in the activation and sensitization of nociceptors.131,222 When released locally from damaged cells, but also from sympathetic, endothelial, or tumor cells, ATP initiates pain pathways by activating P2X3 and P2X2/3 receptors located on sensory fibers in visceral organs, the tongue, and the skin. It depolarizes cell membrane of neurons from trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn, triggering pain sensation in the brain (Figure 5).38,222,225−227 There is experimental evidence for the participation of P2X3 receptors in peripheral painful neuropathies.228−231 Antisense and siRNA strategies have demonstrated that P2X3 receptor participates in hyperalgesic and allodynic responses in a model of nerve ligation in rats.229,231 In addition, an overall increased expression or function of P2X3 and P2X2/3 receptors has been noticed in several models of neuropathic pain.222 P2X3 receptors of presynaptic primary afferent nerve terminals of spinal dorsal horn mediate facilitation of glutamate and probably also ATP release by these terminals (Figure 5).232 In turn, released ATP activates P2X2, P2X4, and P2X6 receptor subtypes, perhaps as heteromultimers, of postsynaptic dorsal horn neurons, which transmit subsequent signals to the brain.233 Co-release of ATP and GABA in spinal dorsal horn neurons has also been reported indicating that co-release of fast-acting excitatory and inhibitory neurotransmitters can act as modulatory pathways of nociceptive information.232,234 P2X4 and P2X7 receptors are expressed in microglia and have also been shown to contribute to neuropathic pain (Figure 5).131,232,235−238 In response to injury to a peripheral nerve, microglia are activated in the dorsal horn of the spinal cord. The mechanism underlying this process has been studied in detail. Released ATP following injury acts on P2X4 receptors on these microglia to elicit the release of brain-derived neurotrophic factor (BDNF). This, in turn, produces a depolarizing shift in the anion reversal potential (Eanion) in nearby neurons through activation of the tyrosine kinase receptor TrkB and reduction in the expression of K+/Cl− cotransporter 2, KCC2 (Figure 5).237,239−242 This shift inverts the polarity of currents activated by GABA, leading to GABAinduced depolarization and hyperexcitability of neurons.239−242 The contribution of P2X7 receptors to neuropathic hypersensitivity has been demonstrated in a partial nerve ligation model with P2X7 knockout mice.243 In addition, microglia activation by these receptors may contribute to spinal LTP induction.244 Finally, a very recent study discovered in humans a genetic association between lower pain intensity and the hypofunctional R270H mutation located in the ectodomain of the P2X7 receptor.245 In this study, authors reported that women carrying this mutation reported lower amounts of pain following breast surgery than did carriers of the R270 allele. Overall, these studies highlight the involvement of specific P2X receptors in neuropathic pain and, therefore, pave the way for innovative therapeutic interventions in pain relief aiming at selectively reducing P2X activity. 4.5.2. iGluRs. Although less well documented than for P2X receptors, participation of NMDA receptors in the molecular mechanisms of neuropathic pain has also been suspected.223 It is known that NMDA receptors mediate fast excitatory neurotransmission in the dorsal horn, and activation of these receptors accentuates sustained depolarization leading to the maintenance of central states of hypersensitivity in animal models of pain.246,247 In addition, it has been shown that the

4.5. Neuropathic Pain

Neuropathic pain is characterized by chronic pain due to an abnormal hyperexcitability of sensitive neurons in response to peripheral or central nerve injury or to diseases.222,223 In this context, amputation, spinal cord injury, viral infection, cancer, and diabetes are often associated with neuropathic pain.223 It involves the activation of sensory nerve fibers from a subpopulation of neurons, which project to the dorsal horn of the spinal cord through dorsal root ganglia (DRG) (Figure 5).222,224

Figure 5. Molecular and cellular mechanisms that involve LGICs underlying neuropathic pain in the peripheral nervous system and spinal cord. 6293


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increase in NMDA receptor function produced by activating the protein tyrosine kinase (Src) participated in the long-lasting enhancement of excitatory transmission (LTP) associated with neuropathic pain at the level of the spinal cord dorsal horn.248,249 More recently, it has been demonstrated that spinal cord GluN2B subunit-containing receptors were required for LTP induction in the nociceptive stimulus transmission associated with neuropathic pain.250,251 Neuropathic pain was attenuated in mice with a knock-in mutation that affected phosphorylation at residue Y1472 of Glu2B (Y1472F-KI) confirming the major role of this subunit in the disease.252,253 Very recently, another study using knock-in mice that specifically abolished zinc binding in NMDA receptors containing GluN1 subunits (H128S-KI) showed that these subunits were involved in pain processing including neuropathic pain and that endogenous zinc alleviated pain perception in the wild-type animals.254 Therefore, targeting NMDA receptors could prove to be useful in the treatment of neuropathic pain, in particular with the possible use of antagonists, which would selectively target GluN2B or GluN1A subunit-containing receptors.

Table 1. Compounds Targeting LGICs Currently in Clinical Use or under Clinical Development for AD, PD, Epilepsy, and Paina compound TC-5619 nicotine ABT-089 TC-6683/AZD1446 ABT-560 AZD-3480 galantamine carbamazepine ETH-0202 muscimol stiripentol

5. LGICS AS ATTRACTIVE TARGETS FOR NEW THERAPEUTIC AGENTS

felbamate


5.1.1. nAChRs as Targets in AD Treatment. Early studies indicated that acute administration of nicotine improved performance of AD patients in cognitive tasks, including verbal learning and memory, attention in a continuous performance task, and accuracy in a visual attention task, whereas acute administration of the noncompetitive (channel blocker) antagonist mecamylamine resulted in dose-dependent impairment of performance in a battery of cognitive tasks.255−261 Pharmacological studies using a range of structurally diverse nAChR agonists or positive allosteric modulators indicated improvement of the cognitive deficits associated with AD.89 The ability of nAChR activation to improve cognitive deficits has recently been reviewed in depth.262 The promising therapeutic potential of nAChR agonists or positive allosteric modulators (Table 1 and Figure 6 for chemical structures) for the treatment of AD patients is not only driven by the procognitive effects of such compounds but also driven by the fact that this kind of candidate drug may possess neuroprotective properties, in particular toward Aβ1−42.89 Several pharmacological options have thus emerged for the development of novel α7 nAChR ligands as candidate drugs against AD: 5.1.1.1. Full Agonists. If neuroprotection and memory enhancement result from desensitization, candidate drugs should mimic ACh with respect to its ability to rapidly desensitize nAChRs. Desensitization should be maintained in any event to reduce the risk of toxicity. Agonists TC-6683 and AZD-3480, which both target α4β2 nAChRs, have reached phase II clinical trials (Table 1 and Figure 6). 5.1.1.2. Partial Agonists. Partial agonists possess many of the properties of endogenous agonists, but due to residual inhibition, problems of toxicity arising from excessive stimulation and Ca2+ entry should be limited.263 TC-5619, a partial agonist of α7 nAChR, and ABT-089 and ABT-560, which are partial agonists of α4β2 nAChRs, have been developed and were tested in clinical trials up to phase II (Table 1 and Figure 6).

topiramate ganaxolone clobazam clonazepam

S-47445/C-X1632 deronpavel aniracetam E-2007/perampanel

topiramate

memantine

amantadine ketamine dextromethorphan methadone

GSK-1482160

disease

ligand type

development stage

nAChR AD partial agonist pain agonist PD cognitive partial agonist dysfunction AD agonist

phase phase phase phase

AD AD AD

phase I phase II launched

partial agonist agonist positive allosteric modulator (AChE inhibitor) epilepsy channel blocker GABAA Receptors AD positive allosteric modulator epilepsy agonist epilepsy positive allosteric modulator epilepsy positive allosteric modulator also NMDA antagonist epilepsy positive allosteric modulator epilepsy positive allosteric modulator pain agonist epilepsy positive allosteric modulator AMPA Receptors AD positive modulator PD antagonist cognitive positive modulator enhancer epilepsy antagonist Kainate Receptors epilepsy inhibitor also AMPA NMDA Receptors AD open channel blocker (also targets nAChR and 5-HT3) pain PD pain open channel blocker PD pain noncompetitive antagonist pain noncompetitive antagonist pain noncompetitive antagonist P2X Receptors pain antagonist

I IV II II

phase II

launched phase II phase I launched launched launched phase II launched launched

phase I phase II launched filed for approval launched

launched launched phase IV launched launched launched launched launched

phase I

a

The table was prepared by searching in www.clinicaltrial.gov website and by consulting the pipeline of major pharmaceutical companies. Molecules with uncertain mechanism of action were excluded.

