Accepted Manuscript Title: Allosteric modulation of nicotinic acetylcholine receptors Author: Anna Chatzidaki Neil S. Millar PII: DOI: Reference:

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Please cite this article as: Chatzidaki Anna, Millar Neil S.Allosteric modulation of nicotinic acetylcholine receptors.Biochemical Pharmacology http://dx.doi.org/10.1016/j.bcp.2015.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Allosteric modulation of nicotinic acetylcholine receptors Anna Chatzidaki and Neil S. Millar Department of Neuroscience, Physiology & Pharmacology, University College London, London, WC1E 6BT, UK Address correspondence to: Neil S Millar, Department of Neuroscience, Physiology & Pharmacology, University College London, London, WC1E 6BT, UK. Tel. +44 (0)20 7679 7241; Email: [email protected] Graphical abstract ABSTRACT Nicotinic acetylcholine receptors (nAChRs) are receptors for the neurotransmitter acetylcholine and are members of the ‘Cys-loop’ family of pentameric ligand-gated ion channels (LGICs). Acetylcholine binds in the receptor extracellular domain at the interface between two subunits and research has identified a large number of nAChR-selective ligands, including agonists and competitive antagonists, that bind at the same site as acetylcholine (commonly referred to as the orthosteric binding site). In addition, more recent research has identified ligands that are able to modulate nAChR function by binding to sites that are distinct from the binding site for acetylcholine, including sites located in the transmembrane domain. These include positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), silent allosteric modulators (SAMs) and compounds that are able to activate nAChRs via an allosteric binding site (allosteric agonists). Our aim in this article is to review important aspects of the pharmacological diversity of nAChR allosteric modulators and to describe recent evidence aimed at identifying binding sites for allosteric modulators on nAChRs. Keywords: Allosteric modulation; Ion channel; Neurotransmitter-gated ion channel; Nicotinic acetylcholine receptor; Receptor

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1. Introduction Nicotinic acetylcholine receptors (nAChRs) are excitatory receptors for the neurotransmitter acetylcholine and play an important role in the central and peripheral nervous system [1-4]. They are members of the ‘Cys-loop’ family of pentameric ligand-gated ion channels (LGICs); a family that includes a subset of receptors for 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA) and glycine [5-7]. Nicotinic receptors are widely expressed in the brain in both pre- and post-synaptic locations [8, 9]. They are also expressed in both sympathetic and parasympathetic ganglia, where they are responsible for fast synaptic transmission. In addition, nAChRs are expressed in skeletal muscle, epithelial and immune cells [10-13]. Nicotinic receptors have been implicated in a number of neuromuscular, neurological and psychiatric disorders. For example, in recent years, neuronal nAChRs have been identified as important targets for therapeutic drug discovery, in connection with disorders such as Alzheimer’s disease and schizophrenia [14, 15]. Sixteen human nAChR subunits (α1-α7, α9, α10, β1-β4, γ, δ and ε) have been identified. The α1, β1, γ, δ and ε subunits are expressed in muscle, whereas the α2-α7, α9, α10 and β1-β4 subunits are expressed more widely and are commonly referred to as neuronal subunits [16, 17]. Receptors expressed at the neuromuscular junction are heteromeric complexes containing two copies of the α1 subunit co-assembled with three non-α subunits, with receptors in embryonic and adult muscle having the subunit composition of (α1)2β1γδ and (α1)2β1δε, respectively [18, 19]. There is, however, considerably greater subunit diversity amongst neuronal nAChRs [8, 17]. Some neuronal nAChR subunits, such as α7, can form functional homomeric [(α7)5] nAChRs [20], whereas the majority of neuronal nAChR subunits form heteromeric complexes, consisting of at least two α-type subunits co-assembled with at least two β-type subunits, for example (α4)2(β2)3 and (α4)3(β2)2 [17]. Each nAChR subunit contains an amino-terminal extracellular domain and four transmembrane helices (TM1TM4). The pore of the channel is lined by the second transmembrane domain (TM2) from five co-assembled subunits [21] (Figure 1).

