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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 Mar 20;175(11):1805–1821. doi: 10.1111/bph.13745

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors

Jingyi Wang 1, Jon Lindstrom 2,✉
PMCID: PMC5980197  PMID: 28199738

Abstract

Heteromeric nicotinic ACh receptors (nAChRs) were thought to have two orthodox agonist‐binding sites at two α/β subunit interfaces. Highly selective ligands are hard to develop by targeting orthodox agonist sites because of high sequence similarity of this binding pocket among different subunits. Recently, unorthodox ACh‐binding sites have been discovered at some α/α and β/α subunit interfaces, such as α4/α4, α5/α4 and β3/α4. Targeting unorthodox sites may yield subtype‐selective ligands, such as those for (α4β2)2α5, (α4β2)2β3 and (α6β2)2β3 nAChRs. The unorthodox sites have unique pharmacology. Agonist binding at one unorthodox site is not sufficient to activate nAChRs, but it increases activation from the orthodox sites. NS9283, a selective agonist for the unorthodox α4/α4 site, was initially thought to be a positive allosteric modulator (PAM). NS9283 activates nAChRs with three engineered α4/α4 sites. PAMs, on the other hand, act at allosteric sites where ACh cannot bind. Known PAM sites include the ACh‐homologous non‐canonical site (e.g. morantel at β/α), the C‐terminus (e.g. Br‐PBTC and 17β‐estradiol), a transmembrane domain (e.g. LY2087101) or extracellular and transmembrane domain interfaces (e.g. NS206). Some of these PAMs, such as Br‐PBTC and 17β‐estradiol, require only one subunit to potentiate activation of nAChRs. In this review, we will discuss differences between activation from orthosteric and allosteric sites, their selective ligands and clinical implications. These studies have advanced understanding of the structure, assembly and pharmacology of heteromeric neuronal nAChRs.

Linked Articles

This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc

Abbreviations

ABT‐594

tebanicline

AChBP

ACh‐binding protein

bPiDI

N,N‐decane‐1,10‐diyl‐bis‐3‐picolinium diiodide

Br‐PBTC

(R)‐7‐bromo‐N‐(piperidin‐3‐yl)benzo[b]thiophene‐2‐carboxamide

dFBr

desformylflustrabromine

LY2087101

[2‐[(4‐fluorophenyl)amino]‐4‐methyl‐5‐thiazolyl]‐3‐thienylmethanone

NAM

negative allosteric modulator

NS206

3‐N‐benzyloxy‐3‐hydroxyimino‐2‐oxo‐6,7,8,9‐tetrahydro‐1H‐benzo[g]indole‐5‐sulfonamide

NS9283

3‐(3‐(pyridine‐3‐yl)‐1,2,4‐oxadiazol‐5‐yl)benzonitrile

OCD

obsessive–compulsive disorder

PAM

positive allosteric modulator

PNU‐120596

N‐(5‐chloro‐2,4‐dimethoxyphenyl)‐N′‐(5‐methyl‐3‐isoxazolyl)‐urea

SCAM

scanning cysteine accessibility mutagenesis

TC‐2559

4‐(5‐ethoxy‐3‐pyridinyl)‐N‐methyl‐(3E)‐3‐buten‐1‐amine

Tables of Links

TARGETS
Ligand‐gated ion channels http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=467&familyId=76&familyType=IC
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=463&familyId=76&familyType=IC http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=468&familyId=76&familyType=IC
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=464&familyId=76&familyType=IC http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=472&familyId=76&familyType=IC
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=465&familyId=76&familyType=IC http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=473&familyId=76&familyType=IC
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=466&familyId=76&familyType=IC http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=474&familyId=76&familyType=IC
LIGANDS
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=294 http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3987
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2585 http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3993
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5348 http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1013
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5347 http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6693
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4006 http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3992
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4005
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These Tables list key protein targets and ligands in this article that are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Introduction

Vertebrate nicotinic ACh receptors (nAChRs) are pentameric ligand‐gated ion channels assembled from homologous subunits – the α1–10, β1–β5, γ, δ and ε subunits (Hurst et al., 2013). The α1, β1, γ, δ and ε subunits form fetal and adult muscle‐type nAChRs. Other subunits form neuronal subtypes such as heteromeric α4β2 and homomeric α7 nAChRs. Agonists bind at subunit interfaces such as α7/α7 and α4/β2. α7 nAChRs have five ACh‐binding sites. Heteromeric nAChRs were thought to have two ACh‐binding sites at α/β subunit interfaces. All nAChRs evolved from a homomeric ancestor (Le Novere et al., 2002), and all of their subunits may retain the potential for activation by a suitable agonist. Recently, a third ACh site was identified at the α4/α4 subunit interface in (α4β2)2α4 nAChRs (Harpsoe et al., 2011; Mazzaferro et al., 2011). Subsequently, additional ACh sites were found at α5/α4, β3/α4 and α4/α5 subunit interfaces (Jin et al., 2014; Wang et al., 2015c; Jain et al., 2016). These sites are referred to as unorthodox sites.

Unorthodox sites substantially changed our understanding of the ligand selectivity, activation mechanism and assembly of heteromeric nAChRs. It was known for more than a decade that (α4β2)2α4 and (α4β2)2β2 stoichiometries differed in agonist sensitivity and rate of desensitization (Nelson et al., 2003). Now, we know that this difference results from an unorthodox α4/α4 ACh site unique in (α4β2)2α4 nAChRs. One orthodox site is sufficient, but not as efficacious as two, to activate nAChRs. Occupying two to three sites is most efficacious to activate α7, but higher occupancy promotes desensitization (Rayes et al., 2009). The additional α4/α4 site increases activation from α4/β2 sites by fivefold (Harpsoe et al., 2011; Wang et al., 2015c) and accelerates desensitization of (α4β2)2α4 (Benallegue et al., 2013). This site also explains partial agonism of sazetidine‐A that activates at α4/β2 but cannot bind to the α4/α4 interface (Eaton et al., 2014; Mazzaferro et al., 2014).

3‐(3‐(pyridine‐3‐yl)‐1,2,4‐oxadiazol‐5‐yl)benzonitrile (NS9283) acts at the unorthodox α4/α4 agonist site with no effect at orthodox α4/β2 sites (Timmermann et al., 2012; Olsen et al., 2013; Wang et al., 2015c). NS9283 alone cannot activate, thus was initially misidentified as a positive allosteric modulator (PAM) (Lee et al., 2011; Rode et al., 2012; Timmermann et al., 2012). Engineering in three NS9283 sites allowed it to activate the nAChR (Marotta et al., 2014; Olsen et al., 2014). As NS9283 is neither allosteric nor modulatory, it is not a PAM. It is an agonist selective for the α4/α4 ACh site.

Non‐canonical sites such as β2/α4, β2/α3 and β2/β2 sites lost ability to bind ACh in vertebrates during evolution. However, ligands acting at these sites are agonists for lower species. For example, morantel is an agonist for nematode nAChRs while, for rat nAChRs, it is a PAM, acting at the β2/α3 site (Wu et al., 2008).