5.1.1.3. Antagonists. Antagonists might theoretically reduce the neuronal accumulation of Aβ1−42 if we admit that they bind 6294


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development of drugs targeting α7 nAChRs because these ligands do not alter the pattern of the physiological response, only its amplitude. One potential disadvantage is that modulation efficacy would decrease upon the progressive loss of endogenous ACh that occurs in AD as the destruction of cholinergic neurons proceeds. Allosteric modulators might be of better use for the treatment of schizophrenia, in which the agonist is present, while exogenous agonists would have the advantage in neurodegenerative disorders of replacing the endogenous agonist, which disappears.264 Galantamine, currently in practice, is a positive allosteric modulator of α7 nAChRs and also possesses inhibitory properties on AChE.265 5.1.2. nAChRs as Targets in PD Treatment. Current evidence suggests that drugs acting on nAChRs may be of therapeutic value in PD to attenuate L-DOPA-induced dyskinesia and for long-term neuroprotection against nigrostriatal damage. Of the multiple nAChRs present throughout the peripheral and central nervous system, the α4β2 and α6β2 subtypes may be of particular relevance as central nervous system drug targets.266 Interestingly, nicotine has recently been pushed to phase II clinical trials to test its effect on motor symptoms in advanced PD. 5.1.3. nAChRs as Targets in Neuropathic Pain Treatment. Interest in the potential analgesic activity of compounds acting at neuronal nAChRs has been stimulated by the discovery that epibatidine (Figure 9), a nonopioid alkaloid isolated from skin of the poison frog Epipedobates tricolor,267 is a potent ligand of nAChRs with analgesic properties.268−271 Unfortunately, epibatidine is quite potent at all subtypes of nAChRs and is toxic at antinociceptive doses.268,271 Structurebased activity relationships on the epibatidine scaffold, however, allowed identification of ABT-594 (also known as tebanicline), a potent α4β2-preferring agonist,272 as the first nicotinic receptor ligand to undergo phase-II clinical trials for analgesic activity.273−277 While the compound had allowed for the clinical proof-of-concept, it was abandoned because of adverse effects, such as emesis and nausea.278 Another source of inspiration on this indication is coming from toxins.279 Cobratoxin, which is used in traditional medicines in China280 and India, has been proposed for pain treatment but, to our knowledge, has not been yet tested in clinical trials.281,282 Finally, nicotine is currently being evaluated for pain in phase IV clinical trials (Table 1). 5.1.4. GABAA Receptors as Targets in Epilepsy Treatment. Blockade of voltage-gated sodium channels is the most common mechanism of action among currently available antiepileptic drugs.283 However, it has been shown that other mechanisms are in play; notably GABAA receptors have provided an excellent target for the development of drugs with an anticonvulsant action.284 Some clinically useful anticonvulsants, such as benzodiazepines and barbiturates, act at the GABAA receptors by increasing opening frequency of the chloride ion pore.283 Benzodiazepines are among the most widely prescribed drugs in the United States, with ∼80 million prescriptions written each year. Besides the use of a drug such as clonazepam in epileptic seizure control (Table 1 and Figure 6), they are also used for sedation, sleep induction, anxiety relief, muscle spasm relief, and treating some forms of depression.285 Benzodiazepines exert their effect by binding to the benzodiazepinebinding site of the GABAA receptor and allosterically modulating the GABA response (Figure 10). For instance, ligands such as diazepam, flurazepam (two typical [1,4]-

Figure 6. Chemical structures of some clinical compounds currently used to treat AD, PD, epilepsy, and pain involving LGICs.

to the same site on nAChRs. However, to our knowledge, no drugs are under clinical testing. 5.1.1.4. Allosteric Modulators. The design of allosteric modulators currently occupies a central position in the 6295


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that AMPA receptor antagonists might provide some neuroprotective effect to prevent loss of the dopaminergic neurons through excitotoxicity.289,292 5.2.3. AMPA Receptors as Targets in Epilepsy Treatment. AMPA antagonists, such as the noncompetitive talampanel, have been shown to have anticonvulsant properties,293,294 strengthening the involvement of AMPA receptors in epilepsy and therefore supporting the fact that they represent potential important targets for drug development.292 Talampanel was tested in a phase II trial, but not pursued. Other competitive antagonists have also been developed, such as NS1209,295 displaying anticonvulsant effects in mice.292,296,297 The antagonist E2007/perampanel has been recently filed for approval for the treatment of epilepsy (Table 1). 5.2.4. NMDA Receptors as Targets in AD Treatment. GluN2B-containing NMDA receptors are well-established targets in AD.298 As mentioned in section 4.1, NMDA receptors are thought to play a major role in excitotoxicity causing cell death in AD because of their high permeability to calcium. Consequently, memantine, which is an NMDA lowaffinity open channel blocker in clinical use,96,299 provides some relief of AD symptoms.292 Accordingly, synthetic compounds targeting NMDA receptors in clinical use are mainly lowaffinity channel blockers (ketamine, amantadine) but also compounds antagonizing receptor activity (felbamate) (Table 1).300 5.2.5. NMDA Receptors as Targets in PD Treatment. Considering the crucial role of NMDA receptors in neurotoxicity and in L-DOPA-induced motor side effects, it has been proposed that the design of memantine-based drugs and other NMDA receptor antagonists could provide successful treatment of PD. These new treatments would slow the neuronal degeneration and relieve motor symptoms.127,301 In agreement, antagonists of NMDA, AMPA, and metabotropic glutamate receptors (mGluRs) have been tested or are currently being evaluated in patients with PD for the treatment of dementia, motor signs (including dyskinesia), and gait disorders (Table 1).302 Amantadine and memantine, which are channel-blockers of NMDA receptor, are currently in clinical use and under clinical development, respectively, for PD treatment. 5.2.6. NMDA Receptors as Targets in Epilepsy Treatment. It is tempting to consider that targeting NMDA receptors is a potential strategy for the control of seizures because (i) glutamatergic and GABAergic systems share, as previously mentioned, the control of the excitatory/inhibitory balance in the central nervous system and (ii) there is some evidence for the involvement of NMDA receptors in the pathophysiology of epilepsy.189 However, when one considers anti-NMDA receptor encephalitis, attention must be given to the fact that all procedures leading to excessive reduction or dysfunction of NMDA receptors may have detrimental consequences at the behavior level. That is particularly true for epilepsy, for which despite potential beneficial effects of modulators of NMDA receptors, adverse effects of antagonists have been reported in clinical settings.189 Conantokins G and T have also been noticed to display antiepileptic properties.303,304 However, none of the currently available antiepileptic drugs exerts its effects solely by an action on the glutamate system. Blockade of the NMDA receptor is, nevertheless, believed to contribute to the pharmacological profile of felbamate.283,305 However, the ability of these agents to produce neurotoxicity in adult rats and psychosis in adult humans compromises their clinical usefulness. The antagonist

benzodiazepines), or zolpidem (a compound of the imidazopyridine class) are positive allosteric modulators that potentiate the GABA response (also known as benzodiazepine agonists), while others like 3-carbomethoxy-4-ethyl-6,7-dimethoxy-βcarboline (DMCM, Figure 10B) are negative allosteric modulators that inhibit the GABA response (known as benzodiazepine inverse agonists). A third family of compounds such as Ro15-1788 (or flumazenil) are zero modulators; they bind to the benzodiazepine site but have no effect on the GABA response (known as benzodiazepine antagonists). Other anticonvulsant drugs have been developed. Stiripentol, which has been described as a novel antiepileptic chemically unrelated to other existing drugs used to treat seizure disorders, acted directly on the GABAA receptors as a positive allosteric modulator with a preference for α3β3γ2-containing receptors.286 Felbamate and topiramate also modulate GABA responses at the GABAA receptor.283 Extrasynaptic GABAA receptors have been proposed to be a therapeutic target for treatment of epilepsy.138 Ganaxolone, which is a positive allosteric modulator of most GABAA receptors with greater potency at δ subunit containing receptors, enhances tonic conductance. The molecule is in clinical trials for the treatment of catamenial epilepsy (Table 1). But as stated above, enhancing tonic inhibition induced by these receptors is not a universal strategy to fight all epilepsies. 5.1.5. GABAA Receptors as Targets in AD Treatment. It has been proposed that enhancing GABA signaling is a potential strategy to treat or prevent AD related to apoE4, the major known genetic risk factor for AD.111 In agreement with this idea, the positive allosteric modulator ETH-0202, which targets α1β3γ2 GABAA receptors, has entered phase II clinical trials (Table 1). 5.1.6. GABAA Receptors as Targets in Pain Treatment. In the past decade, the identification of separable key functions of GABAA receptor subtypes287 suggests that receptor subtypeselective compounds could overcome the limitations of classical benzodiazepines, for example, providing the anxiolytic properties without the sedative effect. These compounds might be valuable for novel indications such as chronic pain.287 The ideal candidate would be selective to α2βγ2 GABAA receptors.287 The agonist clobazam has entered phase II clinical trials (Table 1 and Figure 6). 5.2. Tetrameric Receptors

5.2.1. AMPA Receptors as Targets in AD Treatment. The only drug on the market targeting AMPA receptors is the positive allosteric modulator aniracetam for the treatment of dementia (Figure 6), but it has been shown that this compound also possesses a wide range of anxiolytic properties, which may be mediated by an interaction with cholinergic, dopaminergic, and serotoninergic systems.288 The positive allosteric modulator CX-1632 is under phase I clinical trials for the treatment of AD. 5.2.2. AMPA Receptors as Targets in PD Treatment. In animal models of PD, it was found that CX-516, a positive modulator of AMPA receptors, increased L-DOPA-induced dyskinesia, while the noncompetitive [2,3]benzodiazepine antagonist talampanel (GYKI-53405) reduced L-DOPAinduced dyskinesia. 289,290 Another [2,3]benzodiazepine AMPA receptor antagonist, GYKI-52466, was able to potentiate the effect of L-DOPA on dopamine turnover.291 This suggests that AMPA receptor antagonists might be useful as an enhancement to L-DOPA treatment. There is also a suggestion 6296


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quinolinic acid produces motor excitement or clonic convulsions. Preconditioning models using NMDA pretreatment have demonstrated to lead to neuroprotection against seizures and damage to neuronal tissue.306 5.2.7. NMDA Receptors as Targets in Pain Treatment. Conantokins G and T have been noticed to display antinociceptive properties.307,308 Some conantokins demonstrated receptor subunit selectivity, which makes them attractive drug candidates.309 To date, the molecules on the market targeting NMDA receptors, memantine and ketamine, are antagonists. 5.2.8. Kainate Receptors as Targets in Epilepsy Treatment. Topiramate is similarly distinguished by an inhibitory action on AMPA and kainate receptors with higher affinity for the latter (Figure 6).310

the field, not only because membrane proteins are notoriously difficult to work with but also because ∼30% of our genetically encoded proteins are membrane proteins and only ∼3% of these receptors have their structures resolved (308 in 2011 from a database of membrane proteins of known structure at http://blanco.biomol.uci.edu/Membrane_Proteins_xtal. html).313 These structures thus offer unprecedented views of the molecular mechanisms of drug−receptor interaction. In the following section, we review the recent structural advances that expand our understanding of the details of the interactions of orthosteric and allosteric ligands acting in the three superfamilies of LGICs. We also describe the chemical tools that have been developed, usually before the knowledge of the three-dimensional structures, to explore structure−function relationships of these critical targets.