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2. Allosteric modulation of nAChRs The endogenous neurotransmitter of nAChRs, acetylcholine, binds in the receptor extracellular domain, at the interface between two subunits [22, 23]. Research conducted over many years has identified a large number of nAChR-selective ligands, including agonists and competitive antagonists that bind at the same site as acetylcholine (commonly referred to as the orthosteric binding site). In addition, more recent research has identified ligands that are able to modulate nAChR function by binding to sites that are distinct from the binding site for acetylcholine, including sites located in the transmembrane domain. As has been discussed previously, there can be some confusion as to the use of the term ‘allosteric’ in receptor pharmacology [24]. We will use the term ‘allosteric site’ to describe any nAChR ligand-binding site that is distinct from the conventional acetylcholine binding site and we will use the term ‘allosteric modulator’ to describe any ligand that alters the functional properties of nAChRs by interacting with a site that is distinct from the orthosteric site. In doing so, we do not necessarily intend to imply a particular mechanism of action for such ligands. Instead, our aim in this review is to describe evidence for receptor modulation that occurs as a consequence of ligands binding to sites that are distinct from that at which acetylcholine acts as an agonist. Allosteric modulators can either potentiate the effects of agonist-activation (positive allosteric modulators; PAMs), or inhibit agonist-activation (negative allosteric modulators; NAMs). Of course, quite correctly, the term non-competitive antagonist is also used extensively to describe ligands that are capable of inhibiting agonist-evoked responses through a distinct binding site. Possible mechanisms for modulation of nAChRs by allosteric ligands have been discussed previously [25-28]. For example, it has been proposed that PAMs that potentiate lower agonist concentration but have minimal effect on higher agonist concentrations may do so by facilitating agonist binding. PAMs may also increase agonist efficacy by reducing the energy barrier between closed and open states or by increasing the energy barrier between open and desensitised states (Figure 2). In addition, an increase in the apparent peak response and a large steady-state current might be a consequence of PAMs destabilising desensitised states. It has also been suggested that large molecules such as ivermectin may act as PAMs by binding at an inter-subunit transmembrane site and preventing the transition from the open to the closed state [28].

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Allosteric modulators often have no intrinsic activity and only modulate the effects of an agonist. However allosteric ligands that can induce nAChR activation in the absence of an orthosteric agonist have been reported and referred to as either allosteric agonists or agoallosteric modulators [29, 30]. In addition, to PAMs and NAMs, compounds referred to as silent allosteric modulators (SAMs) have also been reported for nAChRs, as they have for other receptors [31-33]. Such compounds do not potentiate or inhibit responses to orthosteric agonists but they can block the effect of other allosteric modulators (PAMs, NAMs or allosteric agonists) by binding competitively at an allosteric binding site. One of the goals of therapeutic drug discovery is the identification of receptor ligands with high subtype selectivity. Although high subtype and species selectivity can be achieved with ligands binding to the orthosteric binding site (see, for example [34]), it is possible that receptor subtype selectivity might be achieved more easily by ligands binding to an allosteric site. This is because, whereas the acetylcholine-binding site is conserved within all nAChRs, there may be less need for conservation of structure or amino acid composition at other receptor sites. In addition, the low intrinsic activity of PAMs, in comparison to agonists, may be useful in reducing toxicity and off-target effects. This may be advantageous because it can enable the spatial and temporal pattern of signalling of endogenous acetylcholine-evoked responses to be retained [27, 35]. A number of previous reviews have discussed the structural diversity of nAChR allosteric modulators, including many compounds that have been described in patent applications [3640]. In addition, previous reviews have discussed the possible therapeutic uses of nAChRs allosteric modulators [27, 41-43] for example in the treatment of cognitive deficits [44]; depression [35]; pain [45, 46] and cancer [47]. In contrast, the present review does not attempt to provide a comprehensive overview of nAChR allosteric modulation but will focus on important aspects of the pharmacological diversity of allosteric modulators and on recent evidence aimed at identifying binding sites for allosteric modulators on nAChRs.

