1521-0111/90/3/288–299$25.00 http://dx.doi.org/10.1124/mol.116.104240MOLECULAR PHARMACOLOGY Mol Pharmacol 90:288–299, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

MINIREVIEW—A LATIN AMERICAN PERSPECTIVE ON ION CHANNELS

Understanding the Bases of Function and Modulation of a7Nicotinic Receptors: Implications for Drug Discovery

Jeremías Corradi and Cecilia BouzatInstituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur, CONICET/UNS, Bahía Blanca,Argentina

Received March 7, 2016; accepted May 5, 2016

ABSTRACTThe nicotinic acetylcholine receptor (nAChR) belongs to asuperfamily of pentameric ligand-gated ion channels involvedin many physiologic and pathologic processes. Among nAChRs,receptors comprising the a7 subunit are unique because of theirhigh Ca21 permeability and fast desensitization. nAChR agonistselicit a transient ion flux response that is further sustained by therelease of calcium from intracellular sources. Owing to the dualionotropic/metabotropic nature of a7 receptors, signaling path-ways are activated. The a7 subunit is highly expressed in thenervous system, mostly in regions implicated in cognition andmemory and has therefore attracted attention as a novel drugtarget. Additionally, its dysfunction is associated with severalneuropsychiatric and neurologic disorders, such as schizophre-nia and Alzheimer’s disease. a7 is also expressed in non-neuronal

cells, particularly immune cells, where it plays a role in immunity,inflammation, and neuroprotection. Thus, a7 potentiation hasemerged as a therapeutic strategy for several neurologic andinflammatory disorders. With unique activation properties, thereceptor is a sensitive drug target carrying different potentialbinding sites for chemical modulators, particularly agonists andpositive allosteric modulators. Although macroscopic and single-channel recordings have provided significant information aboutthe underlying molecular mechanisms and binding sites of mod-ulatory compounds, we know just the tip of the iceberg. Furtherconcerted efforts are necessary to effectively exploit a7 as a drugtarget for each pathologic situation. In this article, we focusmainlyon the molecular basis of activation and drug modulation of a7,key pillars for rational drug design.

IntroductionNicotine has been a key molecule for the advancement of

pharmacology since the beginning of the 20th century, whenLangley (1905), through fundamental experiments, concludedthat muscle contraction was mediated by a “receptive sub-stance” present on the muscle. The muscle nicotinic acetylcho-line receptor (nAChR) was thus a pillar in the discovery ofneurotransmitter receptors (Langley, 1905). Still, it was not until1970 that the first neurotransmitter receptor, nAChR, wasidentified (Changeux et al., 1970;Miledi and Potter, 1971).Withthe later advent of themolecular biology revolution in the 1980s,the nAChR family was first identified and an extended family ofhomologous pentameric receptors was revealed (Patrick et al.,

1983; Le Novère and Changeux, 1995). This class of receptorswas first known as Cys-loop receptors because all family sub-units contain a conserved pair of disulfide-bonded cysteinesseparated by 13 residues. The discovery of orthologs in pro-karyotes (Tasneem et al., 2005), which lack the double cyste-ines, has extended the Cys-loop family to the superfamily ofpentameric ligand-gated ion channels (pLGIC).In vertebrates, the pLGIC superfamily includes cationic chan-

nels, nAChRs and serotonin 5-HT3 receptors, and anionic chan-nels activated by GABA or glycine (Le Novère and Changeux,2001; Lester et al., 2004; Sine andEngel, 2006;Bartos et al., 2009).Their vital role in converting chemical recognition into anelectrical impulse makes these receptors prime loci for learn-ing, memory, and disease processes, as well as targets for clini-cally relevant drugs.The nAChR is widely distributed throughout the animal

kingdom, from nematodes to humans (LeNovère and Changeux,1995). nAChRs are expressed in many regions of the central

This work was supported by grants from Universidad Nacional del Sur(UNS), Consejo Nacional de Investigaciones Científicas y Técnicas (CONI-CET), FONCYT, and the Bill and Melinda Gates Foundation to C.B.

dx.doi.org/10.1124/mol.116.104240.

ABBREVIATIONS: ACh, acetylcholine; a-BTX, a-bungarotoxin; ECD, extracellular domain; JAK, Janus kinase; LY-2087101, (2-amino-5-keto)thiazole), [2-[(4-fluorophenyl)amino]-4-methyl-5-thiazolyl]-3-thienyl-methanone; nAChR, nicotinic acetylcholine receptor; NAM, negative allostericmodulators; NS-1738, 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea; PAM, positive allosteric modulator; pLGIC, pentamericligand-gated ion channels; PNU-120596, 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; SAM, silent allosteric modulator;STAT, signal transducer and activator of transcription; TMD, transmembrane domain; TQS, 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide.

