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Tocris Bioscience Scientific Review Series

Susan Wonnacott and Jacques BarikDepartment of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UKSusan Wonnacott is Professor of Neuroscience in the Department of Biology and Biochemistry at the University of Bath. Her research focuses on understanding the roles of nicotinic acetylcholine receptors in the mammalian brain and the molecular and cellular events initiated by acute and chronic nicotinic receptor stimulation. Jacques Barik was a PhD student in the Bath group and is continuing in addiction research at the Collège de France in Paris.

Nicotinic ACh Receptors

IntroductionThe nicotinic acetylcholine receptor (nAChR) is the prototype of the cys-loop family of ligand-gated ion channels (LGIC) that also includes GABAA, GABAC, glycine, 5-HT3 receptors, and invertebrate glutamate-, histamine-, and 5-HT-gated chloride channels.1,2 nAChRs in skeletal muscle have been characterised in detail whereas mammalian neuronal nAChRs in the central nervous system have more recently become the focus of intense research efforts. This was fuelled by the realisation that nAChRs in the brain and spinal cord are potential therapeutic targets for a range of neurological and psychiatric conditions. The generation of transgenic mice with deleted or mutated nAChR subunits3 and the development of subtype-selective ligands to complement the generous armamentarium of natural products that target nAChRs,4 support this research. Progress is being made in understanding the physiological roles of nAChRs in the brain and the underlying molecular and cellular mechanisms, and the contribution of nAChRs to pathological conditions.

Muscle nAChRnAChRs in vertebrate skeletal muscle have been studied for over a century; this preparation was pivotal in Langley’s formulation of the concept of a ‘receptive substance’.5 In these studies he showed that ‘nicotine causes tonic contraction of certain muscles of fowl, frog and toad, and that this contraction is prevented .... by curare’. This was the first notion that the action of a neurotransmitter or pharmacological agonist is transduced into an intracellular response by interaction with a molecular entity (‘receptor’) in the membrane of the responsive cell. Dale distinguished the actions of muscarine and nicotine, leading to the recognition of two pharmacologically distinct (and structurally and functionally unrelated) families of receptors for the neurotransmitter acetylcholine (ACh), that take their names from these natural products.6 Neuromuscular and ganglionic preparations lend themselves to physiological and pharmacological investigations,

and there followed detailed studies of the properties of nAChRs mediating synaptic transmission at these sites. nAChRs at the muscle endplate and in sympathetic ganglia could be distinguished by their respective preferences for C10 and C6 polymethylene bistrimethylammonium compounds, notably decamethonium and hexamethonium.7 This provided the first evidence that muscle and neuronal nAChRs are structurally different.

In the 1970s, elucidation of the structure and function of the muscle nAChR, using biochemical approaches, was facilitated by the abundance of nicotinic synapses akin to the muscle endplate in electric organs of the electric ray, Torpedo, and eel, Electrophorus. High affinity snake α-toxins, including α-bungarotoxin (α-Bgt), enabled the nAChR protein to be purified and subsequently resolved into 4 different subunits, designated α,β,γ and δ.8 An additional subunit, ε, was subsequently identified in adult skeletal muscle. In the early 1980s, these subunits were cloned and the era of the molecular analysis of nAChRs commenced. The muscle endplate nAChR has the subunit combination and stoichiometry (α1)2β1εδ, whereas the extrajunctional nAChR (α1)2β1γδ predominates in foetal or denervated muscle, and (muscle-derived) electric organs. The high density of nAChRs in Torpedo electric organ has facilitated high resolution structural studies using electron microscopy.9 Together with biochemical and biophysical approaches to studying structure-function relationships, this has resulted in a detailed molecular description of the nAChR.1

Molecular Architecture of the nAChR (Figure 1)Each of the five subunits comprising the nAChR span the lipid bilayer to create a water-filled pore. Each subunit consists of 4 transmembrane segments, the second transmembrane segment (M2) lines the ion channel. The extracellular N-terminal domain of every subunit contains a ‘cys-loop’ that is the signature sequence of this LGIC family: two cysteine residues, separated by 13 amino acids (Cys 128, 142, Torpedo α subunit numbering), form a disulphide