5.3. Trimeric Receptors


To date, no drug targeting P2X receptors is on the market,311 and to our knowledge, only very few clinical trials have been performed, such as CE-224535 and GSK-1482160, which target P2X7 receptor (Figure 6). However, P2X receptors have aroused much interest from pharmaceutical companies as shown by the significant number of patents recently filled.312 As we reviewed in the previous sections, activation of P2X receptors, in most cases, leads to neurological disorders including neuropathic pain, and accordingly all of the compounds proposed to target these receptors are antagonists.

6.1.1. Orthosteric-Binding Sites. Within the family of Cys-loop receptors, biochemical data accumulated since the 1970s wonderfully anticipated the first crystal structure reported in 2001 of a protein homologous to the extracellular domain of these receptors.15 In this section, we give a brief historical review of these studies and show how chemical tools, in particular photoaffinity labeling, have helped in the absence of high-resolution crystal structures to give invaluable information about the mode of agonist and antagonist binding. Affinity and photoaffinity labeling are experimental approaches that directly identify amino acids contributing to a drug-binding site without previous assumptions about the protein points of contact. A strong limitation of these techniques is the large quantities of receptor that are needed to identify labeled residues. In the Cys-loop receptors, however, this was made possible because of the plentiful source of nAChRs in the electric organs of Torpedo rays and electric eels. The basis of these approaches relies on the establishment of a covalent bond formed between the protein target and a high-affinity ligand. In the case of affinity labeling, the probe carries an electrophilic moiety that is reactive toward nucleophilic amino acid residues (cysteine, aspartate, glutamate, tyrosine). Upon binding of the ligand, if the electrophilic group is correctly oriented and spatially close to the nucleophilic residue, a covalent bond is likely to be formed (Figure 8A). The nucleophilic residue may either be a native amino acid that belongs to the binding site or an introduced residue engineered by site-directed mutagenesis.314 In the case of photoaffinity labeling, the probe carries a photosensitive moiety that can be activated by light to give a highly reactive species that may react covalently with any amino acid residue surrounding the binding site, leading to the topographical mapping of the binding site (Figure 8B).315 In addition, the probe usually carries a radioactive isotope to identify labeled residues. Affinity labeling was first reported on the electroplax nAChR from Electrophorus electricus (electric eel), using the reactive affinity markers p-(trimethylammonium) benzenediazonium fluoroborate (TDF) and 4-(N-maleimido) phenyltrimethylammonium iodide (MPTA) (Figure 9A).316,317 These probes, however, were not efficient enough to allow the identification of labeled residues at the amino acid level, except for 4-(Nmaleimido) benzyltrimethylammonium iodide (MBTA), a MPTA analog, which identified on the α1-subunit two vicinal cysteines residues C192−193.318 A strong limitation of the use of MBTA was that reaction with the maleimide group occurred only if the electroplax has been exposed to the reducing agent

6. THREE-DIMENSIONAL STRUCTURES ILLUMINATE THE MECHANISMS OF LIGAND RECOGNITION At the dawn of the 21th century, LGICs entered the poststructure era, when crystal structures of each member of the superfamily were determined (Figure 7).16−18,28 These studies probably represent the most outstanding advances in

Figure 7. Examples of crystal structures for the three families of LGICs: (A) ELIC (PDB code 2VL0) for the pentameric receptors, (B) rat homomeric AMPA receptor type 2 (GluA2) (PDB code 3KG2) for the tetrameric receptors, and (C) zebrafish P2X4 receptor (PDB code 3H9V) for the trimeric receptors. 6297


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Figure 8. (A) Affinity and (B) photoaffinity labeling strategies. (C) Chemical structures of electrophiles, nucleophiles, and photoreactive groups used to probe LGICs.

dithiothreitol, a reagent that breaks the disulfide bridge formed between C192 and C193 and consequently alters the response to agonist.319 An alternative approach was then to use the photoaffinity labeling technique that offers the great advantage to work with native receptors. Pioneering photoaffinity-labeling studies by Changeux and co-workers identified a number of residues that are near the ACh-binding site of the Torpedo marmorata (electric ray) nAChR. When the photoaffinity probe p-(dimethylamino) benzenediazonium fluoroborate (DDF), a dimethylamino photoactivatable analog of TDF, was used and an energy-transfer photolysis procedure was employed,320 several amino acid residues, namely, Y93, W149, Y190, C192, and C193 from the α1-subunit, were successfully identified, including those previously identified by MBTA.321,322 Additional affinity and photoaffinity studies performed for two decades using a series of other competitive antagonists with different chemical structures, such as the alkaloid d-tubocurarine,323 an analog of the coral lophotoxin,324 and 4-[(3trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine),325 and agonists, such as ACh mustard,326 nicotine,327 (diazocyclohexadienoylpropyl)-trimethylammonium (DCTA),328 and 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine),329 not only confirmed these data but also extended the pattern of labeled residues to the γ- and δsubunits,323,325,330 leading to the proposal that the ACh-binding site lies at the interface between two subunits (Figure 9B). Other studies applying engineered affinity labeling with reactive alkyltrimethylammonium agonists and antagonists, such as MBTA (Figure 9A), further mapped the ACh-binding site and proposed orientation requirements for activation by covalent agonists.331−333 Photoaffinity labeling experiments were also carried out on the GABAA receptor334 and more recently on the neuronal α4β2 nAChR.335 Based on all these data, a multipleloop model of the orthosteric-binding site was proposed for Cys-loop receptors.23,336 In this picture, the agonist binds to an intersubunit cavity made by the principal or positive (+) face,

Figure 9. (A) Chemical structures of reactive affinity and photoaffinity markers used to identify the orthosteric-binding site of Cys-loop receptors. (B) Multiple-loop model of the orthosteric-binding site. Residues identified by (photo)affinity labeling and by mutagenesis are indicated. (C) X-ray structure of carbamylcholine (magenta) bound to AChBP (PDB code 1UV6). Residues homologous to those previously identified by photoaffinity labeling are shown in stick representation, and each subunit is depicted in a different color.

which contains loops A, B, and C, and by the complementary or negative (−) face, which contains loops D, E, and F. A striking observation was that almost all identified residues of the binding sites contained aromatic side chains, a fact that suggested at that time the existence of a cation−π interaction.337 This interaction, previously described in supramolecular chemistry,338 is a noncovalent binding force in which the aromatic ring provides a region of negative electrostatic potential that can bind a positive charge (i.e., quaternary ammonium) with considerable strength. The existence of such interaction in nAChR was later demonstrated using another powerful chemical approach that is based on the in vivo incorporation of unnatural amino acids.339 The methodology, which has been described in detail elsewhere,340,341 employs the 6298


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7A),16,28 followed later by that of the eukaryotic Caenorhabditis elegans GluClα channel.359 Prokaryotic receptors were discovered in 2005 using sensitive sequence-profile searches,27 and form functional pentameric ion channels that are activated by a ligand, establishing the prokaryotic origin of the Cys-loop superfamily.360 The ligand can be either a proton for the Gloeobacter violaceus receptor (named GLIC)360 or a class of primary amines that include GABA for the Erwinia chrysanthemi receptor (named ELIC).361 On the other hand, GluClα channel is most similar to the α1 glycine receptor, with which it shares 34% amino acid sequence identity, and is activated by glutamate. Structure comparison of both prokaryotic and eukaryotic structures further confirmed the interfacial location of the orthosteric-binding sites in the extracellular domain359,361 and revealed that the overall architecture of the eukaryotic receptors is similar to that found in the bacterial pentameric receptors, in agreement with a recent report based on the functional analysis of chimeras made from eukaryotic and prokaryotic fragments.362 6.1.2. Allosteric-Binding Sites. Gating of the Cys-loop receptors can be allosterically modulated by a number of clinically important drugs. GABAA receptors are of special interest because they are targeted by a large number of drugs, including benzodiazepines.363 Extensive studies based on chimeric approaches coupled to site-directed mutagenesis,336,364−370 photolabeling experiments,371,372 and substituted cysteine accessibility method (SCAM)373,374 have made significant strides in uncovering the specific amino acid residues that contribute to the binding of classical benzodiazepines. The picture emerged that the site is located on the extracellular surface of the receptor and is formed by residues located in noncontiguous regions at the α(+)/γ(−) interface (Figure 10).336 This position is homologous to the orthosteric