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and peripheral nervous system, in addition to non-neuronaltissues. The muscle nAChR plays a major role in neuromus-cular transmission and is the target of muscle relaxants (Sine,2012), whereas nAChRs in the brain represent a broad hetero-geneous family of ubiquitously expressed receptors. nAChRresponses to endogenous acetylcholine (Ach) and choline andexogenous nicotine are involved in a number of physiologicprocesses and pharmacological effects (Dani and Bertrand,2007; Albuquerque et al., 2009; Hurst et al., 2013).The homopentameric a7, one of the most abundant nAChRs

in the nervous system, is also expressed inmany non-neuronalcells. Its unique activation properties, high calcium perme-ability, ionotropic/metabotropic dual action, ubiquitous distri-bution, and involvement in a range of neurologic, psychiatric,and inflammatory disorders have made a7 an importantemerging drug target; the participation of a7 in pathologicconditions and the therapeutic potential of a7 ligands has beenwell documented (see for example Taly and Changeux, 2008;Wallace and Porter, 2011; Lendvai et al., 2013; Wallace andBertrand, 2013; Uteshev, 2014; Dineley et al., 2015). In thisarticle, we focus on the unique properties of activation and drugmodulation of a7 and its relationship with disease and therapy.

nAChR Structure and FunctionnAChR subunits are classified as two types, a and non-a,

with the a-type containing a disulfide bridge in the agonistbinding site. Five muscular (a1, b, g, «, and d) and elevenneuronal (a2–a7, a9, a10, and b2–b4) nAChR subunits havebeen identified in the mammalian genome (ligand-gatedion channel database, http://www.ebi.ac.uk/compneur-srv/LGICdb/cys-loop.php).nAChRs are assembled from five identical (a7 or a9) or

different subunits (at least two a-type subunits), and can formavariety of different heteromeric receptors with a broad spec-trum of pharmacological and kinetic properties (Fig. 1). Theresolution of the three-dimensional structures of pLGICs hasbeen the subject of intense efforts over the last decade (Brejcet al., 2001; Dellisanti et al., 2007; Hilf andDutzler, 2008, 2009;Bocquet et al., 2009; Hibbs and Gouaux, 2011; Corringer et al.,2012; Hassaine et al., 2014; Miller and Aricescu, 2014; Sauguetet al., 2014; Cecchini and Changeux, 2015). However, no high-resolution structure of the full length a7 has been reported todate; an extracellular domain of a7/AChBP chimera (Li et al.,2011; Nemecz and Taylor, 2011) and a nuclear magneticresonance (NMR) structure of a7 transmembrane domain havebeen described (Bondarenko et al., 2014).All pLGICs share a conserved organization with five

subunits symmetrically arranged around a central ion pore(Fig. 2). Functional domains include the extracellular domain(ECD), which carries the agonist binding sites at subunitinterfaces; the transmembrane domain (TMD), which con-tains the ion pore and the gate; and the intracellular domain(ICD), which contains determinants of channel conductanceand sites for regulation and intracellular signaling (Pauloet al., 2009; Jones et al., 2010; King et al., 2015) (Fig. 2). Theinterface between the ECD and TMD, also referred to as thecoupling region, is important for coupling agonist binding tochannel opening (Bouzat et al., 2004; Lee and Sine, 2005;Castillo et al., 2006; Bartos et al., 2009), as well as fordetermining open channel lifetime and rate of desensitization(Bouzat et al., 2008; Yan et al., 2015) (Fig. 2).

The possible structural events that translate neurotrans-mitter binding at the ECD into opening of the transmembraneion channel 60 Å away is an issue of intense research that hasbeen discussed in recent reviews (Corringer et al., 2012;Althoff et al., 2014; Sauguet et al., 2014; Cecchini and Changeux,2015). On the basis of the Monod-Wyman-Changeux model(Monod et al., 1965), the functional response of a pLGIC can beinterpreted as a selection from a few global and discreteconformations elicited by the binding of agonist: closed, open,and desensitized, the latter showing high agonist affinity at thesame time being impermeable to ions (Zhang et al., 2013) (Fig. 3).Intermediate states between closed and open or open anddesensitized states have been proposed for nAChRs and severalpLGICs (Lape et al., 2008; Corradi et al., 2009; Mukhtasimovaet al., 2009; Cecchini and Changeux, 2015). Therefore, thenumber of states in the main conformations and the rate oftransition between states determine receptor kinetics, and thistunes each receptor to its physiologic role. In turn, drugs, bybinding to different states, can differently modulate receptorfunction.

a7 in the Nervous System in Healthy and DiseaseStates

a7 is one of the most abundant nAChRs in the centralnervous system. It is particularly expressed in regions impli-cated in cognitive function andmemory, such as hippocampus,cortex, and several subcortical limbic regions (see Lendvai

Fig. 1. Models of pentameric arrangements of homomeric and someheteromeric nAChRs. a7 and a9 are the only a-type subunits capable offorming functional homomeric receptors, which contain five identicalbinding sites. In a7, occupancy of only one site is required for activation.Examples of possible combinations of a and non-a subunits in heteromericarrangements are shown. An a-type subunit is required for the principalface of the binding site. Themuscle nAChR contains two functional bindingsites at a/d and a/«(g) interfaces. Some subunits can assemble withdifferent stoichiometries, such as a4 and b2. In addition to the arrange-ment shown, (a4)3(b2)2 receptors are also functional.