There is interest in developing nAChR PAMs because of the success of benzodiazepine drugs, which are allosteric ligands for GABAA receptors. Some nAChR PAMs act at sites equivalent to benzodiazepine sites, that is, non‐canonical sites at the β/* subunit interfaces mentioned above (Sigel and Buhr, 1997; Weltzin et al., 2014; Weltzin and Schulte, 2015). Other PAMs act at extracellular (Seo et al., 2009; Weltzin and Schulte, 2015), or transmembrane domains (Young et al., 2008), their interface (Olsen et al., 2013), or at the C‐terminus (Paradiso et al., 2001; Wang et al., 2015a; Alcania et al., 2017). In this review, we will discuss activation from orthodox, unorthodox and allosteric sites, their selective ligands and clinical implications. We mainly focus on heteromeric nAChRs because allosteric modulation of homomeric nAChRs has been reviewed extensively elsewhere (Williams et al., 2011c; Pandya and Yakel, 2013; Chatzidaki and Millar, 2015).

Assembly of nAChRs

Historically, assembly of heteromeric neuronal nAChRs was predicted from the Torpedo muscle nAChR structure (Bon et al., 1984; Brisson and Unwin, 1984; Brisson and Unwin, 1985). Two pairs of α and β subunits form primary agonist sites at their interface, together with an accessory subunit taking the fifth position (Figure 1A). The primary α subunit can be α2, α3, α4 or α6 and the primary β subunit can be β2 or β4. The fifth accessory subunit can be either an α or β subunit that occupies the primary site, or a β3 or α5 subunit. All α subunits harbour two adjacent cysteines in loop C that are critical for activation of nAChRs by ACh (Figure 2). The subunit interface providing loop C is also called the plus face. In this review, we use α/β to describe the interface where the α subunit provides the plus and the β subunit provides the minus face.

Figure 1.

Figure 1

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Schematic illustration of assembly of heteromeric nAChRs. (A) Empirical assembly of heteromeric neuronal nAChRs. Each nAChR contains two α/β pairs and a fifth accessory subunit (illustrated as black circle). ‘+’ indicates subunits that donate the C‐loop at the subunit interface. Subunit interfaces that form ACh‐binding sites are noted. α5 and β3 subunits form an ACh‐binding site with α4 subunit (Jain et al., 2016), and it is to be determined whether they do so with other α subunits. (B) Illustration of different stoichiometric forms of (α4)2(β2)2α5 expressed from subunit concatamers (Jain et al., 2016). In the ACh‐binding site formed at the interface with α4, α5 can act either as an α subunit when expressing β2‐(AGS)6‐α4 with free α5 subunit (left) or as a β subunit when expressing α4‐(AGS)6‐β2 with free α5 (right) (Jain et al., 2016).

Figure 2.

Figure 2

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Illustration of orthosteric and allosteric sites in heteromeric nAChRs. (A) 3‐D structure of desensitized (α4β2)2β2 nAChRs (pdb.5KXI) (Morales‐Perez et al., 2016). The last five residues of α4 subunit were not resolved in this crystal structure, probably reflecting the mobility of the C‐tail (this mobility might be greatly reduced in the presence of PAMs that interact with the tail) (Morales‐Perez et al., 2016). Zoomed‐in views of the (B) orthosteric and (C) allosteric sites of α4 subunit. (D) Sequence alignments of mature human nAChR subunits using CLUSTAWL‐Q 1.2.2. Nicotine is shown in magenta in sticks. The five aromatic residues that stabilize the charged nitrogen of agonists are shown in sticks in (B) and highlighted in tan or grey in (D). Cysteines forming the Cys‐loop or loop C are highlighted in yellow in (D). H116, which is critical for ligand selectivity of the α4/α4 site is also illustrated in (B). Residues that constitute binding sites for PAMs NS206 (Olsen et al., 2013), Br‐PBTC (Wang et al., 2015a), oestrogen (Paradiso et al., 2001) and LY2087101 (Young et al., 2008) are shown in red orange as sticks (A) or spheres (C). Two sites have been suggested for dFBr: one at the non‐canonical site as illustrated by residues coloured in cyan (Weltzin and Schulte, 2015); the other at the transmembrane domain as illustrated by residues coloured in cornflower blue (Alcania et al., 2017). The non‐canonical PAM sites are ACh equivalent sites at the β/α interface that do not bind ACh. If the β2 subunit on the left of α4 subunit shown in the centre is replaced with α4, this site will become an α4/α4 ACh unorthodox site. Residues constituting the channel gates are coloured in green.

It is challenging to study the pharmacology of heteromeric nAChR subtypes containing more than two kinds of subunits by expressing free subunits, because this results in a mixture of subtypes. Studying the pharmacology of individual subtypes is made possible by expressing subunit concatamers. Linked subunits define both subunit composition and order (Zhou et al., 2003; Kuryatov and Lindstrom, 2011; Kuryatov et al., 2011; Wang et al., 2015a) (Figure 1B). This method has been used to understand the pharmacology of complex nAChRs (Kuryatov and Lindstrom, 2011; Jin et al., 2014; Mazzaferro et al., 2014, 2017) as well as to identify agonist‐binding sites at subunit interfaces (Harpsoe et al., 2011; Mazzaferro et al., 2011, 2014; Wang et al., 2015c; Jain et al., 2016). Although the two β/α subunit interfaces cannot form ACh sites, the accessory subunit can form an ACh site when it is α2, α3, α4, α5, α6 or β3 (Figure 1A).

Until recently, β3 and α5 subunits were thought to assemble only in the accessory position because they did not form functional nAChRs when expressed in pairs with α or β subunits. Because α5 has a shorter C‐loop than the other α subunits and β3 lacks the two critical adjacent cysteines in the C‐loop (Figure 2D), these two subunits were predicted to be unable to form an ACh site. However, Jin et al. used concatamers to show that α5 could also assemble in the primary site as a β subunit (Jin et al., 2014). We found that α5 and β3 act as α or β subunits to form functional ACh‐binding sites with an α4 subunit (Jain et al., 2016) (Figure 1B). These data suggest that α5 and β3 subunits can function and assemble in nAChRs like other α and β subunits. Concatamers were used in these studies to force α5 and β3 to assemble in specific positions. It remains to be determined how these subunits assemble in neurons. Methods such as immunoprecipitation elucidate subunit composition (Gotti et al., 2007; Zoli et al., 2015) not stoichiometry or order. NS9283 enabled the detection of (α4β2)2α4 nAChRs in vivo (Grupe et al., 2015). Additional site‐selective ligands targeting other unorthodox sites such as α5/α4, α4/α5 and β3/α6 could provide more details of assembly of heteromeric neuronal nAChRs.

Structure of nAChRs

To understand the different ligand sites and their mechanisms of action, we will briefly review the structure of neuronal heteromeric nAChRs. High‐resolution interactions between agonists and nAChRs have been predicted by crystal structures of agonists bound to ACh‐binding proteins (AChBP). However, homology between AChBP and nAChR extracellular domains is less than 30%, and AChBPs lack transmembrane domains.