nonsense codon suppression procedure and oocyte expression. Interestingly, authors showed that progressive fluorination at W149 from the muscle nAChR with monofluoro, difluoro, trifluoro, and tetrafluoro derivatives gave a clear linear correlation between the apparent agonist affinity of ACh and the degree of fluorination, known to weaken the cation−π interaction.339 This strategy, successfully extended to other Cys-loop receptors, established in each case that a strong cation−π interaction exists between the receptor and its agonist, but the location of the interaction and the residues involved differed among receptors: loop B for nAChRs, 5-HT3, and glycine receptors, loop A for GABAA receptors, and loop C for MOD-1, the invertebrate 5-HT 3 receptor homologue.342−345 All these biochemical and biophysical data have been collected before the release of the first X-ray structures. In 2001, Sixma, Smit, and co-workers reported the first crystal structure of the acetylcholine-binding protein (AChBP), a soluble surrogate of the nAChR extracellular domain.15,346 AChBP is produced and stored in glial cells from the freshwater snail Lymnaea stagnalis and is released in an ACh-dependent manner in the synaptic cleft where it regulates synaptic transmission.346 Sequence analysis shows 20−25% sequence identity to the extracellular domain of nAChR (notably with α7) and 15−20% identity to other Cys-loop receptors. Unlike the Cys-loop receptors, AChBP lacks the transmembrane domain, a fact that facilitated X-ray investigations. From a structural point of view, this was the first landmark achievement in the Cys-loop receptor field because, for the first time, the three-dimensional organization of the ACh-binding site was revealed. Wonderfully, all accumulated biochemical data, including mutagenesis,347,348 fitted perfectly with the structure.349 The structure revealed an oligomer of five identical subunits arranged around an axis of 5-fold symmetry. Each subunit comprises an N-terminal α helix, two short 310 helices, and a core of ten β strands folded into a twisted β sandwich reminiscent of that of immunoglobulins.23 The canonical cysteine residues of the defining loop, referred to as Cysloop, connect through an intrasubunit disulfide bridge the two twisted β sheets of the β sandwich. As predicted by photaffinity labeling, the ACh-binding sites are located at each interface between AChBP subunits, as revealed by additional structures resolved in the presence of agonists and antagonists (Figure 9C).350,351 The binding sites are lined by residues formerly shown to belong to the ACh-binding site of nAChR on the basis of photoaffinity labeling experiments, thus validating a posteriori the multiple-loop model previously established for nAChRs, GABAA, glycine, and 5-HT3 receptors.349,352 Later, the crystal structure of the extracellular domain of the nAChR α1 subunit,353 and more recently of an α7-nicotinic receptor chimera354 further confirmed the interfacial location of the orthosteric-binding site established by ligand-bound structures of AChBP. It appeared rapidly that one loop of the binding site, namely loop-C, adopted an uncapped conformation in the absence of agonist or in the presence of antagonists,351,355,356 whereas in the presence of agonist,351 it closes or tightens the binding site cavity, thereby trapping bound agonist molecules.357 Whether this “capping” motion initiates receptor activation is not firmly established, but a study showed that capping of loop-C though disulfide trapping evoked long-lived openings.358 The second landmark achievement came with the first X-ray structures of the full-length prokaryotic receptors (Figure

Figure 10. (A) Schematic representation of GABA- and benzodiazepine (BZs)-binding sites in GABAA receptor. (B) Chemical structures of benzodiazepine agonist, inverse agonist and antagonist.

(GABA)-binding site but is situated at a different subunit interface, a hypothesis that had been proposed earlier.375 Although, no crystal structure of the GABAA receptor has been reported, homology models on the extracellular domain of the receptor based on crystal structures of AChBP not only confirmed these assumptions336,376,377 but also enabled further investigations into the mechanisms underlying benzodiazepine binding and modulation.378−380 In addition, using another chemical approach that combines affinity labeling and sitedirected mutagenesis,314 new insights into the relative positioning of ligands in the benzodiazepine site were provided.381−386 In this engineered site-directed affinity labeling approach, which is based ultimately on SCAM studies,387 residues thought to 6299


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reside in the binding pocket are individually mutated to cysteine, and then functional mutants are combined with a high-affinity ligand, which carries a substituent reactive to cysteine. Direct apposition of such reactive substituent with a cysteine residue is expected to lead to a covalent reaction.314 This approach, originally applied to investigate the noncompetitive blockers site of the GABAA receptor,388 has been used to investigate other important sites in LGICs (see the following sections). Beyond the classical benzodiazepine site, a second site for benzodiazepine binding has been recently postulated to occur at the extracellular α(+)/β(−) interface,385,389 distinct from that previously identified in the membrane-spanning region (Figure 10).390 Because drugs interacting with the α(+)/β(−) interface should be able to modulate αβ, αβγ, and αβδ receptors, this raises the possibility that they might exhibit a much broader action than benzodiazepines and might become clinically important molecules. Other natural and synthetic allosteric modulators, including ivermectin (IVM), PNU-120596, barbiturates, neuroactive steroids, ethanol, convulsants, and general anesthetics, that have been developed for their therapeutic potential act in the transmembrane region of the Cys-loop superfamily (Figure 11).89,391,392 The recent crystal structures of GLIC,28 ELIC,16 and GluClα,359 along with that previously obtained of nAChR from cryoelectron microscopy at medium resolution,393 showed that the membrane-spanning region from each subunit is arranged as a four-α-helix bundle (M1−M4) with possible water-filled cavities that may accommodate these allosteric modulators. General anesthetics caught special attention because they cause reversible loss of consciousness, and the underlying mechanism has been a long-standing mystery.394 Almost all general anesthetics have been found to potentiate the GABA response in GABAA receptors, while they inhibit nAChR function.395 Photoaffinity labeling performed on the Torpedo nAChR with 3-azioctanol and the photoreactive etomidate analogs, azietomidate and p-trifluoromethyldiazirinyl ethylbenzyl (TDBzl)-etomidate, identified a first anesthetic site located in the channel lumen between all five subunits.396−398 Interestingly, this site was originally defined by other noncompetitive photoaffinity and affinity blockers of the nAChR such as chlorpromazine,172,399−401 triphenylmethyl phosphonium,402 meproadifen mustard,403 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID),404 a benzoic acid ester analog of TID (TID-BE),405 diazofluorene (DAF),406 and tetracaine.407 Consistent with SCAM studies408 and engineered site-directed affinity labeling with chemically reactive affinity probes derived from noncompetitive blockers of the GABAA receptor,388 the site is located within the ion pore. When an agonist opens the channel, the anesthetic binds with high affinity in the open pore, but once bound, it sterically obstructs the flow of ions through the channel (they are referred to as open-channel blockers). Very recently, this view has been further confirmed by the crystal structure of picrotoxin, an alkaloid convulsant open channel blocker of the GABAA and glycine receptors, bound to GluClα receptor.359 Consistent with previous studies that tentatively located its binding site within the ion channel,409−415 the structure showed a deep picrotoxin location in the channel, at the cytosolic base of the pore on the 5-fold axis of molecular symmetry.359 A second anesthetic-binding site has been located within the four-helix bundle of individual subunits in LGICs. It was identified by photoaffinity labeling in the Torpedo nAChR with

Figure 11. Chemical structures of some allosteric modulators acting in the transmembrane region of the Cys-loop receptors.

halothane416 and has been extensively investigated in the GABAA receptor by mutational analyses417−424 and by the use of sulfhydryl reagents.425,426 These studies identified not only the amino acid residues critical for receptor modulation by anesthetics and long-chain alcohols but also those responsible for subunit selectivity. In particular, position 15′ of M2 is of special interest because drug sensitivities can be reversed by swapping amino acids in this position.419,424 Moreover, the importance of this position for anesthetic modulation has been confirmed in glycine and 5-HT3 receptors417,427 and also in α7 nAChR where it determines the efficacy of the positive allosteric modulator (PAM) PNU-120596.428 A recent study also provided evidence that PAM binding, such as 4-(16300


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and support the notion that a cross talk between both cavities might underlie general-anesthetic-mediated action.438 Finally, a recent study based on a fluorescent quenching and computational approach suggested an additional binding pocket of some anesthetics at the extracellular−transmembrane interface.439 Although, this hypothesis needs further direct evidence, the crucial role of this interface in the allosteric coupling of agonist binding to channel gating has been extensively investigated440−450 and supported by identification of some mutations that lead to human congenital myasthenic syndromes.451 The action of neuroactive steroids has also been investigated in GABAA receptors. Mutagenesis studies indicated that these compounds might have at least two different binding sites within the transmembrane domain of the receptor; one seems to be located within the four-helix bundle, mediating allosteric modulation of the receptor at low steroid concentrations, while the other seems to be located at the β(+)/α(−) interface, below the GABA binding pocket, mediating direct activation at high steroid concentrations.452,453 However, it has recently been shown that a single homology model cannot accommodate an interfacial neurosteroid activation binding site and an interfacial site for the anesthetic etomidate.436 Other studies are awaited to resolve this intriguing issue (for discussion, see refs 21 and 454). Divalent ions are known to modulate the function of pentameric receptors. Extracellular Ca2+ was found to be necessary for proper functioning of many nAChR subtypes, including the α7 nAChR. The use of chimeras made from the α7 nAChR and the 5-HT3 receptor, the latter being insensitive to the extracellular Ca2+, revealed that the divalent cation binds in the extracellular domain of the α7 nAChR and interacts with residue E172 localized at the subunit interface.455,456 Other studies also identified the molecular determinants of Zn2+ potentiation in neuronal nAChRs and suggested that these sites are structurally and functionally similar to the classical benzodiazepine-binding sites on GABAA receptors.457 Zinc also affects the function of GABAA receptors formed from binary combinations of α and β subunits through distinct binding sites,458,459 one situated on the internal surface of the ion channel and the other located on the external surface of the receptor at the interfaces between α and β subunits. Finally, zinc exhibits biphasic activity on the glycine receptor; it allosterically enhances channel function at low concentration, whereas it inhibits responses at higher concentrations.460 The inhibitory effect was suggested to occur through Zn2+-chelating residues including a couple of histidine residues located at the subunit interface within the extracellular vestibule lumen.461−463 For the potentiating effect, it was proposed that the divalent cation binds to a site on the extracellular outer face of the receptor and exerts its positive allosteric effect via an interaction with the Cys-loop to increase gating efficacy.464

naphtyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolone-8sulfonamide (TQS), at this intrasubunit transmembrane site is able to activate α7 nAChR in the absence of agonist binding at the orthosteric site.429 A third anesthetic-binding site, located at the subunit− subunit interfaces, has been identified in the GABAA receptor by the photoaffinity label azietomidate.430 The probe photoincorporated within the αM1 transmembrane helix at α1M236, and within the βM3 transmembrane helix at β3M286.430 In the light of a homology GABAA receptor model based on cryoelectron microscopy Torpedo nAChR structure at 4 Å resolution,393 these data demonstrated that the etomidatebinding site lies at the β(+)/α(−) interface in the transmembrane domain. Interestingly, this pocket is at the same interface as the binding pockets for the neurotransmitter GABA ∼50 Å above in the extracellular domain.430 Even more interestingly, the subunit interface location of etomidate site strikingly resembles that of the IVM site, for which the structure has recently been resolved in the GluClα receptor.359 IVM is a semisynthetic macrocyclic lactone that is widely used to treat river blindness in humans and parasitic infections in animals.431,432 It activates invertebrate GluCl channels at nanomolar concentrations to achieve its therapeutic goals.433 It was also found to be able to activate or potentiate Cysloop391,434 and P2X receptors.435 In the invertebrate receptor, IVM binds at subunit interfaces on the periphery of the transmembrane domains, proximal to the extracellular side of the membrane bilayer.359 It appeared that the IVM-binding site in GluClα is shared, at least in part, by many anesthetic modulators of vertebrate receptors. Yet, photoincorporation of etomidate seems to be allosterically inhibited by neurosteroids, barbiturates, and the intravenous anesthetic agent propofol, indicating that these drugs bind to a site that is not overlapping with that of etomidate.436,437 In agreement with this idea, the recent crystal structure of propofol bound to the prokaryotic GLIC receptor indicated that the general anesthetic binding occurs in an intrasubunit transmembrane pocket, lined by hydrophobic residues from M1 to M4 (Figure 12).438 Interestingly, this study also revealed narrow tunnels of less than 3 Å in diameter that link the inter- (where etomidate binds) and intrasubunit (where propofol binds) cavities.438 These new data thus may reconcile some of the discrepancies

6.2. Tetrameric Receptors

At the end of the last century, biophysical and bioinformatics techniques combined with extensive mutational analyses had already revealed the characteristic modular architecture of iGluRs.465,466 They showed that the extracellular part of these receptors was composed by the amino terminal domain (ATD) (for eukaryotic iGluRs), a domain that is structurally related to the bacterial leucine-binding protein,467 and by the LBD, which shares sequence homologies with bacterial periplasmic ligandbinding proteins.468,469 These techniques had also allowed the generation of computer-assisted structural models of crucial

Figure 12. X-ray structure of propofol bound to GLIC (PDB code 3P50). (left) General view of GLIC and (right) enlarged view of the general anesthetic-binding cavity. This last view is looking down the pore from the extracellular side with the extracellular domain removed for clarity. The intersubunit cavity is joined to the intracavity by linking tunnel as indicated. The cavities are underlined in blue dashes. 6301


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chain of helix D, and this interaction is crucial for the highaffinity binding of glutamate (or glycine for GluN1) (Figure 13A). In a similar manner, the ammonium function of glutamate also forms a salt bridge with a conserved glutamate or aspartate side chain of the D2 domain of AMPA/kanaite or NMDA receptors, respectively. Finally, the γ-carboxylate function of glutamate makes hydrogen bond contacts with a conserved threonine side chain and with the main chain of helix F. From the compilation of these structures with agonists, partial agonists, and antagonists, five key themes emerged for the orthosteric-binding site:489 (i) the ligand-binding core is much larger than necessary to accommodate glutamate, except for GluN1, which has the right size to accommodate glycine and excludes glutamate by steric hindrance;480 (ii) the subtype selectivity arises from residue differences in the D2 domain with a key role of steric effects for selective agonist binding; (iii) the extent of agonist-triggered domain closure is variable, between receptor subtypes and between agonists, partial agonists, and antagonists in a given subtype (Figure 13B); however, subsequent studies on GluN1 NMDA receptors suggested that different mechanisms can underlie partial agonist activity;490 (iv) competitive antagonists are working by a foot-in-the-door mechanism leading to a wide-open conformation; (v) trapped water molecules have a key role in agonistbinding mechanisms. The development of competitive antagonists for iGluR subtypes is of special interest because they can be viewed as neuroprotective agents in the treatment of a variety of neurological disorders (see above). Today structures of ATPO (Figure 14), CNQX, DNQX, FQX, NS1209, UBP277,

elements involved in ligand binding. However, during the last 14 years, X-ray crystallography provided unprecedented advances of ligand recognition at the atomic level of detail in iGluRs. 6.2.1. Orthosteric-Binding Sites. The first breakthrough occurred in 1998, when the X-ray structure of kainate bound to the AMPA GluA2 receptor LBD was resolved.14 This achievement was facilitated first by the finding that fusion of S1 and S2 with a hydrophilic linker generates a water-soluble construct that retains wild-type ligand binding affinities470−472 and second by the development of large-scale folded GluA2 S1S2 construct production as inclusion bodies in Escherichia coli.473 This first structure enabled localization of the agonist binding site on iGluRs, confirming the general principle that glutamate (or glycine in the case of NMDA GluN1 receptors) binds in the cleft of a clamshell-like motif formed from the S1− S2 segments of a single iGluR subunit (Figure 13A). Of note,

Figure 13. (A) X-ray structure of glutamate (Glu) bound to GluA2S1S2 (PBD code 1FTJ). (B) X-ray structures of antagonist (left) and agonist (right) bound to GluA2-S1S2 showing the tightening of the clamshell formed from S1 and S2, depicted in different color (DNQX PDB code 1FTL).

this fact had been previously predicted from structural analysis of bacterial periplasmic ligand-binding proteins.468,469 The development of facile expression and purification methods for various GluA2-S1S2 constructs has rapidly enabled the determination of more than 80 crystal structures,474 with agonists, partial agonists, or competitive antagonists or without ligands (apo structure).475−477 Thus, among the 18 iGluR subunits encountered in mammals, the crystal structure of 11 of the LBDs of these subunits has now been reported: GluK1,478,479 GluK2,478 GluN1,480 GluN2A,481 GluN3A,482 GluN3B, 482 GluNA3, 483 GluA4, 484−486 GluK3, 487 and GluN2D.488 In all the structures, the α-carboxylate function of glutamate forms a bidentate salt bridge with an arginine side

Figure 14. Chemical structures of some iGluR agonists, competitive antagonists, and allosteric modulators.

UBP289, and ZK 200775 GluA and GluK antagonists in complex with the GluA2 LDB have been solved.474 However, the highly conserved nature of the antagonist-binding domain usually diminishes the prospect that strong subunit selectivity can be achieved through conventional modifications of competitive antagonist structure. For example, classical NMDA iGluR antagonists, like selfotel491 and midafotel 6302


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(CPPene),492 which have been examined for their clinical potential, showed in most cases severe side-effects preventing their clinical uses.493 Therefore, an alternative strategy recently focused on GluN1-selective antagonists for their interesting subtype selectivity. In this respect, crystal structure determination of the competitive antagonist 5,7-dichlorokynurenic acid (DCKA) bound to GluN1 LBD should allow rational design of potent and specific GluN1 competitive antagonists.480 Of note, the binding mode of the antagonist in the crystal structure was anticipated precisely by the use of the engineered site-directed affinity labeling approach with DCKA-based reactive ligands (Figure 15).494

6.2.2. Allosteric-Binding Sites. Many allosteric modulators that regulate iGluR function have been described so far, and for some of them, their binding site locations have been unveiled by recent crystal structures. These studies thus make possible rational drug design of potent and new compounds targeting allosteric sites for treating iGluR-associated diseases. Many iGluR channel blockers, such as PCP, ketamine, and memantine (the latter is still used for AD treatment) bind to the channel, but since this transmembrane region is highly conserved among iGluRs, these blockers showed little subtype selectivity, thus limiting their clinical use. A wealth of information based on spectroscopic measurements, electrophysiological recordings, and site-directed mutagenesis provided new insights into the action of some allosteric modulators on the iGluR LBDs. These domains are important not only because they bind agonists but also because they regulate gating and desensitization.496 Therefore, positive allosteric modulators, such as CX-691, S-18986, and LY450108, targeting the LBD of the AMPA receptor have been developed (Figure 14).497−502 We can anticipate that within a few years such approaches should lead to the discovery of new drugs treating AMPA receptor-associated diseases. Before the release of the full-length GluA2 receptor structure, structures of the ATDs of the GluA2 and GluK2 receptors expressed as soluble glycosylated proteins were solved.503,504 This was followed by other structures: GluN2B ATD resolved in the apo and Zn2+ bound states,505 GluK3 and GluK5 ATDs,506 GluA1 ATD,507 GluA3 ATD,508 the heterodimeric GluN1/GluN2B ATD,509 and the heterodimeric and heterotetrameric assemblies of GluK2/GluK5 ATDs.510 Key features emerging from these high-resolution structures combined with dimer formation studies by sedimentation velocity measurements are an atomic level understanding of how the ATD plays a role in facilitating the efficient assembly of heteromeric iGluRs. Currently many efforts are undertaken to understand the mechanisms of action of the allosteric modulators acting at the ATD. This represents a special interest because the ATD is structurally and functionally the most divergent region of the iGluR subunits, and thus one may expect that these modulators may display subtype selectivity. Up to date, ligand binding to this allosteric domain in iGluRs has been associated exclusively with the NMDA type receptors. This NMDA ADT’s feature has been revealed for the first time using electrophysiological experiments on a series of chimeric NMDA receptors with variable GluN2D and GluN2A ATD’s.511 Although there is no ATD-targeted ligand or evidence for the ATD-mediated functional regulation in AMPA and kainate iGluRs, there is a rich spectrum of ligands that bind to the ATD of NMDA receptors.512 These ligands are small molecules or ions able to regulate receptor activity in a subunit-specific manner. Ions are typically protons and Zn2+ cations that bind specifically to GluN1 and GluN2A/GluN2B ATDs, respectively. In particular, zinc ions found at many excitatory synapses in the brain have recently been shown to be involved in pain pathways through GluN2A binding.254 X-ray crystallography unveiled the atomic details of the interaction between Zn2+ and the ATD of the NMDA receptor GluN2B subunit. 505 From a clinical perspective, small molecules acting as ATD-mediated allosteric modulators of NMDA receptors have recently evoked considerable interest.513 These include polyamines514,515 and the neuroprotectant ifenprodil,516,517 which are positive and negative allosteric modulators, respectively, of receptors

Figure 15. (A) Chemical structures of DCKA and two related probes that are reactive to cysteine (7-NCS and m1-NCS). (B) Close-up view of DCKA bound to GluN1 LBD structure (PDB code 1PBQ). Residues labeled by 7-NCS and m1-NCS when mutated into cysteines494 are indicated in gold and magenta, respectively.