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et al., 2013). It is also expressed on non-neuronal cells,including astrocytes, microglia, oligodendrocyte precursorcells, and endothelial cells, where it plays a role in immunity,inflammation, and neuroprotection (Shytle et al., 2004; Shenand Yakel, 2012; Dineley et al., 2015).In neurons,a7 receptors localize presynaptically onGABAer-

gic and glutamatergic terminals in the hippocampus and otherregions to facilitate release of neurotransmitters. Postsynapti-cally, a7 receptors mediate fast synaptic transmission, and inperisynaptic locations they modulate other inputs to neurons andactivate a variety of signaling pathways through volume trans-mission (Gotti and Clementi, 2004; Jones and Wonnacott, 2004;Dani and Bertrand, 2007; Dickinson et al., 2008; Albuquerqueet al., 2009; Sinkus et al., 2015) (Fig. 4).a7 contributes to cognitive functioning, sensory processing

information, attention, working memory, and reward path-ways, and a large body of evidence shows that enhancing a7activity improves attention, cognitive performance, and neu-ronal resistance to injury (reviewed in Uteshev, 2014).Significant reduction of a7 in the brain, particularly in thehippocampus, has been reported in Alzheimer disease (Guanet al., 2000; Kadir et al., 2006) and schizophrenic patients(Schaaf, 2014; Dineley et al., 2015). The a7 gene, CHRNA7 onchromosome 15, is genetically linked to multiple disorderswith cognitive deficits, including schizophrenia, intellectualdisability, bipolar disorder, autism spectrumdisorders, attention

deficit hyperactivity disorder, epilepsy, Alzheimer disease, andsensory processing deficit (Sinkus et al., 2009, 2015; Schaaf,2014; Dineley et al., 2015; Deutsch et al., 2016). A partial dupli-cation of CHRNA7 resulting in the chimeric gene CHRFAM7A,which is present only in humans, has been associated withschizophrenia (Sinkus et al., 2009; Neri et al., 2012). Its geneproduct, dupa7, lacks part of the binding site and may act as anegative modulator of a7 (Wang et al., 2014).Despite its homomeric character, a7 can assemble with

other subunits to form heteromeric receptors. In particular,a7b2 heteromeric receptors have been detected in several

Fig. 2. Structural model of pLGICs. The ECD is folded into ahighly conserved immunoglobulin-like b-sandwich. The agonistbinding site is found in a cavity at an interface between twoadjacent subunits (Sine, 2012). The principal or positive face isprovided by an a-type subunit and includes three loops thatspan b strands (named as Loops A–C) that harbor predomi-nantly aromatic residues essential for binding and gating. Theadjacent subunit, which forms the complementary or minusface, contributes with residues clustered in segments calledLoops D–F (Brejc et al., 2001; Sine, 2012). ACh docked into thebinding site is shown (a7/AChBP chimera, pdb code 3SQ6). Keyaromatic residues from the principal face are Tyr188, Trp149,and Tyr93, and from the minus face, Trp55. The transmem-brane domain (TMD) is composed of four transmembrane-spanning helices (TM1–TM4). The TM2 forms the walls of theion pore, which contains the gate (ring of leucines at 99 position)and determinants for selectivity. The outer ring of fifteena-helices (TM1, TM3, and TM4) shields the inner TM2 ringfrom the lipids (reviewed in Althoff et al., 2014). The interfacebetween the ECD and TMD, also named as coupling region,includes the conserved Cys-loop (b6b7 loop), b1b2 and b8b9loops from the ECD, and the M2–M3 linker from the TMD. Thelong intracellular region (ICD) between TM3 and TM4 is highlyvariable and particularly short in prokaryotic pLGICs. It isthought to be associated with cytoskeletal proteins and in-volved in channel modulation in all pLGICs (Kabbani et al.,2013). As shown in the figure, in a7, this region containsdeterminants of channel conductance (Andersen et al., 2011,2013) and mediates several intracellular signals (Paulo et al.,2009; Jones et al., 2010).

Fig. 3. Minimal model of nAChR activation. pLGICs have three mainclasses of conformational states: Closed (C), open (O), and desensitized (D).Intermediate states have also been detected.

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brain areas (Liu et al., 2009, 2012; Moretti et al., 2014;Thomsen et al., 2015; Zoli et al., 2015). Interestingly, a7b2are highly sensitive to blockade by Ab1–42, suggesting thatthey may play a unique role in the neuropathology of Alzheimerdisease (Liu et al., 2009). Additionally, these heteromericreceptors exhibit high sensitivity to volatile anesthetics, andtherefore could be targets for anesthetic action (Mowrey et al.,2013). Thus, a7b2 nAChR may represent a novel moleculartarget requiring selective a7b2 ligands.

Extraneuronal a7 and Its Pleiotropic Rolesa7 is present in various non-neuronal tissues, such as glia

(Sharma and Vijayaraghavan, 2001), blood cells (Kawashimaand Fujii, 2004; De Rosa et al., 2005; Báez-Pagán et al., 2015),keratinocytes (Maus et al., 1998), epithelial cells and fibro-blasts (Zia et al., 1997), endothelial cells (Macklin et al., 1998),cells of the digestive system and lung cells (reviewed inWessler and Kirkpatrick, 2008), spermatogonia, spermato-cytes, and seminiferous tubular and Sertoli cells (Schirmeret al., 2011). The functional role of a7 in these cells is beingintensively investigated and has been associated with differ-entiation, migration, adhesion, cell contact, apoptosis, andangiogenesis processes (Ni et al., 2010; Egea et al., 2015;Zdanowski et al., 2015).In particular, a7, present in all types of immune cells,