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bond to create a loop that has been implicated in the transduction of agonist binding into channel opening.10 The principal agonist binding site resides in the N-terminal domain of α subunits, close to a pair of adjacent (‘vicinal’) cysteine residues (Cys 192, 193, Torpedo numbering) that define an α subunit. Mutagenesis and photoaffinity labelling experiments have highlighted the importance of 4 aromatic residues (Tyr 93, Trp 149, Tyr 190, Tyr 198, Torpedo numbering), consistent with 3 polypeptide loops of the α subunit (loops A-C) contributing to the primary agonist binding site (see Figure 1).11 The adjacent subunit (γ/ε or δ) also contributes to the binding site (complementary site: ‘loops’ D-F, now recognised to be mostly β strands). One consequence of this is that the αγ/ε and αδ binding sites are not identical with respect to ligand affinity.1 However, occupancy of both binding sites is required to open the channel.

Knowledge of ligand binding to nAChRs has been greatly augmented by the crystal structure of an ACh binding protein first identified in the snail Lymaea stagnalis and subsequently also cloned from Aplysia

californica and Bulinus truncatus.12,13 Each subunit of this pentameric secreted protein is homologous to the N-terminal domain of a nAChR subunit, with conservation of all the residues implicated in ACh binding to muscle nAChRs. These proteins provide a high resolution view of the extracellular portion of the receptor, notably of the binding sites at the interface between adjacent subunits, and the interaction of agonists with these sites.10

Upon agonist binding, nAChRs undergo an allosteric transition from the closed, resting conformation to an open state that allows an influx of Na+, and to a lesser extent Ca2+, and an efflux of K+ under normal physiological conditions. In the closed state the ion channel is occluded by a ‘hydrophobic girdle’ that constitutes a barrier to ion permeation. Agonist binding in the extracellular domain promotes a conformational change that results in a rotational movement of the M2 helices lining the pore. Twisting of the girdle widens the pore by ~3 Å, sufficient for ion permeation.9 At the muscle endplate, the ensuing depolarisation elicits muscle contraction. Despite the

Figure 1 | General structure of nAChRs1

M2 linesthe channel

ACh bindingprotein

M2 M3 M4

C

CC

N

Cys-loop

Primarybinding site: α

Complementarybinding site: γ/(δ)

Agonist / CompetitiveAntagonist

Non-competitiveAntagonist

Positive AllostericModulator

Channel BlockerB

CNic

A

E

F

DW55/(57)

Y117Y111

Y151

Y190

Y198 Y93

C192C193

W149

W86

D180/(182)

M1

Ca2+, Na+

K+

a) b)

c)

a) Schematic of a nAChR with one subunit removed to reveal the ion channel lumen. Notional sites of action of interacting drugs in the extracellular domain or within the channel lumen are indicated. b) Agonist binding site loop model. The agonist binding site is enlarged to show the contributing polypeptide loops forming the primary and complementary components, with key amino acids indicated on the loops. c) The topography of a single subunit.

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Nicotinic Receptors

presence of agonist, the nAChR channel closes within seconds to minutes, to enter a desensitised state. In this condition, the nAChR is refractory to activation. Multiple desensitised states have been proposed to exist.14 In the active (open) conformation, the nAChR binds agonists with low affinity (Figure 2; e.g. Kd for ACh ~50 μM). The desensitised states display higher affinity for agonist binding (Kd for ACh ~1-5 μM), thus the desensitised nAChRs can retain bound agonist despite its non-conducting state.