Recently, structures of nAChRs and related receptors with both extracellular and transmembrane domains have been determined in resting, open or closed conformations. These have been discussed extensively elsewhere (Changeux and Christopoulos, 2016; Morales‐Perez et al., 2016; Nemecz et al., 2016). Overall, Cys‐loop receptors including nAChRs undergo stepwise global conformational change including un‐blooming and twist of the extracellular domain and tilting of transmembrane domains to open the channel pore (Gupta et al., 2017). Similar global conformational changes are induced by neurotransmitters binding at subunit interfaces in the extracellular domain and allosteric agonists such as ivermectin binding at the top of the transmembrane domain (Hibbs and Gouaux, 2011; Althoff et al., 2014; Du et al., 2015). Several sequences at the interface of extracellular and intracellular domains are important for this signal transduction and may contribute to the similar conformational change by ligands binding at different sites. These sequences include the M2‐M3 loop, β1‐β2, C‐terminus and Cys‐loops from the plus face (Figure 1C) and the β8‐β9 and β10‐M1 loops in the minus face (Figure 1A). PAMs bind in these regions, which will be discussed later. Electron microscope structures of nAChRs obtained from spray‐freeze‐trapping of Torpedo nAChRs with and without ACh suggest that the resting/open channel gate is located at residues 9′ and 13′ in the middle of the M2 helix (Unwin and Fujiyoshi, 2012) (Figure 1A, C). This is similar to the resting/open channel gate in other Cys‐loop receptors (Nemecz et al., 2016). Binding of agonists eventually leads to desensitization in which the desensitization gate at the −1′ residue of M2 closes while the activation gate in the middle of the channel remains open (Miller and Aricescu, 2014; Morales‐Perez et al., 2016) (Figure 1A, C).

Orthosteric agonists and antagonists contact highly conserved amino acids in the ACh‐binding site (Figure 1D). The smallest agonist to activate nAChRs is tetramethyl ammonium (Horenstein et al., 2008). Its charged nitrogen is stabilized by the C‐loop and an aromatic box (four aromatic residues from the plus side and one aromatic residue from the minus side) (Figure 1B), which has been confirmed by co‐crystallization of various agonists with AChBP, extracellular domain of pentameric α7‐AChBP chimera (Li et al., 2011), extracellular domain of pentameric α2 (Kouvatsos et al., 2016) and (α4)2(β2)3 nAChRs with truncated intracellular domains (Morales‐Perez et al., 2016). The crystal structure of (α4)2(β2)3 nAChRs is the first high‐resolution heteromeric structure in the Cys‐loop family (Morales‐Perez et al., 2016). It reveals that, besides lacking the double cysteine in loop C, the conserved residues orient differently when β2 constitutes the plus face than when α4 does. This may explain why α4/β2 forms an ACh site while β2/β2 and β2/α4 do not. Some PAMs have been suggested to bind at non‐canonical sites at β2/β2 and β2/α4 interfaces (Weltzin et al., 2014; Weltzin and Schulte, 2015). Functional studies suggest that α2 and α4 can form an ACh site with another α2 or α4. This is confirmed by the crystal structure of pentameric α2 with epibatidine bound (Kouvatsos et al., 2016). A conserved histidine 116 in α4 and α2 in loop E is in close proximity to the 2‐chloro‐pyridinyl ring of epibatidine. This histidine is critical for binding of both orthodox and unorthodox agonists at α/α sites (Mazzaferro et al., 2014; Olsen et al., 2014), which will be discussed later.

Activation from orthodox and unorthodox orthosteric sites

Unorthodox ACh sites have been confirmed at α4/α4, α2/α4, α3/α4, α5/α4, α4/α5, α6/α4, β3/α4 and α2/α2 sites (Harpsoe et al., 2011; Mazzaferro et al., 2011; Wang et al., 2015c; Jain et al., 2016; Kouvatsos et al., 2016). All known unorthodox ACh sites promote activation from the orthodox sites (Harpsoe et al., 2011; Wang et al., 2015c; Jain et al., 2016) but differ in sensitivity to ACh (Table 1). The α4/α4 site has 100‐fold lower sensitivity than α4/β2 sites and increases activation from the two α4/β2 sites by fivefold (Harpsoe et al., 2011; Wang et al., 2015c). (α3β4)2α3 and (α6β2)2α6 nAChRs appear less sensitive to agonists than their two α stoichiometric forms (Krashia et al., 2010; Henderson et al., 2016). This results from the low sensitivity of the additional ACh site at the α3/α3 or α6/α6 subunit interface that potentiates their response to high ACh concentrations. They retain high sensitivity low amplitude responses from their two high affinity ACh sites (Harpsoe et al., 2011; Eaton et al., 2014; Wang et al., 2015c). As β3 has high sequence similarity to α5, it may be that β3 can act like a β2 or β4 subunit to form an ACh site at the α4/β3 interface. β2 and β4 are unable to act as a primary α subunit to form an unorthodox ACh site (Wang et al., 2015c; Jain et al., 2016; Morales‐Perez et al., 2016).

Table 1.

Sensitivity to ACh of known orthodox and unorthodox sites. Potencies are collected from papers using voltage‐clamp in heterologous expression systems

nAChR subtype Orthodox site Unorthodox site Reference
EC50 (μM) EC50 (μM)
α2* α2/β2 1–5 α2/α2 26, 40, 99–130 (Khiroug et al., 2004; Dash and Li, 2014)
α2/β4 10
α3* α3/β2 66a α3/α3 310 (Krashia et al., 2010; Cesa et al., 2012)
α3/β4 138
α4* α4/β2 1.0–3.2 α4/α4 83–153 (Nelson et al., 2003; Zhou et al., 2003; Harpsoe et al., 2011; Mazzaferro et al., 2011; Wang et al., 2015c) (Blum et al., 2013; Jin et al., 2014; Jain et al., 2016)
α4/β4 15 α2/α4 120
α3/α4 101
α6/α4 109
α5/α4 22.6
α4/α5 1.2,9
β3/α4 1.4
α6* α6/β2 0.17–1.3 α6/α6 ~36 (Gerzanich et al., 1997; Kuryatov et al., 2000; Dash et al., 2011; Kuryatov and Lindstrom, 2011; Ley et al., 2014; Henderson et al., 2016)
α6/β4 18–38a
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a

The potencies of α3/β2 and α6/β4 sites were estimated from studies expressing α and β at 1:1 ratio because we did not find any study that reported agonist potency of different stoichiometric forms

Unorthodox sites synergize with orthodox sites to promote activation. To evaluate the contribution of unorthodox sites to channel activation, we engineered a cysteine into an agonist site (Wang et al., 2015c; Jain et al., 2016). Application of a thio‐reactive agent prevents ACh activation from that site (Papke et al., 2011; Wang et al., 2015c). α2/α4 and α6/α4 sites contribute to more than 64% of the total activation of (α4β2)2* AChRs, slightly more efficacious than the α4/α4 site, which contributes to more than 53% of total activation (Wang et al., 2015c). α3/α4, α5/α4 and β3/α4 sites are less efficacious than α4/α4 sites and contribute to 23–41% of total activation (Wang et al., 2015c; Jain et al., 2016).