including lymphocytes (T and B cells), dendritic cells andmacrophages (reviewed in Egea et al., 2015), has attractedconsiderable attention as an important drug target for in-flammation. a7 is an important player in the “cholinergic anti-inflammatory pathway,”which is a link between vagal efferentfibers and innate immune system (Martelli et al., 2014). a7modulates intracellular signal pathways [Janus kinase 2(JAK2)/signal transducer and activator of transcription(STAT3) and PI3K/Akt] in immune cells, which results inpotent anti-inflammatory effects through cytokine productioninhibition and overexpression of heme oxygenase 1 (Báez-Pagán et al., 2015; Egea et al., 2015) (Fig. 5). A brain cholinergicpathway also exists that regulates microglial activationthrough a7 (Shytle et al., 2004). This pathway is crucial forneuroprotection (Park et al., 2007) and is probably importantin Parkinson disease (Stuckenholz et al., 2013), oxygen andglucose deprivation (Parada et al., 2013), and global ischemia(Guan et al., 2015).

Therefore, a7 nAChR is emerging as an important drugtarget for the modulation of inflammation in different path-ologic contexts, including sepsis, ischemia/reperfusion, rheu-matoid arthritis, and pancreatitis.

a7 Pharmacology and Ion SelectivityHallmark features of a7 receptors include high Ca21

permeability, relatively low sensitivity to ACh, full activationby choline, high-affinity for a-bungarotoxin (a-BTX), relative-ly low affinity for nicotine, and fast desensitization that occurson the submillisecond time scale.Dose-response curves show EC50 values of ∼100–200 mM for

ACh [Hill coefficient (nH) ∼1] (Andersen et al., 2013), 0.4–1.6mM for choline, and 18–91mM for nicotine (Wonnacott andBarik, 2007). Several a7-specific agonists have been synthe-tized, including PNU-282987 (EC50 128 nM), AR-R17779(EC50 ∼10–20 mM), compound A (EC50 ∼14 nM to 0.95 mM),and partial agonists GTS-21 (EC50 ∼6–26mM), and SSR180711(EC50 ∼1–4 mM). Selective competitive antagonists are a-BTX(IC50 ∼1–100 nM), which has been widely used to detect a7in tissues, and methyllycaconitine (MLA, IC50 10–200 nM)(Wonnacott and Barik, 2007).a7 allows flux of Na1 and K1 and is highly permeable to

Ca21. The PCa21/PNa1 ratio is ∼10–20, which is greater thanthat of other nAChRs and similar to N-methyl-D-aspartatereceptors (Séguéla et al., 1993; Albuquerque et al., 1997). Thehigh Ca21 permeability underlies most of a7 functions: facil-itation of neurotransmitter release, depolarization of post-synaptic cells, and initiation ofmany cellular processes throughits action as a second messenger (Gotti and Clementi, 2004).The transient increase in intracellular Ca21 is converted into asustained, wide-ranging phenomenon by calcium release fromintracellular stores through a calcium-induced calcium releasemechanism, a process involving IP3 and ryanodine receptors(Tsuneki et al., 2000; Dajas-Bailador et al., 2002; Guerra-Álvarez et al., 2015) (Fig. 5).The concept of a7 as a dual metabotropic/ionotropic receptor

is attracting increasing attention (Kabbani et al., 2013). a7binds both Ga and Gbg proteins through the M3–M4 loop andenables a downstream calcium signaling response that canpersist beyond the expected time course of channel activation(Fig. 5) (Kabbani et al., 2013; King et al., 2015). Moreover,in lymphocyte T cells, mobilization of Ca21 through the a7channel is not necessarily required for the nicotine-inducedrelease of Ca21 from the internal stores (Razani-Boroujerdiet al., 2007). Channel-independent signal transduction hasbeen related to the role of a7 in inflammation (de Jonge andUlloa, 2007; Thomsen and Mikkelsen, 2012) and in neuritegrowth (Nordman and Kabbani, 2012; Kabbani et al., 2013).a7 is not only permeable to Ca21 but is also modulated by

the ion; Ca21 has been shown to regulate agonist efficacy andcooperativity (Bonfante-Cabarcas et al., 1996; Albuquerqueet al., 1997). As in other pLGICs (Zimmermann et al., 2012),the divalent modulatory sites may be located at the ECD.

a7 Channel KineticsHeterologous expression of a7 in oocytes and mammalian

cells combined with electrophysiological experiments hasprovided information about the receptor’s unique activationproperties. The surface expression of recombinant a7 requires

Fig. 4. Neurotransmission mediated by a7 in the mammalian brain. a7receptors can be postsynaptic, presynaptic (with a role in regulation ofneurotransmitter release), or perisynaptic when they are involved involume transmission.