The second landmark achievement came in 2009, when the first X-ray structure of an intact iGluR, the rat GluA2 receptor, was solved at 3.6 Å (Figure 7B).18 Because the structure was solved with a bound antagonist lodged in the clamshell of each LBD, this study provided further evidence that the agonist/ competitive antagonist binding site is located within subunits. The transmembrane segments, in which the channel lies, share a conserved global architecture and 4-fold symmetry with that of tetrameric voltage-gated pore loop ion channels but with inverted topology.495 However, the full-length GluA2 structure revealed numerous unexpected features not anticipated from previous work. First, an unexpected cross over of subunits forming dimer pairs in the ATD and LBD layers was described. Second, multiple conformations are adopted for the ATD/LBD and the LBD/ion channel connecting linkers, leading to the amazing observation that different conformations are possible for identical subunits. Third, molecular symmetry switches from 4- to 2-fold between the ion channel and the extracellular domain, respectively. Fourth, no packing contacts are observed between the ATD and the LBD on the central axis of symmetry of the tetramer assemblies, leading to an enormous chaliceshaped void. These unexpected features have many potential consequences for the assembly, activation, and especially allosteric regulation of iGluRs. 6303


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high-resolution crystal structure. It is known that binding of ATP phosphates often involves polar and charged residues and that adenine and ribose moieties may bind to aromatic residues. Therefore, the search for the ATP binding site started with a focus on conserved positively charged,522−524 polar,525−528 and aromatic residues.524,529 By use of site-directed mutagenesis combined with electrophysiological characterization, analysis of 11 conserved positively charged lysine or arginine residues in the extracellular loop of P2X1 receptor pointed to four basic residues (K68, K70, R292, and K309), contributing to ATP potency.522 Other works confirmed their roles for ATP action in other P2X subtypes: P2X2,525,527 P2X3,530 and P2X4.527 SCAM approaches employing positively and negatively charged methanethiosulphonate (MTS) reagents led to the conclusion that these residues contribute to the binding of the ATP phosphate tail.526,531 However, single channel recordings in a P2X2 receptor bearing a channel mutation showed that one of these residues (K308, homologous to K309 in P2X1) is important for gating, thus elegantly distinguishing residues involved in binding from those involved in gating.532 Consistent with this finding in the P2X2 receptor, another study in P2X4 suggested that K313, homologue of K308, might not contribute directly to ATP binding.533 As performed on charged residues, conserved polar/aromatic residues were also subjected to similar investigation, that is, mutagenesis and cysteine alkylation combined with electrophysiological recordings. The results have strongly suggested contributions of two aromatic (F185 and F291) and polar residues (T184 and N288, P2X1 numbering) to ATP action in P2X receptors.525,526,529 Based on SCAM studies, it has been proposed that residues N288 and T184 were involved in ATP binding but not F291 and F185.527,528 Based on these data, a common ATP-binding site model for the P2X receptors has been proposed. However, the proposed binding model remained speculative because of the lack of a crystal structure with bound ATP. In the absence of structural information, the most direct evidence for the location of the agonist binding site in these receptors was provided by a study in which the mouse P2X7 residue R125 was ADP-ribosylated, a post-translational modification that covalently tethered to this residue an ADP-ribose moiety,534 leading to an irreversible channel opening.535 Nonetheless, because ADP-ribose is larger than ATP, this raises the possibility that R125 might not be located within but at the periphery of the ATP-binding site. A landmark achievement in P2X receptors made by Gouaux and co-workers came in 2009 when the first X-ray structure of the zebrafish, Danio rerio, P2X4 receptor was resolved at 3.1 Å (Figure 7C).17 This structure represents one of the greatest advances in the field since the first cloning of the P2X receptor in 1994.536,537 In agreement with previous biochemical and biophysical data,538−541 the structure confirmed the trimeric assembly of the ATP-gated P2X receptors. Each subunit of the trimer has two transmembrane α-helices (M1 and M2), two intracellular N- and C-termini, and a large ectodomain. Note, however, that a truncated form of the receptor, in which the Nand C-termini were shortened, was used for crystallization. The two transmembrane helices extend ∼28 Å through the membrane and the extracellular domain projects 70 Å above the cell surface. Although the crystal structure of the zebrafish P2X4 receptor was resolved in the absence of ATP and thus represents a closed channel state, it clearly shows that the above-mentioned eight conserved residues line a large and deep intersubunit cavity; there are three cavities per trimer, each

containing GluN2B subunits. A recent study based on a combination of biochemical and electrophysiological analyses proposed that polyamines bind to a site located at the bottom lobes of the GluN1−GluN2B ATD dimer interface and act by shielding ionic interactions between residues that influence receptor function.518 Another recent work reported the crystal structure of the GluN1/GluN2B ATD heterodimer assembly in complex with ifenprodil.509 Surprisingly, the ligand binds in a hydrophobic interface between the upper lobes of the GluN1 and GluN2 subunits and not within the cleft of the NR2B clamshell as previously suggested on the basis of homology modeling combined with engineered site-directed affinity labeling experiments performed with reactive analogs of ifenprodil.519 However, it should be noted that some of the labeled residues519 are found in the crystal structure to be in close contact to the ifenprodil molecule (Figure 16). In

Figure 16. X-ray structure of ifenprodil bound to GluN1/GluN2B ATD (PDB code 3QEL). Residues identified by affinity labeling519 are indicated.

conclusion, the atomic level understanding of small molecule ATD allosteric modulators should lead to the rational design of new drugs specifically targeting specific NMDA subunits and should allow the development of clinical, well-tolerated GluN2B-selective negative allosteric modulators.511 6.3. Trimeric Receptors

The understanding at the atomic level of the ATP-binding mechanism would provide a rational basis for the design of new drugs able to treat P2X-related diseases, including pain.232 We review the current understandings about molecular mechanisms of agonist/antagonist and allosteric modulator binding in P2X receptors and how the recent crystal structure of a P2X receptor allowed illumination of such mechanisms. 6.3.1. Orthosteric-Binding Sites. Several consensus amino acid motifs, such as the Walker motif520 and Qmotif,521 may account for ATP binding in a wide range of ATPsensitive proteins. However, such consensus motifs for ATP binding are absent in the P2X receptor family. Furthermore, sequence analysis of P2X subunits showed no homology to other ion channels or proteins, thus precluding homology modeling. Such facts have made the identification of the ATPbinding site particularly difficult, especially in the absence of a 6304


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shaped like an open jaw. The jaw is framed on its upper side by the “head” domain and on its bottom side by the “dorsal fin” domain of one subunit and the left flipper of another, with residues K70, K72, F188, and T189 from one subunit and N296, F297, R298, and K316 (P2X4 numbering) from the adjacent subunit (Figure 17). The structure also confirmed

Figure 18. NCS-ATP identified the ATP-binding site in a P2X2 receptor homology model built from the P2X4 crystal structure. Two residues, N140 (left) and L186 (right), when mutated into cysteines (indicated in yellow), are covalently labeled by NCS-ATP.548 Distances between the sulfur atom of NCS-ATP and that of the sulfhydryl group of the mutated cysteines are shown in red dashed lines, and residues previously identified and proposed to contribute to the binding site are also indicated in green.

Figure 17. X-ray structure of the zebrafish P2X4 receptor (PDB code 3H9V). Putative ATP-binding site is shown enlarged in inset. Residues previously identified and proposed to contribute to the ATP site are shown in stick representation. Note that because the electron density for the side chain of K70 is weak it was built as an alanine.17 Note also that the transmembrane helices are angled nearly 45° from the membrane normal.