Because orthodox and unorthodox sites have evolved from ACh sites of their homomeric ancestor, they probably share similar paths to opening the activation gate and closing the desensitization gate. As with orthodox sites (Papke, 2006; Rayes et al., 2009), higher occupancy of unorthodox sites increases channel open probability and desensitization rate (Wang et al., 2015c). One orthodox site is sufficient to activate heteromeric and homomeric nAChRs (Rayes et al., 2009; Williams et al., 2011a; Jain et al., 2016), two and three sites are more efficacious, but four or five sites promote desensitization (Rayes et al., 2009). Therefore, (α4β2)2α4 nAChRs exhibit higher maximum absolute current but desensitize faster than (α4β2)2β2 (Tapia et al., 2007; Harpsoe et al., 2011; Wang et al., 2015c; Mazzaferro et al., 2017). Incorporation of β3 and α5 also increases the desensitization rate of α4β2* nAChRs (Kuryatov et al., 2008).

The very low affinity interaction of ACh at α4/α4 sites is insufficient to trigger a global conformation change to the activated state unless one or two orthodox sites are activated (Wang et al., 2015c). The α4/α4 ACh site site‐selective agonist NS9283 (Timmermann et al., 2012; Wang et al., 2015c) cannot activate nAChRs with one or two α4/α4 sites, but can when three α4/α4‐like ACh sites are present due to mutating three amino acids in β2 subunits (illustrated as * in Figure 2D) (Marotta et al., 2014; Olsen et al., 2014). Current evidence suggests that three unorthodox sites are required to activate nAChRs, but there may be agonists that are efficacious enough to activate from a single unorthodox site.

Why have unorthodox sites evolved? These sites increase the range and amplitude of sensitivity to ACh of nAChRs subtypes (Table 1). Unorthodox sites can exhibit different sensitivity to ACh from that of orthodox sites (Table 1). nAChRs with only high‐sensitivity orthodox sites such as (α4β2)2β2 may be activated through volume transmission. nAChRs with low‐sensitivity unorthodox sites such as (α4β2)2α4 may function best post‐synaptically where they are exposed to higher ACh concentrations. (α4β2)2α4 can be activated by low concentrations of ACh just like (α4β2)2β2 (Harpsoe et al., 2011; Mazzaferro et al., 2011; Wang et al., 2015b). They may also exist pre‐ or extra‐synaptically and be modestly activated by volume transmission. (α4β2)2α4 and (α4β2)2α5 nAChRs have high permeability to calcium, which increases their efficacy at promoting presynaptic transmitter release (Tapia et al., 2007; Kuryatov et al., 2011). Their fast desensitization may limit the net calcium influx thus avoiding cytotoxicity. Various nAChR subtypes are expressed in various parts of the brain (Gotti et al., 2007; Zoli et al., 2015), which contribute to the addictive and aversive effects of nicotine as well as its related psychiatric disorders (Hurst et al., 2013; Picciotto and Kenny, 2013; Picciotto et al., 2015; Wang et al., 2015b). The physiological significance of the alternative stoichiometries remains to be determined.

Modulation from allosteric sites

ACh‐binding sites only account for a small fraction of drug‐interaction sites present in nAChRs (Figure 1A). There are many potential allosteric sites to target (Williams et al., 2011c; Wang et al., 2015a). Allosteric ligands can activate, inhibit or modulate activation of nAChRs by ACh (Williams et al., 2011c; Hurst et al., 2013; Chatzidaki and Millar, 2015; Grupe et al., 2015). We will mainly focus on PAMs and only briefly discuss negative allosteric modulators (NAMs).

Three types of potentiation from PAM sites have been identified in nAChRs. Type I PAMs increase the peak response of agonists without altering desensitization (Gronlien et al., 2007). In addition to increasing peak responses, type II PAMs slow desensitization and reactivate desensitized nAChRs (Gronlien et al., 2007; Williams et al., 2011c). The third type of PAMs are allosteric agonists, which not only increase agonist activation but also activate nAChRs directly without agonists (Gill et al., 2011).

Both types I and II PAMs have been discovered for heteromeric nAChRs. Type I PAMs such as [2‐[(4‐fluorophenyl)amino]‐4‐methyl‐5‐thiazolyl]‐3‐thienylmethanone (LY2087101) bind in an intrasubunit vestibule in the transmembrane domain near the channel gate (Young et al., 2008). α7 type II PAM N‐(5‐chloro‐2,4‐dimethoxyphenyl)‐N′‐(5‐methyl‐3‐isoxazolyl)‐urea (PNU‐120596) acts at a similar site. Known heteromeric nAChR type II PAMs influence desensitization from distant sites. (R)‐7‐bromo‐N‐(piperidin‐3‐yl)benzo[b]thiophene‐2‐carboxamide (Br‐PBTC) acts from the extracellular C‐terminus of α2 or α4 in association with nearby parts of the α subunit (Wang et al., 2015a). Desformylflustrabromine (dFBr) was initially reported to act at the extracellular domain (Weltzin and Schulte, 2010; Hamouda et al., 2015; Weltzin and Schulte, 2015), but has recently been reported to act in the transmembrane domain near the C‐tail of α4 near where Br‐PBTC and estradiol act (Alcania et al., 2017). Type II PAMs for heteromeric nAChRs that act directly at or close to the desensitization gate may be found. Other heteromeric nAChR PAMs also act from the C‐terminus or non‐canonical sites (Paradiso et al., 2001; Seo et al., 2009; Weltzin et al., 2014) or at the interface of extracellular and transmembrane domains (Olsen et al., 2013), but their pharmacology has not been categorized.

Occupying one PAM site is sometimes sufficient to potentiate activation of heteromeric nAChRs. For example, oestrogen and Br‐PBTC significantly increase activation of chimeric α4β2 with only one free α4 C‐terminus, and their efficacy escalates with increasing numbers of PAM sites (Jin et al., 2014; Wang et al., 2015a). HEPES, a β2/β2 ACh homologues site PAM, augments activation of (α4β2)2β2 nAChRs (Weltzin et al., 2014). Some homomeric PAMs such as PNU‐120596 require at least four PAM sites (daCosta and Sine, 2013). The unique property of C‐terminus PAMs such as oestrogen and Br‐PBTC makes them promising lead compounds for targeting α5* nAChRs, which often incorporate one α5 subunit per nAChR.