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coexpression of the chaperone RIC-3 (Williams et al., 2005), atransmembrane protein that is required for efficient receptorfolding, assembly, and functional expression (Castillo et al.,2005; Millar, 2008). Recently, another chaperone, NACHO,has been identified. NACHO is a transmembrane protein ofneuronal endoplasmic reticulum that mediates assembly ofa7 by promoting protein folding, maturation through theGolgicomplex, and expression at the cell surface (Gu et al., 2016).Typical a7 receptor-mediated currents decay rapidly in the

presence of agonist as a result of desensitization (Fig. 6A).Given the fast kinetics, the temporal resolution of agonistexchange and most recording systems limit the accurateestimation of the true desensitization rate (Zhou et al., 1998;Lovinger et al., 2002; Bouzat et al., 2008), which may partiallyaccount for the great variability of desensitization rates foundin the literature. Outside-out patches rapidly perfused withACh, which allows for a more accurate determination of thedesensitization rate, show current decay time constants of∼0.4 milliseconds (Bouzat et al., 2008). Owing to their fastdecay, a7 peak responses occur in advance of complete solu-tion exchange. Therefore, more accurate EC50 values areobtained if the net charge, which represents the time in-tegration of all channel activation, rather than the peakcurrent, is used for the analysis of the concentration-effectrelationship (Papke and Porter Papke, 2002) (Fig. 6A).At the single-channel level, channel activity appears as

isolated brief pulses (0.1–0.3 milliseconds) flanked by longclosed periods and, less often, as two or three brief pulses in

quick succession (bursts) (Fig. 6B). Thus, a7 has a very lowopen probability. Single-channel openings exhibit a broaddistribution of current amplitudes, probably owing to limitedtime resolution of the brief openings, with amaximum of∼10 pAat –70mV (Mike et al., 2000; Bouzat et al., 2008; Andersen et al.,2013; daCosta and Sine, 2013; Yan et al., 2015).Although there is no general consensus for an a7 kinetic

model, an interesting aspect is that the temporal pattern ofsingle ACh-activated currents is similar at 10 mM or 1 mMACh (Bouzat et al., 2008) (Fig. 6B). This lack of concentration-dependence combined with the fact that most receptor acti-vation episodes consist of a single opening with a durationsimilar to the desensitization time constant suggests thatdesensitization is the predominant pathway for channelclosing, a unique feature among nAChRs. Control of open-channel lifetime through desensitization has potential conse-quences for inter-response latency at a synapse where theneurotransmitter pulse is transient. Recovery from desensiti-zation depends on agonist concentration and exposure dura-tion, since different desensitized states may exist. Desensitizeda7 receptors expressed in human embryonic kidney cellsrecover with a time constant of ∼1 second (Bouzat et al.,2008), whereas 15–30 seconds are required for full recovery inthe hippocampus (Frazier et al., 1998). Thus, after a7 briefresponse, a latency of several seconds is required to generateanother response of full amplitude. The fast desensitizationand brief open duration may avoid cell toxicity caused byincreased intracellular Ca21 owing to a7 overstimulation.

Fig. 5. Dual ionotropic/metabotropic nature of a7: Intracellular pathway signaling mediated by a7 activation. a7 allows transient flux of Na+, K+, andCa2+. The transient increase of calcium may also lead to a sustained calcium response by a calcium-induced calcium release (CICR) mechanism throughIP3 receptors. As ametabotropic receptor, a7mediates intracellular signals by binding to Ga andGbg proteins and several other partners (Kabbani et al.,2013; King et al., 2015). In immune cells, a7 has been shown to be involved in several intracellular pathways; although not yet fully decipheredmechanistically, these pathways lead to potent anti-inflammatory effects. For example, a7 has been shown to activate JAK2/STAT3 in some immunecells, which leads to blockade of nuclear factor (NF)-kB nuclear translocation and inhibition of NF-kB, resulting in inhibition of proinflammatory cytokineproduction. Also, a7 has been shown to activate the PI3K/Akt pathway that promotes Nrf-2 translocation to the nucleus and overexpression of hemeoxygenase 1 (HO-1), resulting in potent anti-inflammatory effects (see de Jonge and Ulloa, 2007; Báez-Pagán et al., 2015; Egea et al., 2015). DAG,diacylglycerol; GPCR, G protein-coupled receptor; PKC, protein kinase C; ROS, reactive oxygen species.

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Another intriguing aspect of a7 activation is the relation-ship between ACh occupancy and activation. We set out todetermine howmany of the five identical agonist binding sitesare required to activate a7 by developing a strategy thatutilizes coexpression of an inactivated binding-site subunitand a reporter amplitude subunit. This allows for the de-termination of the number of ACh-occupied sites from theamplitude of each individual single-channel opening (Rayeset al., 2009; Andersen et al., 2011, 2013). The results revealedthat ACh occupancy of only one of five a7 binding sites isnecessary for activation, and that open-channel lifetime ofa single-occupied receptor is indistinguishable from that ofreceptors containing five intact binding sites (Andersen et al.,2013). Also, occupancy of a single site by the antagonistmethylcaconitine (Palma et al., 1996) or a-BTX (daCostaet al., 2015) will inhibit a7 function.Whole-cell experiments have revealed the physiologic con-

sequence of having more sites than those required foractivation; saturation of receptors in cells expressing a wild-type and a binding-site mutant subunit (a7Y188T) is achievedat higher ACh concentrations when the proportion of recep-tors with fewer functional binding sites increases (Andersenet al., 2013; Williams et al., 2011a). This suggests that a7 isa highly sensitive sensor of ACh and is therefore adapted tofunction with submaximal occupancy of its sites, a propertyappropriate for volume transmission.The unique activation properties of a7 also suggest that any

slight change in the energy barriers between active, closed,and/or desensitized statesmay have a deep impact on receptorfunction, which makes these receptors very sensitive drugtargets.