Sites on the Muscle nAChR for Ligand Interactions (Figure 1)In addition to agonists binding to the agonist binding sites in the extracellular domain, competitive antagonists also bind at or close to these sites, preventing access to agonists. Their antagonism can be overcome by increasing the agonist concentration (unless the antagonist binds irreversibly, as is the case for α-Bgt), hence competitive antagonism is referred to as ‘surmountable’. The concentration of competitive antagonist necessary for nAChR blockade will depend on the experimental conditions. Non-competitive antagonists bind to sites distinct from

agonist binding sites, and include channel blocking drugs that occlude the channel. Their inhibition is not surmountable with increasing agonist concentration. In addition to compounds that interact specifically with residues in the mouth or lumen of the pore, any small positively charged species may be predicted

Figure 2 | Relationship between the major conformational states of a nAChR

RESTING

Channel closed

ACTIVE

Channel open

DESENSITISED

Fast onset

DESENSITISED

Slow onset

agonist

Agonist bindswith low affinity

Agonist bindswith high affinity

RESTING

Channel closed

ACTIVE

Channel open

DESENSITISED

Fast onset

DESENSITISED

Slow onset

agonist

Agonist bindswith low affinity

Agonist bindswith high affinity

Table 1 | Selected compounds that interact with mammalian muscle nAChRs

Drug Comment Potencya

Agonists(±)-Anatoxin A A bicyclic amine from blue-geen algae that is a potent, enantio-selective ACh-like agonist lacking

significant activity at muscarinic receptors or AChE.88,237EC50 = 50 nM

(-)-Nicotine The natural tobacco alkaloid is ~6 times more potent than its unnatural enantiomer at muscle nAChRs.238

ED20 = 20 µM

Competitive AntagonistsBenzoquinonium A classical neuromuscular blocking agent,239 also used for invertebrate preparations.240 More

recently reported to act as an allosteric potentiating ligand and open channel blocker of muscle and neuronal nAChR subtypes.241

α-Bungarotoxin (α-Bgt)

Polypeptide snake toxin from Bungarus multicinctus; most potent of the ‘long’ α-neurotoxins. Binds pseudo-irreversibly, reflecting very slow dissociation kinetics. Interacts potently with α subunit sequence around Tyr190-X-Cys192-Cys193.1,242

Kd = 0.01-10 nM

α-Conotoxin MI One of several α-conotoxins from Conus sp. that specifically block muscle nAChRs. MI exhibits a 10000-fold preference for the α/δ versus the α/γ agonist binding site interface of mammalian muscle nAChRs.139,243

Kd ~ 0.1-1 nM

Decamethonium Often used as a competitive antagonist but it produces a depolarising neuromuscular block akin to nicotine and other agonists. It is more accurately classified as a partial agonist.7,244

ED95 = 0.12 µmol/kg;0.03 mg/kg

Pancuronium Used clinically as a non-depolarizing muscle relaxant.245,246 IC50 ~ 5 nM

d-Tubocurarine As a photoaffinity label, it discriminates αδ and αγ agonist binding sites.1,246,247 Kd1 = 30 nM, Kd2 = 8 µMIC50 ~ 50-100 nM

Channel BlockersChlorpromazine This neuroleptic drug also blocks the nAChR channel, interacting with hydrophobic residues in the

Torpedo M2 channel lining.248,249,250IC50 >300 nM

Histrionicotoxin First isolated from frogs of the dendrobatid genera.251,252,253 Ki ~ 0.1-1 µM

(Bold Text Denotes Compounds Available From Tocris)

aEC50, effective concentration producing 50% of maximum activation; ED20, effective dose producing 20% of maximum response (activation); ED95, effective dose producing 95% of maximum response (blockade); Kd, Afinitiy for binding to muscle or Torpedo preparations or purified nAChR; IC50, concentration producing 50% inhibition; dependent on experimental conditions; Ki, Inhibition constant

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to channel block, and many agonists including ACh do this at high concentrations.15 The efficiency of channel blockade is ‘state dependent’; access to the channel requires the channel to be open. Hence the speed of block will be influenced by the state of the receptor: resting, open or desensitised (Figure 2). A selection of compounds acting at such sites on the muscle or Torpedo nAChR is listed in Table 1. Allosteric modulators can act at a number of sites to influence agonist interactions or channel function.