18). Interestingly, L186 and N140 are separated by about 18 Å, a distance that is compatible with the length of ATP in an extended conformation, suggesting that ATP spans the site with two distinct orientations or the conformation of the jaw changes dramatically upon ATP binding. Multiple orientations of ATP are possible for entry into the binding pocket and to induce differential functional consequences. The hypothesis of alternative binding modes deserves further investigation but finds precedent in the pentameric nAChRs, where two bound conformations of nicotinic agonists with inverted orientations relative to each other were established in AChBP by X-ray crystallography.549 During revision of the present review, the ATP-bound crystal structure of the zfP2X4 receptor was published.550 ATP binds to the expected site located at the subunit interface, in the jaw, and of note, the adenine base of ATP makes hydrophobic interactions with nonpolar residues including residue L191, which is homologous to L186 labeled by NCS-ATP. P2X antagonists have been considered as new therapeutic agents of potential interest in various types of pain, including visceral, neuropathic, and inflammatory pain.232 Structure-based design of subtype-selective P2X antagonists in silico requires structural and mechanistic insights into the P2X antagonism. However, in contrast to the pentameric and tetrameric receptors, in which several X-ray structures have been determined with bound antagonists, little is known about the principle of receptor inhibition by antagonists in P2X receptors. The fluorescent synthetic compound 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (Figure 19) has been shown to act as an antagonist on P2X receptors, being more potent when blocking ATP-induced currents at P2X1 and P2X3 receptors (IC50 in low nanomolar range) than at P2X2, P2X4, and P2X7 (IC50 in low micromolar range).38,551,552 Other related TNPATP analogs (e.g., TNP-ADP, TNP-AMP, and TNP-GTP) are also potent antagonists on P2X receptors, especially on P2X1, P2X3, and the heteromeric P2X1/3.551 Kinetic, pharmacological, and site-directed mutagenesis experiments have suggested that TNP-ATP acts in a competitive fashion, thus likely targeting the ATP-binding jaw to inhibit ATP action.532,553,554 It would not be surprising that TNP-ATP shares common binding residues with ATP; nevertheless it remains possible that TNP-ATP has a distinct binding site within the ATP binding jaw.

earlier predictions in which the putative ATP binding site has been suggested to lie at the subunit interface.523,542,543 In particular, the proximity of K68 and F291 was established by disulfide bond trapping experiments in the P2X1 receptor, in which cysteine mutants of K68 and F291 from two different subunits were cross-linked in the absence of ATP, whereas cross-linking was prevented by the presence of ATP.542 In good agreement with this study, the structure showed that the two residues were indeed from two adjacent subunits and were separated by only ∼8 Å (from their respective Cα atoms). It also revealed that the side chains of both F188 and F297 (homologous to F185 and F291 in P2X1, respectively) were oriented away from the cavity, again consistent with data obtained from SCAM studies. Consequently, the jaw has been proposed as the putative ATP-binding site.17,544−546 As previously mentioned by Colquhoun, “perhaps the most direct method of all for location of an agonist binding site is to use an agonist that can label the site covalently”.547 Such efforts were made in the identification of the ATP site in P2X receptors. Direct evidence for the orientation of ATP in the binding site was provided by our recent study using the sitedirected affinity labeling approach.548 In this study, we employed the sulfhydryl-reactive 8-thiocyano-ATP (NCSATP), a P2X2 agonist, to covalently cross-link the ATP moiety in an affinity-dependent manner to single engineered cysteine mutants (Figure 18). Out of 26 positions tested around the putative binding jaw, only residues L186 and N140 when individually mutated into cysteines were specifically labeled by NCS-ATP. This work thus provided direct evidence that the adenine base of NCS-ATP is close to these previously unidentified residues, and allowed us to dock confidently NCS-ATP within the binding site of a P2X2 homology model using the geometrical constraints established by the engineered affinity labeling data.548 Note that this constraint was restricted only to the adenine ring but not to the phosphate tail for which docking of this part of the ATP molecule was uncertain. Consistent with previous studies, the eight conserved residues were found in close proximity to docked NCS-ATP (Figure 6305


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model of thromboembolism, consistent with blockade of the P2X1 receptor function.561 Surmountable shifts of the dose− response curves for ATP led to the suggestion of a competitive antagonism mechanism for suramin on P2X receptors560,562 (but see also refs 554 and 563). However, it has been found that residues involved in ATP binding (K68, K70, and R292) had no or a minor effect on suramin potency in P2X1 receptor.522 In support of this observation, suramin was reported to be able to activate a P2X2 mutant bearing a channel mutation, and the removal of the ATP binding residue K69 (homologous to K68 in P2X1) had no effect on the agonist-like action of suramin.532 By contrast, K138, which does not line the ATP binding site but faces away from it, is involved in suramin sensitivity.564 Recently, the use of chimeric receptors further revealed that clustered positively charged residues (i.e., K136, K138, R139, and K140, P2X1 numbering) at the base of the cysteine-rich head domain were found to be responsible for the nanomolar sensitivity and high selectivity of suramin in P2X receptors.565 Another study using in silico docking and sitedirected mutagenesis of suramin analogs displaying nanomolar affinity for the P2X2 receptor rather suggested a strong overlapping binding area between suramin- and ATP-binding sites.566 Taken together, these data suggest that additional experiments are needed to establish the competitive mechanism of suramin and the fine orientation(s) of the antagonist within the ATP-binding jaw. Other compounds structurally unrelated to ATP that may target the ATP binding jaw include A-317491 and PSB-1011. A-317491 is a competitive non-nucleotide antagonist displaying high potency on P2X3 and P2X2/3 receptors.230 This compound has been found to effectively reduce nociception in inflammatory and neuropathic pain models.230 The newly available PSB-1011 is the first selective and potent competitive antagonist for P2X2 receptors;567 it is followed by optimization of anthraquinone derivatives that have been previously found to be P2 antagonists.568 Taken together, the jaw forming the ATPbinding site represents a very important target in P2X receptors, an ideal place for blocking or triggering the function of the receptor. 6.3.2. Allosteric-Binding Sites. As for other LGICs, there are many allosteric modulators that regulate the function of the P2X channel. Among them, PPADS, a sulfonic acid derivative, is known to act as a nonselective antagonist (Figure 19). It was also used as the first pharmacological agent to assess native P2X receptors.569 PPADS potently inhibits currents mediated by P2X1, P2X2, P2X3, and P2X5 receptors, but less those mediated by P2X4 and P2X7 receptors.570 The PPADS analog pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4,8-disulfonate) (PPNDS) has later proven to be more specific on P2X receptors compared with P2Y receptors.571 PPADS has been shown to reduce nociceptive sensitivity in animal models, including inflammatory and neuropathic pain.572,573 PPADS acts noncompetitively on P2X receptors to inhibit ATP induced-currents.574 It has been shown that residue K246 located at the periphery of the jaw accounted for PPADS sensitivity in the rat P2X2 receptor, and replacing the homologous E249 by a lysine in the rat P2X4 conferred high PPADS sensitivity to the receptor.575 Identification of the PPADS site thus remains to be firmly established. Trace metals, including Zn2+, Cu2+, Co2+, and Ni2+, modulate P2X receptor activities,576 among which the best studied is zinc. Functional modulation of P2X receptors by Zn2+ depends not only on receptor subtypes34 but also on species.577 For

Figure 19. Chemical structures of some P2XR agonists, antagonists, and allosteric modulators.

Suramin has long been known as a nonselective antagonist of P2 receptors (P2X and the metabotropic P2Y receptors), and has been used during the course of discovery of P2X receptors to define native P2X receptors naturally expressed in different tissues.555−557 Subsequently, several analogs of suramin have been explored, aiming to increase potency and selectivity. The suramin-related NF023 was first identified as a P2X selective antagonist,558 which was later shown as a P2X1 antagonist, with selectivity over P2X3, P2X2/3, P2X2, and P2X4 receptors.559 For NF279, another suramin analog, a greater potency on P2X1 was found, with increased selectivity over P2X3 and P2X4.560 More recently, it has been shown that another suramin-derived analog, NF449, reduced in vivo platelet aggregation in a mouse 6306


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another residue, close to E98, was involved in determining noncompetitive antagonist sensitivity in P2X7 for molecules such as N2-(3,4-difluorophenyl)-N1-(2-methyl-5-(1piperazinylmethyl)phenyl)glycinamide dihydrochloride (GW791343) and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580).583 As we reviewed in other LGICs, many allosteric regulators bind to the transmembrane region of the receptors. The crystal structure of P2X4 showed that the shape of this region is reminiscent of an hourglass and is formed by six transmembrane α-helices, two from each of the three subunits.17 Within a subunit, the transmembrane helices are oriented approximately antiparallel to one another and are angled nearly 45° from the membrane normal. Amazingly, comparison of the P2X structure with that of acid-sensing ion channel (ASIC), another trimeric ion channel activated by protons for which the crystal structure has also been resolved,584 revealed similarity in pore architecture and aqueous vestibules.585 In both channels, M2 helices line the ion channel pore, whereas M1 helices make most contacts with the lipid bilayer.585 Although, there is no significant amino acid sequence identity between ASIC and P2X receptors, the transmembrane domains of the two proteins thus adopted a remarkably similar fold, a hypothesis that we challenged before the release of the P2X crystal structure.586 The structure of the P2X4 thus provides invaluable data to understand the action of allosteric regulators that may bind to the transmembrane region. For P2X receptors, the most studied compounds that are suspected to bind to the transmembrane domains are IVM and alcohols. IVM is a positive allosteric modulator of mammalian homomeric and heteromeric P2X4 receptors, but not of P2X2, P2X3, and P2X7 receptors. 435 Single-channel analysis showed that IVM stabilized the open state of the human P2X4 receptor channel relative to both desensitized and closed states, which suggests that the allosteric modulator binds more tightly to the open conformation of the channel.587 Chimeras with transmembranes swapped between P2X2 and P2X4 subunits along with tryptophan-scanning mutagenesis experiments have led to the conclusion that IVM interacted with both M1 and M2, and this interaction, in turn, revealed widespread rearrangements during gating motions of P2X4 receptors.588 Ethanol also potentiates ATP-evoked currents in the rat589 but not in the human P2X3 receptor.590 In contrast, it differentially inhibits ATP-evoked currents in both the rat P2X2 and P2X4 receptors.591 Molecular studies showed that residues located at ectodomain−transmembrane interfaces592,593 are critical for ethanol sensitivity in P2X receptors. Interestingly, these residues are close to the fenestrations revealed by the X-ray structure, raising the possibility that these fenestrations might be interesting targets for the allosteric regulation of P2X receptors (Figure 20). These fenestrations (there are three per receptor) are formed at the interfaces of adjoining subunits just above the outer leaflet of the membrane, and recent studies provided strong evidence that they are lateral portals involved in ion access.594,595 In support of the allosteric regulation site hypothesis, a very recent study showed that these lateral portals are involved in the allosteric regulation of calcium influx and channel gating by IVM in P2X4 receptors (Figure 20).596 Thus, the lateral portals could represent a novel target for drugs in the treatment of P2X-associated diseases including neuropathic pain.