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3. Positive allosteric modulators (PAMs) and allosteric agonists 3.1. Homomeric α7 nAChRs Much of the recent work concerning nAChR PAMs has focussed on the homomeric α7 receptor [36, 37, 40, 48], one of the main nAChR subtypes expressed in the mammalian brain. The α7 subtype is somewhat atypical in that it has a relatively low sensitivity to acetylcholine, high calcium permeability and exhibits very fast desensitisation [20]. In addition to potentiating agonist peak responses, some PAMs acting on α7 nAChRs have been reported to cause a slowing of receptor desensitisation, whereas other PAMs have little or no effect on the rate of receptor desensitisation. As a consequence of this differing effect on desensitisation, α7-selective PAMs have been classified into two categories: type I and type II [26, 49]. Type I PAMs increase peak current in the presence of an orthosteric agonist, without having an effect on receptor desensitisation. Type II PAMs, on the other hand, significantly reduce the fast desensitisation of α7 receptors. A further difference between α7-selective type I and type II PAMs is the ability of type II PAMs to allow receptor reactivation from the desensitised state [49, 50]. It is possible that the mechanism of action of type I PAMs consists of facilitating the transition to the open state by reducing the energy barrier between closed and open states. In addition, type II PAMs may cause destabilisation of the desensitised state. Although classifying PAMs acting on α7 nAChRs as either type I or type II can be useful in some circumstances, it is clear, as will be discussed later, that this is an over-simplification and that PAMs with intermediate properties have been identified. An early demonstration of allosteric modulation of nAChRs came from the observation that calcium potentiates α7 nAChR currents in a voltage-independent manner [51, 52]. Early studies with α7 nAChRs also demonstrated the ability of ivermectin, a large macrocyclic lactone (Figure 3), to potentiate agonist-evoked responses [53]. In addition, the acetylcholinesterase inhibitor galantamine has been reported to act as a relatively weak and non-selective PAM of α7 nAChRs [54]. Similarly, genistein (a tyrosine kinase inhibitor) and also 5-hydroxyindole (5-HI) are weak and relatively non-selective potentiators on α7 nAChRs [49, 55]. A number of proteins have been shown to enhance agonist responses on the α7 receptors, including a secreted mammalian Ly-6/uPAR-related protein (SLURP-1) and bovine serum albumin (BSA) [56, 57]. All of these compounds have only minimal effects on the

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rapid desensitisation of α7 nAChRs and, in most cases, do not display high selectivity for this receptor subtype. Over the past decade there has been considerable interest from pharmaceutical companies and academic research groups in developing PAMs of α7 nAChRs that display higher potency and greater subtype selectivity. PNU-120596 was the first α7-selective PAM to be described in detail that causes a dramatic reduction in the rate of receptor desensitisation [50]. It is also one of the best-studied PAMs in pre-clinical models of pain, ischaemia, schizophrenia and cognitive deficits [50, 58-63]. Other α7-selective PAMs that cause a dramatic reduction in receptor desensitisation and, consequently, have been described as type II PAMs include TQS [49] and A-867744 [64, 65]. A feature of compounds such as PNU-120596, TQS and A-867744 is that they cause little or no receptor activation in the absence of an orthosteric agonist. However, a derivative of TQS (4BP-TQS) has been shown to activate recombinant and native α7 nAChRs in the absence of an orthosteric agonist [30, 66, 67]. In contrast to the activation of α7 nAChRs by acetylcholine, activation by 4BP-TQS occurs with minimal desensitisation [30]. In addition the agonist dose-response curve with 4BP-TQS is steeper and maximal responses are substantially larger than observed with orthosteric agonists, all of which argues that activation by 4BP-TQS occurs by a different mechanism of action [30]. As will be discussed later, there is evidence that 4BP-TQS causes receptor activation via a distinct allosteric site and, as a consequence, it has been described as an allosteric agonist [30]. Subsequently, a number of other TQS derivatives with either PAM or allosteric agonist effects have been reported [68, 69]. Interestingly, very minor changes to ligand structure, such as altering the size of a single halogen atom or the pattern of methyl substitution of an aromatic ring, can convert type II PAMs into potent allosteric agonists [33, 68]. In addition to allosteric modulators that reduce levels of agonist-induced desensitisation, a number of α7-selective type I PAMs have been described in recent years as a consequence of high-throughput compound screening. Amongst the first to be examined in detail were NS1738 [70] and CCMI (also described as ‘Compound 6’) [71]. Other compounds acting as type I PAMs of α7, but displaying less subtype selectivity, are LY-2087101, LY-2087133 and LY1078733 [72]. In addition to potentiating α7 nAChRs, these compounds also potentiate α2β4, α4β2, α4β4 subtypes [72].