Neuronal nAChRHeterogeneity of Subtypes (Figure �)In addition to their presence in skeletal muscle, nAChRs in autonomic neurones were implicit, and recognised to be pharmacologically distinct, since the studies of Paton and Zaimis.7 The existence of nAChRs in the brain was controversial, but realisation that the tobacco smoking habit is underpinned by the psychoactive actions of nicotine

necessitated the presence of specific receptors. In the early 1980’s it was apparent that [125I]-α-Bgt and [3H]-nicotine labelled distinct sites that differ in their pharmacological profiles and anatomical distributions in rodent brain.16,17 This raised the (then) novel and controversial prospect of nAChR heterogeneity in the brain. Since the first publication of a cloned neuronal nAChR subunit (α3) in 1986,18 eleven neuronal nAChR subunits have been identified in mammals (α2-α7, α9, α10, β2-β4),4,19 with an additional subunit, α8, cloned from avian species.20

α Subunits are defined by the presence of a pair of vicinal cysteines equivalent to those that characterise the muscle α1 subunit. This led to the supposition that all α subunits could constitute the primary agonist binding subunit in neuronal nAChRs. However, the α5 subunit is not capable of fulfilling this role as it lacks the critical tyrosine from loop C (Tyr190, Torpedo α1 labelling, Figure 1).21 β Subunits lack the N-terminal vicinal cysteines but β2 and β4 subunits contain the

Figure 3 | Heterogeneity of vertebrate nAChRs Phylogenetic relationship of all vertebrate nAChR subunits cloned to date, adapted from ref. 342. (For complete phylogenetic tree including invertebrate subunits see ref. 127.) Viable subunit combinations are indicated on the right. Putative agonist binding sites are indicated by small dark circles between adjacent subunits.