To the best of our knowledge, no allosteric agonists have been found for heteromeric nAChRs, while several α7‐selective allosteric agonists have been identified (Gill et al., 2011; Gill‐Thind et al., 2015; Horenstein et al., 2016). One reason could be that there are not as many known heteromeric nAChR PAMs as for α7 PAMs. Another reason is that in homomeric nAChRs, there would be five sites at which an allosteric agonist could act. Various analogues of LY2087101, dFBr and Br‐PBTC have been developed (Broad et al., 2006; German et al., 2011; Kamenecka et al., 2016), but none of them showed agonist activity. Additional novel scaffolds of PAMs may lead to allosteric agonists for heteromeric nAChRs. PAMs acting through influencing the opening of the activation gate and destabilizing the desensitization gate may activate heteromeric nAChRs when there are sufficient PAM sites. Other PAMs may act only through destabilizing the desensitization gate. Such PAMs may not be able to open the activation gate, so cannot act as agonists. Allosteric agonists may even arise from known PAMs of other receptors because subtle effects from identical or homologous sites can produce potentiation or outright activation. For example, ivermectin is a PAM for α7 nAChRs (Krause et al., 1998; Collins and Millar, 2010; Tillman et al., 2014) and some GABAA receptors (Sigel and Baur, 1987; Krusek and Zemkova, 1994), but it allosterically activates glycine receptors (Du et al., 2015), an invertebrate glutamate‐gated chloride channel (Hibbs and Gouaux, 2011) and GABAA receptors having an γ2L/β2 interface (Estrada‐Mondragon and Lynch, 2015).

Some ligands act at the same site as PAMs but inhibit activation of nAChRs by agonists. These ligands are NAMs. The antihelminthic drug oxantel is a PAM for the β2/α3 non‐canonical site but a NAM for the β2/α4 non‐canonical site (Cesa et al., 2012). Other inhibitory allosteric sites have been identified in α7 nAChRs at the extracellular domain including one close to the N‐terminus, another in an intrasubunit vestibule, a third one in a pocket below the orthosteric site (Spurny et al., 2015) and a fourth one at the transmembrane domain (Gill‐Thind et al., 2015). These NAM sites may also exist in heteromeric nAChRs.

Channel blockers are noncompetitive antagonists but not NAMs. They block the channel pore, rather than modulate channel gating like NAMs. All sorts of charged ligands, including ACh and PAMs, can act as channel blockers at sufficient concentrations (Weltzin and Schulte, 2010). Therefore, most channel blockers are non‐selective, such as the well‐known non‐competitive antagonist mecamylamine (Papke et al., 2013; Bondarenko et al., 2014). Recently, an α6‐subtype selective channel blocker, N,N‐decane‐1,10‐diyl‐bis‐3‐picolinium diiodide (bPiDI), has been developed (Wooters et al., 2011). This indicates that the channel pore can be targeted to develop subtype‐selective antagonists.

Small molecules with different site selectivity

Prior to discovery of unorthodox sites, agonist selectivity was categorized by subunits. This may misrepresent pharmacology of some site‐selective agonists. For example, sazetidine‐A fully activates (α4β2)2β2 nAChRs but cannot achieve the same fivefold increase in response of (α4β2)2α4 as seen when activated by ACh. This results from sazetidine‐A not being able to activate the α4/α4 site because it cannot bind to it (Mazzaferro et al., 2014). Ligands with known site selectivity are categorized in Tables 2 and 3.

Table 2.

Summary of selectivity and site of action of orthosteric nAChR ligands

Ligand Site‐selectivity Pharmacology Reference
Active Inactive
Orthosteric site non‐selective
ACh α(2,3,4,6)/β(2,4)
α(2,3,4,5,6)/α4
β3/α4, α4/α5
α6/α6,α3/α3
α7/α7		 β(2,4)/α4
β(2,4)/β2
β2/α5
β2/β3		 agonist (Krashia et al., 2010; Harpsoe et al., 2011; Mazzaferro et al., 2011; Jin et al., 2014; Wang et al., 2015c; Henderson et al., 2016; Jain et al., 2016)
Epibatidine α(2,3,4,6)/β(2,4)
α4/α4		 α7/α7 agonist (Ahring et al., 2015)
Cytisine α(2,3,4,6)/β(2,4)
α4/α4
α7/α7		 partial or full agonist (Campling et al., 2013; Wang et al., 2015c)
DHβE α4/β(2,4), α4/α4, α6/β2 α3/β(2,4) antagonist (Mazzaferro et al., 2011; Xiao et al., 2011)
MLA α4/α4, α7/α7 α(2,3,4,6)/β(2,4) antagonist (Absalom et al., 2013)
Orthodox site site‐selective
Sazetidine‐A α(2,3,4,6)/β(2,4)
α7/α7		 α4/α4, α5/α4 agonist (Campling et al., 2013; Mazzaferro et al., 2014; Jain et al., 2016)
TC‐2559 α(2,3,4)/β(2,4) α4/α4 agonist (Mazzaferro et al., 2014)
Unorthodox site site‐selective
NS9283 α(2,3,4,6)/α4
α2/α2		 β(2,4)/α(3–4)
β(2,4)/β(2,4)
α3/α3, α3/β2, α3/β4
α5/α4, β3/α4, α4/α5
α6/β2, β3/α6		 agonist (Timmermann et al., 2012; Wang et al., 2015c; Jain et al., 2016)
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Table 3.

Summary of selectivity and site of action of allosteric nAChR ligands

Ligand Site‐selectivity Pharmacology EC50 (μM) Reference
Active Inactive
17β‐estradiol α4 PAM 10 (Paradiso et al., 2001)
Br‐PBTC α2, α4 α3, α7, β2, β4 PAM 0.162 (LS), 0.274 (HS), 0.446, 0.660 (Wang et al., 2015a)
dFBr β2/α4, β2/β2, α4
α7/α7		 β(2,4)/α3
α3/α3, α3/β2, α3/β4		 PAM for α4β2; Antagonist for α7 0.398 (LS), 2.5 (HS), 0.331, 1.33; 44 (α7) (Weltzin and Schulte, 2010; Hamouda et al., 2015; Weltzin and Schulte, 2015; Alcania et al., 2017)
Galantamine β(2,4)/α(3,4,6)
β(2,4)/ β(2,4)
α7/α7		 PAM ~0.1 (Samochocki et al., 2003; Hamouda et al., 2013)
HEPES β2/β2 α4/α4 PAM 7.1 (Weltzin et al., 2014)
LY2087101 α2, α4, α7 α3, β2, β4 PAM ~2–5 (Broad et al., 2006; Young et al., 2008)
Morantel β2/α3 β2/α4 PAM N.A. (Seo et al., 2009; Cesa et al., 2012)
NS206 α4 α3 PAM 2.2 (LS), 4.2 (HS) (Olsen et al., 2013)
Oxantel β2/α3, β2/α4 PAM for α3β2; NAM α4β2 N.A. (α3β2); ~3 (α4β2) (Cesa et al., 2012)
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Some ligand active sites are predicted from mutagenesis studies of other nAChRs subtypes such as Torpedo nAChRs. These sites are underlined. Some potencies of PAMs are not published. N.A. indicates that no dose‐response curve is available. Some dose‐response curves are reported but not fitted to calculate EC50; we estimated and displayed their approximate values as ‘~’. Some PAMs are evaluated on different stoichiometries of α4β2 nAChRs. ‘LS’ refers to EC50 for (α4β2)2α4. ‘HS’ refers to EC50 for (α4β2)2β2.