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Whilst the distinction between type I and type II PAMs can be useful, there is increasing evidence that it is an over-simplification. A number of studies have reported α7-selective PAMs with effects on desensitisation that are intermediate between classical type I and type II PAMs [73, 74]. A good example of this is a recently described series of structurally-related α7-selective PAMs (TBS-345, TBS-346, TBS-516, TBS-546 and TBS-556) with a range of effects on receptor desensitisation [75]. Another α7-selective PAM, RO5126946, has effects on desensitisation that are typical of a type II PAM [76]. However, it lacks the ability to facilitate reactivation of desensitised α7 nAChRs, a feature that is normally considered to be characteristic of type II PAMs [76]. 3.2. Heteromeric nAChRs Heteromeric α4β2-containing nAChRs are an abundant receptor subtype expressed in the human brain and have been a target for the development of orthosteric ligands. For example, the partial agonists varenicline and cytisine have been approved as aids for smoking cessation [77]. In addition, there has been considerable interest in the identification of PAMs that are selective for heteromeric nAChR subtypes such as α4β2. Steroids are among compounds that act as endogenous PAMs of nAChRs. 17β-estradiol increases acetylcholine-evoked currents in the human α4β2 receptor and, more modestly, in the α4β4 receptor [78-80]. Galantamine also potentiates α4β2 nAChRs, in addition to a number of other heteromeric nAChR subtypes, including α6β4 and α3β4 nAChRs [81]. Both estradiol and galantamine increase the potency (i.e. they reduce the EC50 value) of the orthosteric agonist, without having a significant effect on the size of the maximum response, which could be the result of enhanced agonist binding. In contrast, desformylflustrabromine (dFBr) and some of its derivatives are α4β2-selective PAMs that increase the size of the maximum response and have very modest effect on the potency of the agonist [82, 83]. LY2087101 is another relatively non-selective nAChR PAM that increases maximum response size as well as potency of acetylcholine on α4β2, [72]. Other compounds that have been reported to potentiate α4β2 and α4β4 nAChRs include the muscarinic antagonists atropine and scopolamine [84, 85]. Receptors containing α4 and β2 subunits can exist in two different stoichiometries with distinct functional properties. The (α4)3(β2)2 subtype has a lower sensitivity to acetylcholine

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and it displays higher permeability to calcium and faster desensitisation kinetics in comparison to the (α4)2(β2)3 subtype [86-89]. In addition, the two subtypes display some ligand selectivity. NS9283 (alternatively A-969933) is an α4β2-selective PAM with in vivo efficacy in models of pain [90, 91] and is selective for the (α4)3(β2)2 subtype [92-94]. As will be discussed in more detail in section 6, NS206 is a PAM that is thought to act through a distinct binding site and can potentiate both the (α4)3(β2)2 and (α4)2(β2)3 subtypes [93]. In contrast, HEPES is a selective PAM for the (α4)2(β2)3 subtype [95]. In recent years, PAMs of α3-containing nAChRs have also been reported [96-98]. For example, the anthelmintic compounds levamisole and morantel potentiate acetylcholine response on α3β2 and α3β4. Zinc potentiates a number of nAChR subtypes, including both α3- and α4-containing subtypes [99, 100] but, interestingly, shows selectivity for α4β2 nAChRs with the (α4)3(β2)2 stoichiometry [101, 102]. Recent studies aimed at identifying binding sites for modulators such as zinc and morantel are discussed below.