instance, zinc inhibits the human P2X2 receptor activity, while it strongly potentiates the ATP response in the rat P2X2 receptor.577 In contrast, P2X1 and P2X7 receptors can only be negatively modulated by zinc.576 The zinc potentiating site in the rat P2X2 receptor has been identified,578 which is formed by H120 and H213 from two adjacent subunits. Interestingly, these residues are located at the head and dorsal fin domains and therefore are positioned on the two “lips” of the open jaw in the presumably resting state of the receptor. Recently, we have shown that zinc binding at this site favors the conformational changes induced by ATP to facilitate channel opening.579 Since the zinc-binding site is in close proximity to the ATP site, it remains possible that a direct interaction between zinc and part of the ATP molecule may account for the potentiation of ATP response observed in the rat P2X2 receptor. In addition, the two histidines involved in the zinc potentiating site are also important for positive modulation of the rat P2X2 by Cu2+.580 Even more interestingly, it has been shown that the absence of potentiation seen in the human P2X2 receptor is due to the presence of an arginine residue (R225), which is located precisely at the position homologous to rat H213, that abolished the potentiation site.577 The second, low-affinity zinc inhibitory site in the rat and human P2X2 receptors remains to be determined but is unlikely related to the extracellular histidines.581 The physiological relevance of zinc modulation on P2X receptors has started to emerge, as it has been shown that zinc enhances LTP in the hippocampal CA1 region through the P2X receptors.582 Four Gd3+ binding sites were resolved in the crystal structure of the zebrafish P2X4 receptor.17 Three of them were located at the periphery of the receptor (one site for each subunit), whereas the fourth Gd3+ ion was located in the middle vestibule, on the noncrystallographic axis of the 3-fold symmetry, and was coordinated by the carboxylate groups of residues E98 from each of the three subunits (Figure 20).

Figure 20. Locations of the antagonist Gd3+ binding site and of the lateral fenestration in a slab mode view of the P2X4 crystal structure.

Functional studies have shown that Gd3+ antagonized the zebra fish P2X4 receptor function.17 Interestingly, residue E98 belongs to a loop that forms part of the backside of the ATP-binding site. This raises the possibility that close contacts located on the axis of symmetry are important for mediating the allosteric conformational changes that are initiated from the ATP-binding sites. In support of this, it has been shown that 6307


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7. CONCLUSION AND PERSPECTIVE In this review, we attempted as much as possible to review the molecular aspects of LGICs and the involvement of these receptors in major neurological disorders. Now it appears that the general principles of agonist, antagonist, and allosteric modulator recognition in LGICs are beginning to emerge. These invaluable data may thus serve as template to design original compounds that will be refined through medicinal chemistry into potent therapeutics with high degrees of subunit selectivity. However, the highly conserved nature of some binding site domains reduces the prospect that strong subtype selectivity can be achieved through conventional modifications of ligand structure. The recent identification of new binding sites located in less conserved portions of the receptors may thus represent an attractive solution of the subtype-selectivity issue. We expect that in the near future, more effective therapeutic agents will be designed to treat LGIC-related diseases.

Ruotian Jiang completed his undergraduate studies in biochemical engineering at the Sichuan University in Chengdu (China) and then completed in 2011 his Ph.D. thesis in molecular neurobiology in the laboratory of Thomas Grutter at the University of Strasbourg (Illkirch, France). His main topic focused on structure−function relationships of ATP-gated P2X receptors using electrophysiology and molecular biology. After a short stay (6 months) as a first postdoctoral research

AUTHOR INFORMATION

fellow in the Grutter laboratory, he joined the laboratory of Baljit

Corresponding Author

Khakh at University of California, Los Angeles (USA), for a second

*Mailing address: Laboratoire de Biophysicochimie des Récepteurs Canaux, UMR 7199 CNRS, Conception et Application de Molécules Bioactives, Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, 67400 Illkirch, France. Tel: 33-368-854-157. Fax: 33-368-854-306. E-mail: [email protected].

postdoctoral fellowship. He is now studying the active roles of astrocytes in neuronal networks with imaging methods.

Notes

The authors declare no competing financial interest. Biographies

Antoine Taly graduated from Orsay University (France) in 1999. He received a Ph.D. degree in experimental and computational biophysics from Orsay University and Heidelberg University (Germany) and joined in 2003 the laboratory of Jean-Pierre Changeux in Paris (France), where he modeled the structure and gating mechanism of the nicotinic receptor. From 2006 to 2009, Taly has been pursuing this Damien Lemoine completed his undergraduate studies in chemistry

project in the laboratory of Martin Karplus at the University of

and biology at the University of Strasbourg (France). Since October

Strasbourg (France). In 2009, he obtained a tenure research position

2010, he has been a Ph.D. student in the laboratory of Thomas Grutter

from the CNRS in the laboratory of Thomas Grutter. Currently, Taly

at the University of Strasbourg. His main topic focuses on structure−

is a CNRS researcher focusing on the modeling of ligand-gated ion

function relationships of ATP-gated P2X receptors by using electrophysiology and molecular biology and by developing, in

channels (nicotinic, GABAA, NMDA, and P2X receptors). He has a

collaboration with the group of Alexandre Specht, original photo-

general interest in the modeling of protein structure/function

chemical tools.

relationships including allostery. 6308


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Thomas Grutter studied chemistry and biology at the Louis Pasteur University in Strasbourg (France). He received in 2000 his Ph.D. in bioorganic chemistry in the laboratory of Maurice Goeldner at the Louis Pasteur University where he carried out photoaffinity labeling of nicotinic receptors. He then had a postdoctoral position at the Pasteur Institute (Paris, France) in the laboratory of Jean-Pierre Changeux where he studied structure−function relationships of pentameric ligand-gated ion channels by using molecular biology, biochemistry, and electrophysiology in collaboration with Lia Prado de Carvalho. He obtained a tenure research position from the CNRS in 2003 in the laboratory of Jean-Pierre Changeux and received his Habilitation in 2007. In 2008, he returned to Strasbourg to set up his own group at the faculty of Pharmacy (Illkirch, France) and got a directorship position at the CNRS in 2012. He is now heading a laboratory whose main interest is studying the structure and function of ATP-gated P2X receptors by employing different techniques from single-channel recordings to molecular biology and biochemistry and by developing original chemical biology tools.

Thierry Chataigneau received in 1996 his Ph.D. degree in physiology and membrane pharmacology at the Poitiers University (France). As a postdoctoral fellow, he has worked as pharmacologist in the laboratories of Rudi Busse in Frankfurt (Germany), Paul M. Vanhoutte, and Michel Félétou in Suresnes (Servier Research Institute, France). In 2003, he obtained a tenure position as Associate Professor in Pharmacology at the Faculty of Pharmacy at the Louis Pasteur University (Illkirch, France). He has worked on endothelial function and especially on EDHF, the endothelium-derived hyperpolarizing factor, for about 10 years. In 2012, he joined the laboratory of Thomas Grutter, and his main interest now deals with the molecular pharmacology and electrophysiology of ligand-gated ion channels.

ACKNOWLEDGMENTS We thank Prof. M. Goeldner and Dr. B. Foucaud for the valuable comments on the manuscript. We acknowledge funding from the Centre National de la Recherche Scientifique, the Ministère de la Recherche, the International Center for Frontier Research in Chemistry, and the Agence Nationale de la Recherche (Grants ANR JCJC 06-0050-01 and ANR 11 BSV5 001-01). REFERENCES Alexandre Specht studied chemistry and biochemistry at Louis Pasteur

(1) Bernard, C. Leçon sur les effets des substances toxiques et médicamenteuses; Baillière: Paris, France, 1857. (2) Langley, J. N. J. Physiol. 1905, 33, 374. (3) Langley, J. N. J. Physiol. 1907, 36, 347. (4) Elliott, T. R. J. Physiol. 1905, 32, 401. (5) Feldberg, W.; Fessard, A.; Nachmansohn, D. J. Physiol. 1940, 97, 3. (6) Lillie, R. S. Science 1909, 30, 245. (7) Del Castillo, J.; Katz, B. Proc. R. Soc. London, Ser. B 1957, 146, 369. (8) Changeux, J. P.; Kasai, M.; Lee, C. Y. Proc. Natl. Acad. Sci. U.S.A. 1970, 67, 1241. (9) Neher, E.; Sakmann, B. Nature 1976, 260, 799. (10) Noda, M.; Takahashi, H.; Tanabe, T.; Toyosato, M.; Furutani, Y.; Hirose, T.; Asai, M.; Inayama, S.; Miyata, T.; Numa, S. Nature 1982, 299, 793. (11) Noda, M.; Takahashi, H.; Tanabe, T.; Toyosato, M.; Kikyotani, S.; Furutani, Y.; Hirose, T.; Takashima, H.; Inayama, S.; Miyata, T.; Numa, S. Nature 1983, 302, 528.

University in Strasbourg (France). He received in 2003 his Ph.D. in bioorganic chemistry in the laboratory of Maurice Goeldner at the Louis Pasteur University. He was then awarded a postdoctoral fellowship from the Howard Hughes Medical Institute to work in the laboratory of Howard R. Kaback at the University of California, Los Angeles (USA). In 2004, he returned to Strasbourg and obtained a tenure research position from the French academic system (CNRS) in the laboratory of Maurice Goeldner, where he received his Habilitation in 2009. Since September 2011, he has been leading the bioorganic chemistry group at the Faculty of Pharmacy (Illkirch, France). His research topics deal with the synthesis and applications of chemical and photochemical tools to study the dynamics of neurobiological responses. 6309


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NOTE ADDED AFTER ASAP PUBLICATION Figure 19 was incorrect in the version published to the Web on September 18, 2012. This was corrected in the version published on October 23, 2012.

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