Radioligand competition assays are often used to evaluate ligand‐binding sites. Binding of nAChR radioligands to unorthodox sites is not well studied. Epibatidine binding at the α4/α4 site is 500 fold less potent than the α4/β2 site (Ahring et al., 2015). It is not feasible to interpret unorthodox site selectivity from competition assays in which low concentrations of radioligand were used to target orthodox sites. In the future, development of high‐affinity radioligands for unorthodox sites may facilitate identification and localization of AChRs with these sites. Crystallography is also good for predicting ligand binding. However, high‐resolution crystal structures of neuronal heteromeric nAChRs were not available until last year (Kouvatsos et al., 2016; Morales‐Perez et al., 2016). This will greatly help elucidate nAChR ligand‐binding sites in the future.

Some interpretations of ligand selectivity are based on crystal structures of ligand‐receptor complexes, but most are based on functional, mutagenesis, photolabeling assays, scanning cysteine accessibility mutagenesis (SCAM) and computational modelling. All of these methods have limitations. Potency and efficacy change of ligands from mutating residues of nAChRs and SCAM may reflect either direct disruption of ligand‐binding sites or indirect disturbance of ligand‐induced conformational changes. Photolabeling reveals both specific and non‐specific binding sites. Docking is also used to predict ligand binding in nAChRs, whose structures are homology modelled from AChBP, Torpedo nAChRs or other Cys‐loop receptors. Results of docking depend on the homology model used, and docking does not provide functional information.

PAM site analyses from functional studies also depend on the concentrations of agonists. Because activities of PAMs are not detectable alone, they are usually indirectly measured by how PAMs change activity of agonists at a submaximal concentration. Low concentrations of agonists may not give sufficient signal to detect PAM effects reliably. At higher concentrations of agonists, PAM effects are usually lower. In addition, it is unknown whether occupancy of agonist sites changes the binding site or modulation effect of PAMs. These make it hard to compare studies of PAMs reported from different groups. For example, two publications using different concentrations of ACh suggested binding of dFBr at different places (Figure 2A). Mutagenesis studies using an EC75 concentration of ACh suggested that dFBr binds at extracellular β2/β2 and β2/α4 sites (Weltzin and Schulte, 2015). These sites are also supported by photoaffinity labelling of Torpedo nAChRs (Hamouda et al., 2015). Another study using mutagenesis, SCAM and docking indicates that dFBr binds at the interface between the extracellular and transmembrane domains when testing its PAM effect at an EC10 concentration of ACh (Alcania et al., 2017). These suggest that the agonist occupancy may affect where PAMs bind or act to potentiate receptor function. There are other methodological differences in these contradictory studies, but we cannot attribute those to their different conclusions. Weltzin and Schulte (2015) found that the β2 Y102A (Y94 in Figure 2D) mutation eliminated the PAM effect of dFBr on (α4β2)2β2 and (α4β2)2α4. Alcania et al. (2017) used a mixture of the two stoichiometries and reported ineffectiveness of this mutant on the efficacy and potency of dFBr.

Orthosteric ligands differ in site selectivity (Figure 3 and Table 3). SCAM experiments suggest that the α4/β2 site selectivity of sazetidine‐A and 4‐(5‐ethoxy‐3‐pyridinyl)‐N‐methyl‐(3E)‐3‐buten‐1‐amine (TC‐2559) results from prevention of binding due to histidine 116 on the minus side of α4 at the α4/α4 orthodox site (Mazzaferro et al., 2014). This is consistent with the different efficacy of sazetidine‐A on (α4β2)2β2 and (α4β2)2α4. Similarly, sazetidine‐A cannot bind to α5/α4 or β3/α4 unorthodox sites (Jain et al., 2016). Epibatidine and nicotine efficiently activate both stoichiometries of α4β2 nAChRs (Moroni et al., 2006; Carbone et al., 2009), indicating that they bind at both orthodox and unorthodox sites. Most known nAChR agonists and antagonists are like epibatidine and nicotine (Figure 3). To evaluate site selectivity, we designed mutants to enable selective block of the desired ACh site (Wang et al., 2010; Papke et al., 2011; Wang et al., 2015c). We have shown that blocking the α4/α4 ACh site inhibits potentiation by NS9283 (Wang et al., 2015c), which suggests that NS9283 binds at the α4/α4 ACh site. This is consistent with mutagenesis (Olsen et al., 2013), crystallography data with AChBP (Olsen et al., 2014) and photolabeling data of an analogue of NS9283 (Hamouda et al., 2016). Similarly, using a cysteine‐reactive derivative of methyllycaconitine suggests that this antagonist binds at α4/α4 and α7/α7 sites (Absalom et al., 2013).

Figure 3.

Figure 3

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Chemical structures of orthosteric ligands. Nitrogen that is more than 50% positively charged at pH = 7 is noted.

Some PAMs bind at sites evolved from ACh sites in homomeric AChRs such as β/* subunit interfaces (Figures 1 and 3, Table 3). HEPES potentiates activation of (α4β2)2β2 via the β2/β2 subunit interface at 0.001–1 mM concentrations (Weltzin et al., 2014). Galantamine, an ACh esterase inhibitor, also potentiates α4β2, α3β4 and α6β4 nAChRs at 0.1–1 μM concentrations (Samochocki et al., 2003). Photoaffinity labelling suggests that galantamine binds at three sites on Torpedo nAChRs including two non‐canonical sites, one equivalent to the β/α site and the other one at the β/β site similar to the benzodiazepine site in GABAA receptors (Hamouda et al., 2013). The antihelminthic cholinergic agonist morantel enhances channel gating through β2/α3 non‐canonical sites (Wu et al., 2008; Seo et al., 2009). dFBr selectively potentiates α4β2 nAChRs (Hamouda et al., 2015; Weltzin and Schulte, 2015). At concentrations more than 10 μM, dFBr inhibits activation as a channel blocker (Weltzin and Schulte, 2010). It also inhibits α7 and muscle type nAChRs (Kim et al., 2007; German et al., 2011; Hamouda et al., 2015).

Structural modification of non‐canonical site PAMs alters their site selectivity and pharmacology. Reduction of the olefinic side chain of dFBr retained its PAM effect and increased selectivity of dFBr for α4β2 nAChRs versus α7 (German et al., 2011). Oxantel, an analogue of morantel, noncompetitively inhibits α4β2 nAChRs through a β2/α4 site (Cesa et al., 2012). Therefore, there is promise for developing subtype‐selective allosteric modulators by targeting non‐canonical sites.