4. Negative allosteric modulators (NAMs) Receptor antagonism can occur by a variety of mechanisms. A common distinction is made between antagonists that bind either competitively or non-competitively with respect to an orthosteric agonist. However, the terms ‘non-competitive antagonist’ and ‘negative allosteric modulator’ (NAM) are frequently used interchangeably. A distinction is also sometimes made between, on the one hand, antagonists that bind in the channel pore and thereby occlude the flow of ions (open-channel blockers) and, on the other hand, antagonists that modulate receptor properties such as gating or desensitisation equilibrium. A large number of compounds can act as open-channel blockers, including agonists at high concentrations. In addition, channel-blocking ligands frequently show relatively low receptor selectivity. Such compounds will not be discussed in detail but have been reviewed elsewhere [103, 104]. Many compounds have been reported to act as non-competitive antagonists of nAChRs. These include psychoactive drugs such as mecamylamine and phencyclidine [105-109], philanthotoxin [110], general anaesthetics [111], antidepressants [112-114], monoterpines such as camphor, menthol and propofol [115-119], aminoglycoside antibiotics [120] antipsychotics [121], the fluorophore crystal violet [122]; zinc [99, 123] and some detergents [124]. Several endogenous ligands have also been reported to have negative allosteric effects

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on nAChRs. These include progesterone and neurosteroids [103, 125] and also proteins such as Lynx-1 [126, 127] and the cannabinoid anandamide [128].

5. Silent allosteric modulators (SAMs) The term ‘silent allosteric modulator’ (SAM) has been used to describe compounds on a variety of receptors that interact with a site that is distinct from the conventional allosteric site but do not exert a modulatory effect on responses evoked by an orthosteric ligand (i.e. neither a positive or negative allosteric effect) [31, 32, 129]. More recently, compounds with such properties have been reported for nAChRs [33]. Such compounds do not potentiate or inhibit responses to orthosteric agonists but they can influence allosteric modulation by blocking the effect of other allosteric modulators (PAMs, NAMs or allosteric agonists) by binding in a mutually exclusive manner at a common or overlapping allosteric site [33]. In addition, recent studies have demonstrated that relatively small changes in chemical structure, such as methyl substitution of a single aromatic ring, can determine whether a compound acts as a PAM, NAM or SAM on nAChRs [33].

6. Binding sites for allosteric modulators Several experimental techniques have been employed with the aim of identifying the binding sites of allosteric modulators on nAChRs (Table 1). The availability of a readily purified preparation of nAChR, for example from the electric organ of the marine ray Torpedo, has facilitated affinity labelling experiments that have provided direct evidence for the binding of ligands to sites other than the orthosteric site. This approach has identified sites for a variety of non-competitive antagonists interacting with the transmembrane domain [130-135]. Affinity labelling studies have also identified binding sites for cholesterol within the nAChR transmembrane domain [136]. In addition, more indirect approaches, such as computerdocking studies, have been used. For example, docking studies with cholesterol have predicted binding sites in cavities within the transmembrane region [137]. Other experimental techniques aimed at identifying binding sites have included the construction of recombinant subunit chimeras, site-directed mutagenesis, and the substituted cysteine accessibility method (SCAM). However, it is important to use a degree of caution when interpreting such studies. For example, if the mutation of an amino acid results in a change in the pharmacological properties of a ligand, it is not necessarily appropriate to conclude that this is a consequence of a change to the binding site [24]. Nevertheless, the combined use of a variety of