a7 Modulation as a Therapeutic StrategyIn addition to agonists and antagonists that bind to the

orthosteric site, a large number of compounds modulate a7function by binding to allosteric sites. These compounds mayact as: 1) positive allosteric modulators (PAMs) that potenti-ate currents only in the presence of the agonist; 2) allostericagonists that activate receptors from nonorthosteric sites; 3)negative allosteric modulators (NAMs), which either act asopen-channel blockers by binding to the pore or inhibitactivation allosterically; and 4) silent allosteric modulators(SAMs) that have no effect on orthosteric agonist responsesbut block allosteric modulation (Fig. 7).Since stimulation of a7 improves attention, cognitive per-

formance, and neuronal resistance to injury in addition toeliciting robust analgesic and anti-inflammatory effects, a7potentiation has emerged as a potential therapeutic strategy,and the search for novel potentiators is an active researchfield. The potential therapeutic use of several a7 partialagonists and PAMs on animals and humans has been docu-mented in several recent reviews (e.g., Wallace and Porter,2011; Thomsen and Mikkelsen, 2012; Lendvai et al., 2013;Dineley et al., 2015) (Table 1). Still, no drug has reached phaseIII clinical stage.Compared with agonists, PAMs are promising therapeutic

tools because they: 1) better maintain the temporal spatialcharacteristics of endogenous activation; 2) show higherselectivity, since the orthosteric site is more conserved amongnAChRs than allosteric sites (Yang et al., 2012); 3) allowgreater diversity in structure and final effects; 4) reducetolerance attributable to a7 desensitization; and 5) act asneuronal protectors (Kalappa et al., 2013; Sun et al., 2013;Uteshev, 2014). In particular, because neuronal damageelevates choline levels near the site of injury, the presence ofa PAM may not require coapplication of an agonist for themediation of a local neuroprotective effect (Uteshev, 2014).Therefore, it is increasingly accepted that targeting allostericsites can provide novel medications with greater structuraldiversity and specificity.PAMs have been classified on the basis of their macroscopic

effects as type I or type II (Fig. 8). Type I PAMs mainlyenhance agonist-induced peak currents without significantlyaffecting current decay and do not reactivate desensitizedreceptors, whereas type II PAMs delay desensitization andreactivate desensitized receptors (Bertrand and Gopalakrishnan,2007; Arias and Bouzat, 2010; Williams et al., 2011b). Theratio of the changes in net charge/peak current induced bytype I PAMs is close to one, whereas it is higher than one inthe presence of type II PAMs (Andersen et al., 2016; Williamset al., 2011c).Single-channel recordings provide an invaluable tool for un-

derstanding the foundations of these macroscopic effects. Inthe presence of either type I or type II PAMs, ACh-activated a7channels show prolonged open durations and appear in longeractivation episodes, revealing that both PAM types affectactivation kinetics (Andersen et al., 2016) (Fig. 8). The mostefficacious PAM to date is PNU-120596, a type II PAM (Hurstet al., 2005). This compound elicits significantly prolongedopenings that appear grouped in bursts, which in turn coalesceinto long activation periods of several seconds, referred to asclusters (daCosta et al., 2011, 2015; Williams et al., 2011b;Pałczy�nska et al., 2012; Andersen et al., 2016). However, the

Fig. 6. a7 single-channel andmacroscopic currents. (A) Typical whole-cellcurrents elicited by different ACh concentrations (from 10 to 1000 mM)from cells transfected with human a7. At left, a schematic representationof amacroscopic current with themeasured parameters. Both themaximalcurrent (peak) and the net charge (net) can be used to construct dose-response curves as shown in the figure. (B) Single-channel activity fromcell-attached patches of cells expressing human a7 appears as very brief(∼0.1–0.3 milliseconds) and isolated openings or less often as short bursts.Channel openings are shown as upward deflections. Membrane potential,–70 mV; filter, 9 kHz. At right, a typical open duration histogram (Bouzatet al., 2008).

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Open-channel blockers

Fig. 7. Allosteric modulatory sites in a7. Structural model of the a7 receptor viewed from the side representing several potential sites for allostericmodulators. At the TMD, the intrasubunit transmembrane cavity is a site for a great variety of compounds that may elicit different pharmacologicaleffects (potentiation (PAMs), inhibition (NAMs), no effect (SAMs), or activation (allosteric agonists)). PNU-120596 docked into this cavity is shown. Open-channel blockers inhibit response by transiently blocking the flux of ions through the pore. At the ECD, different potential sites for inhibitors andpotentiators have been proposed (Ludwig et al., 2010; Spurny et al., 2015).

TABLE 1a7 modulators with potential clinical applications.

Drug Potential Therapeutic Effects and Uses References

Agonists GTS-21/DMXB-A Cognitive disorders, schizophrenia, Alzheimerdisease, attention deficit hyperactivity disorder

Olincy et al. (2006)

Pain van Westerloo et al. (2006)Inflammatory processes Vukelic et al. (2013)

Terry et al. (2015)AR-R17779 Cognitive disorders Levin et al. (1999)

Inflammatory processes Hashimoto et al. (2014)Atherosclerotic vascular diseases Van Kampen et al. (2004)

PNU-282987 Cognitive disorders Hajós et al. (2005)Inflammatory processes Terry et al. (2015)

SSR180711 Cognitive disorders Pichat et al. (2007)Terry et al. (2015)

ABBF Cognitive disorders Boess et al. (2007)Terry et al. (2015)