Other known heteromeric PAMs act at the extracellular C‐terminus, transmembrane domain or the interface of the two domains (Figure 1). 17β‐estradiol, dFBr and Br‐PBTC all act close to the C‐terminus at the extracellular end of the fourth transmembrane domain of α2 and α4 subunits (Paradiso et al., 2001; Wang et al., 2015a; Alcania et al., 2017). However, they bind differently because Br‐PBTC can potentiate α4* nAChRs with a constrained C‐tail while 17β‐estradiol cannot (Zhou et al., 2003; Wang et al., 2015a). Docking of dFBr suggests that it binds at the intrasubunit vestibule of α4 close to the extracellular domain (Alcania et al., 2017). Our unpublished studies suggest that Br‐PBTC binds near this site. Br‐PBTC is slightly more potent than dFBr (Table 3) (Weltzin and Schulte, 2015; Wang et al., 2015a; Alcania et al., 2017). The C‐terminus is critical for the formation of this site. Engineering the last six amino acids of α4 into β2 or α3 subunits retained the PAM effect of 17β‐estradiol and dFBr (Jin and Steinbach, 2011; Alcania et al., 2017). This suggests that non‐α4 subunits have all the elements for potentiation by these PAMs except the C‐tail. Another compound targeting the C‐tail of β2, α5 or α6 may potentiate these subunits selectively. The small synthetic molecule LY2087101 binds to the intrasubunit vestibule in the transmembrane domain of α4 subunits (Broad et al., 2006; Young et al., 2008), which is equivalent to the site where many of the α7 nAChR PAMs bind such as PNU‐120596 and 4‐(1‐napthyl)‐3a,4,5,9b‐tetrahydro‐3H‐cyclopenta[c]quinoline‐8‐sulfonamide (Williams et al., 2011c). Another novel PAM, 3‐N‐benzyloxy‐3‐hydroxyimino‐2‐oxo‐6,7,8,9‐tetrahydro‐1H‐benzo[g]indole‐5‐sulfonamide (NS206), acts at the interface of α4 extracellular and transmembrane domains (Olsen et al., 2013). All of these PAMs bind at sites close to structures implicated in signalling between ACh‐binding sites and the activation gate such as the Cys‐loop, M2‐M3 loop and the β10‐M1 loop at the extracellular and transmembrane domain interface (Figures 2A and 3C). These elements are important for coupling with the agonist to open the channel gate (Gupta et al., 2017).

There is no simple correlation between the structure of the ligand and its binding site (Figures 3 and 4). Orthosteric sites bind a variety of molecules (Table 2 and Figure 3). All nAChR orthodox agonists and antagonists have a positively charged amine. Some of them, such as epibatidine and cytisine, bind at both orthodox and unorthodox sites. It remains to be determined what structural elements contribute to the efficacy difference of the two agonists. The orthodox α4/β2 site selective agonists sazetidine‐A and TC‐2559 both contain long and bulky hydrophobic groups at the opposite end of the charged nitrogen, which may prevent them from binding at the α4/α4 site. The unorthodox α4/α4 site of (α4β2)2α4 binds the uncharged molecule NS9283 probably primarily through characteristic features of the minus side of α4, like the histidine that inhibits binding of sazetidine‐A (Olsen et al., 2013; Olsen et al., 2014). The heteroatoms of NS9283 interact with the aromatic box of nAChRs forming a bridge between α4 and α4 subunit like agonists at orthodox sites (Olsen et al., 2014). Conversely, some allosteric ligands are positively charged under physiological pH (Figure 4) but fail to bind at orthosteric sites. Because of the variety of known PAMs structures, it is hard to predict whether a new compound will be a nAChR PAM or not. This is not unexpected as PAMs bind at many different sites. Most PAMs have one to three aromatic rings. Some PAMs, such as NS206, LY2087101 and 17β‐estradiol, lack a charged nitrogen. Even the same ligand site accommodates structurally distinct compounds. The PAMs 17β‐estradiol, dFBr and Br‐PBTC act near the α4 C‐terminus, but the extent of overlap of their binding sites remains to be determined.

Figure 4.

Figure 4

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Chemical structures of allosteric ligands. Nitrogen that is more than 50% positively charged at pH = 7 is noted. Ligands that are PAMs for some nAChRs but NAMs for other subtypes are underlined. *: dFBr has been suggested to bind at two different allosteric sites.

Clinical implications

Full and partial agonists of heteromeric neuronal nAChRs proved beneficial in clinical and preclinical studies for CNS disorders such as addiction (Hendrickson et al., 2013; Hurst et al., 2013; Litten et al., 2013; McKee et al., 2016), pain (Hurst et al., 2013), schizophrenia (Hurst et al., 2013; Dineley et al., 2015), autism (Dineley et al., 2015), attention deficit/hyperactivity disorder (Schuch et al., 2016; Takechi et al., 2016), obsessive–compulsive disorder (OCD) (Tizabi et al., 2002) and Parkinson's and Alzheimer's disease (Srinivasan et al., 2014; Dineley et al., 2015). Potentiators increase activation of nAChRs by neurotransmitters. Therefore, nAChR potentiators, such as unorthodox site‐selective agonists and PAMs, may be useful for diseases where agonists have shown efficacy.

There are a few advantages of nAChR potentiators versus agonists. It is challenging to achieve selective drugs via targeting the orthodox agonist sites, because of the variety and high homology of heteromeric neuronal nAChRs. There is potential for designing subtype‐selective drugs by targeting non‐conserved unorthodox or PAM sites. These selective potentiators may have fewer side effects than traditional agonists. PAMs are better than agonists because they neither activate nor desensitize, just amplify the endogenous pattern of ACh activation without preventing communication of the information that it contains. In the following paragraphs, we will review effects of potentiating nAChRs in pain, addiction, cognitive impairment and OCD because current preclinical studies of selective potentiators focus in these areas (Table 4).

Table 4.

Potential clinical applications of unorthodox site site‐selective agonists and PAMs of heteromeric neuronal nAChRs

CNS disorder nAChR subunits implicated Examples of ligands and preclinical evidence Reference
Addiction α4,α6,β2,α3,β4,α5 dFBr and NS9283 reduce nicotine self‐administration in a rat model (Fowler et al., 2011; Hendrickson et al., 2013; Hurst et al., 2013; Picciotto and Kenny, 2013; Wang et al., 2015b) (Liu, 2013; Maurer et al., 2017)
ADHD α4,β2,α3,β4,α5 NS9283 improves attention in rat drug discriminative model and a rat two‐tone auditory discrimination task (Dineley et al., 2015; Schuch et al., 2016; Takechi et al., 2016) (Mohler et al., 2014) (Grupe et al., 2014)
Alzheimer's disease α4,β2 Galantamine is approved by FDA for Alzheimer's disease. NS9283 enhances cognition in multiple rodent models (Hurst et al., 2013; Dineley et al., 2015) (Timmermann et al., 2012; Grupe et al., 2014)
Autism α3,α4,β2 Not available yet. (Dineley et al., 2015)
OCD α4,β2 dFBr attenuates mouse compulsive behaviours (Mitra et al., 2017)
Parkinson's disease α4,α6,β2 Not available yet. (Hurst et al., 2013; Srinivasan et al., 2014; Dineley et al., 2015)
Pain α3,α4,α5,β2 NS9283 potentiates agonists to reduce neuropathic pain in rodent models (Lee et al., 2011; Zhu et al., 2011; Rode et al., 2012; Hurst et al., 2013).
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Application of nicotinic agonists such as tebanicline (ABT‐594) in pain is limited by the side effects such as gastrointestinal problems (Rowbotham et al., 2009). The unorthodox site‐selective agonist NS9283 potentiated the analgesic effect in rodent models without exaggerating the emetic effect when co‐administrated with ABT‐594 (Lee et al., 2011). In addition, NS9283 did not increase the cardiovascular and hypothermia effects of ABT‐594 (Lee et al., 2011; Zhu et al., 2011). NS9283 is selective for α2 and α4 containing nAChRs without affecting the α3β4 ganglionic subtypes prevalent in the autonomic system. This may explain potentiation of desirable ABT‐594 effects but not side effects.