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approaches, including electrophysiological, pharmacological, biochemical and computational techniques can provide strong evidence for the location of ligand binding sites [138]. Various sites and amino acids have been shown to be important for allosteric modulation. Studies using α7-5-HT3 subunit chimaeras, site-directed mutagenesis and docking simulations, have led to the proposal that α7-selective allosteric modulators such as NS-1738 (a type-I PAM) and PNU-120596 (a type II PAM) act via a binding site within an intrasubunit cavity that is located between the four transmembrane helices of a single subunit [139, 140]. More recent studies have examined a series of nineteen α7-selective allosteric modulators that differ only in methyl substitution of a single aromatic ring [33]. Despite relatively small changes in chemical structure, the compounds examined displayed five distinct pharmacological effects on α7 nAChRs. These included effects typical of type I PAMs, type II PAMs, NAMs, SAMs and allosteric agonists [33] and it has been proposed that all of these pharmacological effects can arise from ligands binding to a broadly similar or overlapping site located within the previously identified intra-subunit transmembrane cavity [33]. Other studies are consistent with α7-selective allosteric modulators such NS-1738, PNU120596 and A-867744 interacting with a transmembrane site [65, 141, 142]. As would be expected, allosteric modulators that have been proposed to bind in a transmembrane location do not displace the binding of orthosteric radioligands ligands such as [3H]-MLA or [3H]-αbungarotoxin [33, 65, 70, 75]. However, some unexpected results have been reported for the α7-selective PAM A-867744. Although A-867744 does not displace binding of [3H]-MLA from α7 nAChRs, in contrast to other PAMs, it has been reported to displace the binding of another agonist ([3H]-A-585539) that is thought to interact with the orthosteric site [65]. A transmembrane binding site in nAChRs has also been proposed for monoterpine compounds such as menthol and propofol [116, 117] and is consistent with evidence supporting a transmembrane binding site for monoterpines on other pentameric LGICs [143147]. Affinity labelling studies with a photoreactive analogue of propofol identified three sites at which it bound within the transmembrane domain of muscle-type nAChRs, but concluded that the functionally relevant site for the inhibitory action of propofol was an intra-subunit site [117], similar to that described earlier for α7-selective allosteric modulators such as NS-1738, PNU-120596 and 4BP-TQS [30, 139]. Similarly, there is evidence for a transmembrane binding site for nAChR NAMs such as the endogenous cannabinoid anandamide [148-150] and also antihistamine compounds [151].

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As discussed earlier, the α7 subunit forms a homomeric nAChR. However, in addition, it can co-assemble with β2 to form a heteromeric α7β2 complex [152]. One difference that has been reported in the allosteric modulation of these two nAChR subtypes is that α7β2 receptors, but not α7 receptors, are inhibited by the volatile anaesthetic isoflurane [153]. On the basis of mutagenesis and computer docking studies, it has been proposed that isoflurane binds to the β2 transmembrane domain [153]. This is consistent with evidence for a transmembrane binding site for anaesthetics on other LGICs [154]. There is also evidence that the macrocyclic lactone ivermectin acts as a nAChR PAM by interacting with a transmembrane site [155]. Recently, a high resolution X-ray structure was obtained of ivermectin bound to a prokaryotic glutamate-gated chloride channel (GluCl) with close structural similarity to nAChRs [156]. These findings are consistent with ivermectin binding to an inter-subunit transmembrane site, rather than the intra-subunit transmembrane site that has been proposed for smaller allosteric modulators [30, 117, 139]. Spinosad, like ivermectin, is a macrocyclic lactone pesticide that acts as an allosteric modulator of nAChRs [157]. Recent studies of spinosad-resistant insects have identified a resistance-associated point mutation in the nAChR transmembrane domain that is consistent with spinosad binding to nAChRs in a similar inter-subunit transmembrane site to the known binding site of ivermectin in GluCl [158]. It addition to the transmembrane domain, there are many other potential sites at which allosteric modulators might potentially interact with nAChRs, as is the case with other ligandgated ion channels [25, 159]. For instance, a site on the C-terminus of the α4 subunit is believed to be necessary for estradiol PAM activity [78-80]. Whereas there are five potential agonist binding sites in a homomeric nAChR, there are expected to be just two or three functioning orthosteric agonist sites in heteromeric nAChRs. For example, the acetylcholine binding site in neuronal heteromeric nAChRs is at the interface between an α and β subunit, in which the α-type subunit forms the principal face and the β-type subunit the complementary face (designated α(+)/β(-)). However, it is possible that compounds can bind at equivalent positions at other subunit interfaces, for example at a β(+)/α(-) interface. Indeed, this has been proposed as a mechanism by which compounds such as morantel and desformylflustrabromine can potentiate agonist-evoked responses [96, 160-163]. Similarly, compounds interacting with the extracellular α4/α4 interface of (α4)3(β2)2 nAChR (or α/α interfaces in other nAChR subtypes) can act as PAMs [93, 164, 165], just as the inability of