EVP-6124 Cognitive disorders Prickaerts et al. (2012)Terry et al. (2015)

TC-5619 Cognitive disorders Hauser et al. (2009)Terry et al. (2015)

RG3487 Cognitive and sensorimotor gating disorders Wallace et al. (2011)Terry et al. (2015)

PHA-568487 Cognitive disorders Karamihalev et al. (2014)Terry et al. (2015)

CP-810123 Cognitive disorders O’Donnell et al. (2010)Terry et al. (2015)

AZD0328 Cognitive disorders Sydserff et al. (2009)Terry et al. (2015)

Tropisetron Cognitive disorders Shiina et al. (2010)Hashimoto, (2015)Terry et al. (2015)

ABT-107 Cognitive disorders Bitner et al. (2010)Neuroprotection Quik et al. (2015)

Terry et al. (2015)JN403 Cognitive disorders Feuerbach et al. (2007, (2009)

AnticonvulsivePain

Type I PAMs Genistein Neuroprotection Menze et al. (2015, 2016)Memory disorders Terry et al. (2015)

NS-1738 Cognitive disorders Timmermann et al. (2007)Terry et al. (2015)

AVL-3288 Cognitive disorders Ng et al. (2007)Terry et al. (2015)

Galantamine Cognitive disorders Nikiforuk et al. (2015)Alzheimer disease Terry et al. (2015)

Type II PAMs PNU-120596 Cognitive disorders Nikiforuk et al. (2015)Inflammatory processes Callahan et al. (2013)

Terry et al. (2015)PAM-2 Cognitive disorders Targowska-Duda et al. (2016)

Pain Bagdas et al. (2015)Inflammatory processes

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single-channel profile in the presence of a weaker type II PAM,PAM-2 (3-furan-2-yl-N-p-tolyl-acrylamide) (Arias et al., 2011),more closely resembles that of type I 5-HI or NS-1738 PAMsthan that of type II PNU-120596 (Fig. 8). Thus, when analyzedat the molecular level, potentiation is more complex thaninitially believed. Moreover, PAMs showing macroscopic in-termediate type I/II properties were proposed (Dunlop et al.,2009; Malysz et al., 2009; Dinklo et al., 2011; Sahdeo et al.,2014; Chatzidaki and Millar, 2015). Therefore, the classifica-tion of type I and type II appears to be an oversimplificationresulting mainly from macroscopic observations, and a morethorough classification may be required.It is a generally accepted statement that type II PAMs may

increase the energetic barrier for desensitization, which allowssuccessive opening/closing events (daCosta et al., 2011) and/orreversal of some forms of agonist-induced desensitization(Williams et al., 2011b). Also, it has been proposed that PNU-120596 binds predominantly to a fast desensitized state andinduces a set of conformations in which the opening of the poreis energeticallymore favorable (Szabo et al., 2014). On the otherhand, only the decrease in the energetic barrier for opening hasbeen proposed as the underlying mechanism for enhancementof peak currents by type I PAMs (Williams et al., 2011b; Hurst

et al., 2013). Such a decrease might explain the appearance ofbursts of openings owing to rapid reopening of the closedchannel. However, it would only explain the increase in open-channel duration if reopening were so fast that the associatedbrief closings could not be detected, thus making openings ap-pear longer. Alternatively, the increase in open duration couldbe the result of either slight changes in desensitization that arenot detectable from whole-cell macroscopic currents or to theinduction of different open states. Thus, there seems to be morethan one mechanism by which PAMs prolong open-channellifetime and activation episodes.a7 PAM potentiation is particularly dependent on temper-

ature. For PNU-120596, such dependence is revealed bydecreased potentiated macroscopic currents (Dunlop et al.,2009; Williams et al., 2012) and the absence of long clusters(Andersen et al., 2016) at physiologic temperature comparedwith room temperature. Thus, for better extrapolation to thein vivo situation, in vitro studies should be carried out atphysiologic temperatures.

a7 Allosteric Binding SitesComputational studies (Dey andChen, 2011), electrophysio-

logical studies from mutant or chimeric receptors (Bertrandet al., 2008; Young et al., 2008; Collins et al., 2011; daCosta andSine, 2013), crystallographic studies, (Spurny et al., 2015), andNMR studies (Bondarenko et al., 2014) have suggested theexistence of several allosteric binding sites, some of which arecommon to other pLGICs (Sauguet et al., 2014).The Intrasubunit Transmembrane Cavity. In silico

and electrophysiological studies show that several PAMs, in-cluding LY-2087101, PNU-120596, and TQS bind to an intra-subunit transmembrane cavity (Young et al., 2008) (Fig. 7). Ana7 receptor with mutations at five residues lining this cavitywas not potentiated by PNU-120596, PAM-2, or type I PAMNS-1738, indicating common structural determinants fortheir potentiation (daCosta et al., 2011; Andersen et al., 2016).Themacrocyclic lactone ivermectin (a type I PAM) also appearsto bind in close proximity to this intrasubunit site (Collins andMillar, 2010), although it binds at an intersubunit transmem-brane site in the glutamate-activated Cys-loop receptor (Hibbsand Gouaux, 2011).This cavity is also the site for allosteric agonists that me-