Nicotine is rewarding in low doses but aversive in high doses (Fowler et al., 2011). Its aversive effect is mediated by α5* nAChRs in the medial habenula interpeduncular pathway (Fowler et al., 2011). Ligands activating α5* nAChRs may increase the aversive effect of nicotine and be more effective in treating nicotine addiction than current cessation aids. However, the effect of agonists may be limited because they desensitize nAChRs rapidly. Unorthodox agonists and PAMs may be more effective because they are unable to desensitize nAChRs in the absence of agonists. The α4β2* nAChR PAM dFBr reduced rat nicotine self‐administration (Liu, 2013). dFBr acts at α4, the β2/α4 and β2/β2 subunit interfaces, thus is likely to affect α4β2α5 nAChRs. But dFBr also inhibits α7 nAChRs (Kim et al., 2007; Weltzin and Schulte, 2010). It remains to be determined whether more selective potentiators for α4α5* nAChRs will have similar effects. One concern about potentiating α4β2 nAChRs is that it may increase the rewarding effect of nicotine. Though it could benefit heavy smokers by reducing the number of cigarettes consumed daily, it may have limited effect on helping quitting. Site‐selective ligands will facilitate identifying nAChR stoichiometries that contribute to the rewarding effect of nicotine. Gene knockout experiments indicate that α4, α6 and β2 subunits are required for mouse nicotine self‐administration (Pons et al., 2008). NS9283 did not alter nicotine‐induced locomotor sensitization (Zhu et al., 2011), but reduced nicotine self‐administration and reinstatement (Maurer et al., 2017). NS9283 itself is not reinforcing (Maurer et al., 2017). These data suggest that (α4β2)2α4, (α4β2)2β2, (α4β2)2α5 and (α6β2)(α4β2)β3 nAChRs are likely to regulate nicotine self‐administration.

Potentiating heteromeric nAChRs has been shown effective in cognitive enhancement and attention improvement in various preclinical models. The α4/α4 site‐selective agonist NS9283 improved performance in rat models of sustained attention, episodic and reference memory (Timmermann et al., 2012). It also potentiated the discriminative effect of nicotinic agonists (Mohler et al., 2014). The nAChR PAM galantamine is an FDA approved drug for Alzheimer's disease (Mucke, 2015) and galantamine is also an acetylcholinesterase inhibitor that increases activation of nAChRs by increasing the concentration and duration of ACh. The PAM dFBr relieves the inhibition of α2β2 and α4β2 nAChRs by Aβ(1–42) peptide, which forms the amyloid characteristic of Alzheimer's disease (Pandya and Yakel, 2011). These data suggest heteromeric nAChR unorthodox agonists and PAMs as treatments for memory impairment and attention deficit.

Increasing activity of α4β2 nAChRs may also benefit OCD. Smoking prevalence is lower in OCD patients than the general population (Abramovitch et al., 2015). Acute and chronic treatment of dFBr has been shown to reduce male mice compulsive‐like behaviour without affecting their anxiety‐like and locomotor activity in the open field (Mitra et al., 2017).

nAChR potentiators should be used clinically with caution. Some nAChRs have high permeability to calcium and increased intracellular calcium may lead to cytotoxic effects. Such concerns have been raised for α7 nAChR PAMs PNU‐120596. But PNU‐120596 has been shown useful without causing death of rodents or primates in improving schizophrenia‐related deficits in sensory inhibition (Stevens et al., 2015) and enhancing memory and cognition (Callahan et al., 2013; Nikiforuk et al., 2015). One possibility is that PNU‐120596 is unable to keep α7 nAChR open constantly, so the nAChRs eventually desensitize (Williams et al., 2011b) and the resulting calcium influx is insufficient to lead to cell death. Another possibility is that the dosage in the animal model is high enough to show positive behaviour effect but is not high enough to be lethal. PAMs, like all drugs, require appropriate doses for effective use.

Desensitization contributes to the antidepressant and anxiolytic effects of nicotine (Picciotto et al., 2008) and alleviates withdrawal (Brody et al., 2006). Orthosteric and allosteric antagonists may be useful for treating depression, anxiety and addiction. Many orthosteric antagonists have poor subtype selectivity. Conotoxins are antagonists with excellent selectivity but no bioavailability because of their large sizes. bPiDI, a bioavailable allosteric antagonist targeting the cation channel pore of α6, selectively inhibited nicotine induced [3H] dopamine release in rat striatal contributed by α6* without affecting the dopamine release contributed by α4(non α6)* nAChRs (Wooters et al., 2011). This channel blocker inhibited nicotine self‐administration and the reinforcement effect of nicotine (Wooters et al., 2011; Beckmann et al., 2015). If site‐selective antagonists are discovered, they will determine which stoichiometry is responsible for these physiological effects.

Conclusions

A single neuron can express several nAChR subtypes. Subtype‐selective agonists and antagonists have been used to identify subtypes. PAMs and agonists selective for unorthodox ACh‐binding sites will provide further tools for identifying nAChR subtypes in vivo. The unorthodox site‐selective ligand NS9283 facilitated distinguishing (α4β2)2α4 and (α4β2)2β2 nAChRs in brain (Grupe et al., 2015). Subunit‐selective PAMs might be similarly useful. Additional ligands targeting unique subunit interfaces are needed to understand the assembly of nAChRs and their roles in neurological functions in vivo. Among these compounds, unorthodox and PAM site‐selective ligands are promising drug candidates because they do not constantly activate and desensitize nAChRs like agonists and antagonists; instead, they potentiate activation by ACh. It remains to be determined whether these nAChR potentiators will be used clinically.

Conflict of interest

J.W. and J.L. have a patent application for Br‐PBTC and its analogues (patent number: PCT/US16/33774).

Acknowledgements

We thank Dr Alexander Kuryatov and Ms. Akansha Jain for their comments on the paper. This work was supported by the National Institutes of Health National Institute on Drug Abuse (Grants DA030929).

Wang, J. , and Lindstrom, J. (2018) Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors. British Journal of Pharmacology, 175: 1805–1821. doi: 10.1111/bph.13745.

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