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an agonist to interact with this site may explain partial agonist activity [166-169]. In contrast, it has been proposed that HEPES acts as a potentiator of α4β2 nAChRs by interacting with the β(+)/β(-) interface and, as a consequence, is selective for receptors with the (α4)2(β2)3 stoichiometry [95]. Additionally, a binding site for NAMs at the α(+)/β(-) interface of α3β4 and α4β2 nAChRs has been predicted on the basis of computer docking studies and molecular dynamic simulations [170-172]. As described earlier, the acetylcholinesterase inhibitor galantamine is a relatively weak PAM of α7 nAChRs [173] and has been proposed to bind at the extracellular domain at a site that is distinct from the acetylcholine binding site [174, 175]. Galantamine also potentiates α4β2 nAChRs and a number of other nAChR subtypes including α6β4 and α3β4 nAChRs [81], by binding at a non-α subunit interface [176]. In addition, there is evidence that the binding site mediating the potentiating effects of calcium is located on the extracellular site of the α7 nAChR [125, 177]. Another divalent cation, zinc, acts as a PAM of α4β2 nAChRs but does so selectively on receptors with the stoichiometry (α4)3(β2)2 [102]. This has been explained by evidence that it interacts selectively with the α4(+)/α4(-) subunit interface, whilst inhibitory effects on (α4)2(β2)3 nAChRs are thought to be mediated by zinc binding to the α4(-)/β2(+) subunit interface [102].

7. Summary Rather than attempting to provide a comprehensive overview of all aspects of allosteric modulation of nAChRs, our aim in this brief review has been to summarise aspects of the pharmacological diversity of nAChR allosteric modulators and to describe evidence for the location of binding sites for such compounds. Although the focus of this review has been the family of nAChRs, the evidence for the existence of multiple modulatory sites, both in transmembrane and extra-transmembrane locations is a feature that appears to be common amongst neurotransmitter-gated ion channels.

Acknowledgements Research in the authors’ laboratory has been funded in recent years by the Biotechnology and Biological Sciences Research Council (BBSRC), Eli Lilly, the Medical Research Council (MRC), the Royal Society, Syngenta and the Wellcome Trust. Anna Chatzidaki was

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supported by a Medical Research Council (MRC) Industrial CASE PhD studentship [Grant: G1001602] that was co-funded by the MRC and Eli Lilly.

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Figure captions Fig. 1. Cartoon representation of nAChR structure and subunit topology. Five co-assembled nAChR subunits are shown in a lipid bilayer and annotated to show locations that have been proposed as binding sites for allosteric modulators (A). The transmembrane topology of a single nAChR subunit is shown, in which the polypeptide chain is denoted by a blue line and the four transmembrane helices by cylinders (A and B). Also illustrated (C) is the nAChR transmembrane domain, formed by twenty transmembrane helices (four from each of the five co-assembled subunits). The central ion channel pore is lined by the second transmembrane helix (TM2) from each of the five subunits. Fig. 2. Representation of the equilibrium between closed, open and desensitised states of a nAChR. The open state is stabilised by the binding of an agonist, such as the orthosteric agonist acetylcholine (depicted as blue dots). In addition, there is evidence that receptors can enter an agonist-bound, non-conducting, desensitised state. Although only three states are illustrated in this figure, it should be noted that receptors are expected to transition through multiple intermediate states. There is evidence, for example of both short-lived and longerlasting desensitised states. As discussed in the text, the transition between these various states can be influenced by allosteric modulators interacting with a variety of receptor binding sites.

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Fig. 3. Chemical structures of selected nAChR allosteric modulators.

Fig. 1

Fig. 2

Fig. 3

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Table 1 Experimental techniques that have helped to establish the location of binding sites for nAChR allosteric modulators. (Note: References cited are a representative series, rather than a comprehensive list) Experimental Technique

References

Affinity labelling

[117, 130-136, 145, 159]

Computer-docking and molecular dynamics studies

[30, 117, 122, 137, 139, 140, 142, 172, 175, 178]

Subunit chimaeras

[29, 65, 72, 75, 79, 81, 93, 111, 139, 141, 147]

Site-directed mutagenesis (SDM)

[30, 33, 53, 68, 78, 80, 93, 95, 139, 140, 146, 147, 155, 158, 160-163, 172, 174, 175, 177]

Radioligand binding

[29, 33, 65, 70-72, 74-76, 117, 122, 147]

X-ray crystallography and NMR

[143, 153, 156, 176]

25