diate a7 activation in the absence of an orthosteric agonist(Gill et al., 2011, 2012, 2013; Pałczy�nska et al., 2012). Single-channel activity of a7 in the presence of allosteric agonistsresembles that of the receptor in the presence of ACh and aPAM. Both are characterized by long bursts instead of isolatedbrief ACh-elicited openings, indicating activation with signif-icantly reduced desensitization (Pałczy�nska et al., 2012).Moreover, a7-selective allosteric modulators showing subtle

structural changes and displaying distinct pharmacologicaleffects (typical of type I PAMs, type II PAMs, NAMs, SAMs,and allosteric agonists) may bind to this common site (Gillet al., 2013; Gill-Thind et al., 2015). Thus, the intrasubunit trans-membrane cavity appears to be a promiscuous binding sitewith a strategic location for allosteric modulation. Further-more, this cavity may be a commonmodulatory site within thepLGIC superfamily from which a large variety of differentcompounds that bind to and mediate a great spectrum ofallosteric effects (Corradi et al., 2011; Nury et al., 2011;Jayakar et al., 2013; Sauguet et al., 2014).

Fig. 8. Macroscopic and single-channel current profiles of human a7 inthe presence of typical type I and type II PAMs. Macroscopic currentprofiles have been used to classify PAMs into type II (i.e., PNU-120596 andPAM-2), which increase the decay time constant, or type I, which onlyincrease the peak current (5-HI and NS-1738). Left: Macroscopic currentselicited by ACh in the absence (black) and presence of the specified PAM(gray traces). Right: Traces of 50–100 mM ACh-activated single a7channels in the absence or in the presence of 1 mM PNU-120596, 5 mMPAM-2, 2 mM 5-HI, 10 mM NS-1738. Membrane potential: –70 mV.Channel openings are shown as upward deflections. All PAMs enhanceopen channel lifetime and elicit activation episodes formed by successiveopening events.

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Extracellular Allosteric Binding Sites. The putativebinding site of galantamine, an a7 potentiator, is located atthe outer surface of the ECD in the vicinity of the ACh site(Hansen and Taylor, 2007; Ludwig et al., 2010). Threedifferent allosteric sites in the ECD of the a7/AChBP chimerawere also identified by X-ray crystallography (Spurny et al.,2015; Fig. 7). Although all the allosteric binders behaved onhuman a7 as negative allosteric modulators, it was proposedthat their chemical modification could lead to a change infunctional activity.Binding to potential ECD sites has been proposed for the

type I PAMs 5-HI and NS-1738, although other reportssupport a TMD location (Placzek et al., 2004; Bertrand et al.,2008; Hu and Lovinger, 2008; Gronlien et al., 2010; Collinset al., 2011; Andersen et al., 2016). A virtual screening re-vealed that some PAMs that bind to the TMD, such as PNU-120596 and TQS, also dock into potential allosteric sites atthe ECD (Dey and Chen, 2011). Therefore, for any given PAM,multiple binding sites or domains may be involved in theconformational changes associated with potentiation, whichcould account for these controversial results. Also, until thelocation of an allosteric binding site is unequivocally defined,it is advisable to refer to structural determinants of potentia-tion instead of a binding site.Despite the large body of experimental evidence supporting

a7 potentiation as a promising therapeutic strategy, there arestill many unsolved challenges: 1) Potentiation by exogenousagonists may inhibit a7 response owing to desensitization.Thus, PAMs might have therapeutic benefits in situationswhere stronger agonist responses are desirable. 2) Given thepresence of other nAChRs and homologous receptors, highPAM selectivity is required. However, PAMs targeting multi-ple receptors might show better efficacy (Iturriaga-Vásquezet al., 2015; Möller-Acuña et al., 2015). 3) Excessive receptoractivation, particularly with efficacious nondesensitizingPAMs, might lead to cytotoxicity, which is an issue of concernand controversy (Ng et al., 2007; Liu et al., 2009; Williamset al., 2012; Guerra-Álvarez et al., 2015; Uteshev, 2016). 4)The ubiquitous distribution of a7 and its interplay withdifferent signal pathways could make the cell response to agiven PAM variable among cell types or conditions. Given thebroad spectrum of effects andmolecularmechanisms of PAMs,it is probable that each patient or pathologic situation couldrequire a unique PAM.

Concluding Remarksa7 has emerged as an important drug target for improving

cognition and memory in several neuropsychiatric disorders,and as a target for inflammatory processes. a7 is unique owingto its high calcium permeability and fast desensitization, andit behaves as a ACh-sensitive sensor harboring a built-infiltering mechanism against excessive stimulation. Transientcalcium responses are further sustained by the release ofcalcium from intracellular sources, and several signalingpathways are also activated because a7 has a dual ionotropic/metabotropic nature. Its ubiquitous location and pleiotropiceffects make a7 an interesting but complex drug target. A bet-ter understanding of the molecular basis underlying allostericmodulation and its wide spectrum of effects, as well as theavailability of high resolution structures of a7, will help in therational design of therapeutics for the receptor.

Acknowledgments

ASPET thanks Dr. Katie Strong for copyediting of this article.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Bouzat,Corradi.

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Address correspondence to: Dr. Cecilia Bouzat, Instituto de InvestigacionesBioquímicas de Bahía Blanca, CONICET/Universidad Nacional del Sur,Camino La Carrindanga Km 7, Bahía Blanca, Argentina. E-mail: inbouzat@criba.edu.ar

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