ε

Muscle,pCa2+/Na+

0.2

Neuronal,heteromericpCa2+/Na+

~1-1.5

Neuronal,homomericpCa2+/Na+

~10

α 7α 7 α 7

α 7 α 7

α 9α 9 α 9

α 9 α 9

α 7α 8 α 8

α 7 α 7

α 8α 8 α 8

α 8 α 8

α 10 α 10

α 9 α 9

α 9

α 4β2

α 4

β2 xx

α 6β2

α 4

β2 β3

α 6β2

α 6

β2 β3

α 3β2

α 3

β4 Y

α 3β2

α 3

β2 Y

α 3β4

α 3

β4 Y

Y

ε

Muscle,pCa2+/Na+

0.2

Neuronal,heteromericpCa2+/Na+

~1-1.5

Neuronal,homomericpCa2+/Na+

~10

α9

α7

α8

α2

α4α3

α6

α5

α1

β1

β3

β2

β4

α10

ε

Muscle,pCa2+/Na+

0.2

Neuronal,heteromericpCa2+/Na+

~1-1.5

Neuronal,homomericpCa2+/Na+

~10

α 7α 7 α 7

α 7 α 7

α 9α 9 α 9

α 9 α 9

α 7α 8 α 8

α 7 α 7

α 8α 8 α 8

α 8 α 8

α 10 α 10

α 9 α 9

α 9

α 4β2

α 4

β2 xx

α 6β2

α 4

β2 β3

α 6β2

α 6

β2 β3

α 3β2

α 3

β4 Y

α 3β2

α 3

β2 Y

α 3β4

α 3

β4 Y

Y

α 7α 7 α 7

α 7 α 7

α 9α 9 α 9

α 9 α 9

α 7α 8 α 8

α 7 α 7

α 8α 8 α 8

α 8 α 8

α 10 α 10

α 9 α 9

α 9

α 7α 7 α 7

α 7 α 7

α 9α 9 α 9

α 9 α 9

α 7α 8 α 8

α 7 α 7

α 8α 8 α 8

α 8 α 8

α 10 α 10

α 9 α 9

α 9

α 4β2

α 4

β2 xx

α 6β2

α 4

β2 β3

α 6β2

α 6

β2 β3

α 3β2

α 3

β4 Y

α 3β2

α 3

β2 Y

α 3β4

α 3

β4 Y

Y

α 4β2

α 4

β2 xxX

X

α 6β2

α 4

β2 β3β3

α 6β2

α 6

β2 β3

α 3β2

α 3

β4 YY

α 3β2

α 3

β2 YY

α 3β4

α 3

β4 YY

Y

α9

α9

α9α9

α7α7 α7

α7

α7α8α8 α8

α8 α8

α8 α8

α1α1ε/γ

β1

β2

β2

α6

α3 α3 α3 α3α3α3

β3

α6α6α4

α4α4

β2

β2

β2

β2 β2

β4 β4

β4

β2

β2

α7 α7 α7

α10α10

α9 α9 α9

α9

δ

δ

= β2, α4, α5,…

= β2, β4, α5,…Y

γ

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Nicotinic Receptors

tryptophan residue characteristic of loop D (Figure 1); hence these subunits can act like γ and δ muscle subunits to provide the complementary binding site in an αβ pair. The absence of this residue in the β3 subunit makes it the true homologue of the muscle β1 subunit that does not contribute to an agonist binding site. Indeed, the sequence similarity between α5 and β3 subunits (see Figure 3) is consistent with both having this role.22

In contrast, the α7, α8 and α9 subunits are distinguished by their ability to form robust homomeric receptors in expression systems in the absence of a β subunit. Hence these subunits provide both primary and complementary faces of the agonist binding site,19 resulting in five putative binding sites per receptor monomer (Figure 3). It is not known if more than 2 sites must be occupied by the agonist to open the channel, but occupancy of a single site by the antagonist methyllycaconitine (MLA) is predicted to be sufficient to inhibit α7 nAChR function.23 In avian tissues α7α8 heteromers also occur and an association between α7 and β2 subunits has been suggested to occur in mammalian brain.24 A splice variant of the α7 nAChR subunit that incorporates a novel 87 base pair cassette in the N-terminal domain has also been reported to be expressed in rat intracardiac neurones, and possibly other tissues.25 It differs in having slower desensitisation kinetics and more reversible blockade by α-Bgt. The related α10 subunit is only incorporated into a functional nAChR when co-expressed with the α9 subunit.26,27

A distinct but related gene family of α and β subunits has been uncovered in invertebrates. The C. elegans genome sequence incorporates 29 candidate nAChR subunits, Drosophila melanogaster and Anopholes gambiae each have 10 nAChR subunit genes, while the honey bee Apis mellifera has 11 such genes.28,29 Twelve nAChR subunits have been found in the mollusc Lymnaea stagnalis.30

Distribution and Physiological Significance of nAChR SubtypesIn situ hybridisation has shown that nAChR subunits have distinct and often widespread distributions in the vertebrate nervous system. The subunit composition of native nAChRs has proved a more challenging quest. The following methodologies have contributed to the current understanding of subunit composition: subtype-selective radioligand binding (Table 5); pharmacological characterisation; single cell PCR and electrophysiology; immunoprecipitation with subunit-specific antibodies; knock out mice.• Autonomic neurones (including sympathetic

ganglia, parasympathetic innervation, sensory ganglia, chromaffin, neuroblastoma and PC12 cells) typically express α3, α5, α7, β2 and β4 subunits,31,32,33,34 with the likely assembly of α3β4*,

α3β2* and α7 nAChRs (where * indicates the possible inclusion of unspecified subunits; see Figure 3).35 Additional subunits (including α4 and α10) have been reported in dorsal root ganglia;36,37 nAChRs in these sensory neurones are of interest as therapeutic targets for modulating nociceptive signals.

• There is a heterogeneous distribution of α2-α7 and β2-β4 subunits in the mammalian CNS:19 α4, β2 and α7 are the most wide-spread subunits with α4β2* and α7 nAChRs having a somewhat complementary distribution. In contrast to their roles at the neuromuscular junction and in sympathetic ganglia, there are rather few reports of neuronal nAChRs mediating cholinergic synaptic transmission in the CNS. There is abundant evidence in the brain for presynaptic nAChRs that modulate the release of many different neurotransmitters38 and this has led to the unproven supposition that the majority of nAChRs are located presynaptically. However, nAChRs also exist on somatodendritic regions, in perisynaptic or extrasynaptic locations.39,40,41 The current perspective is that presynaptic and extrasynaptic nAChRs serve to modulate short and longer term neuronal activity in response to non-synaptic (‘paracrine’) levels of ACh (or choline, in the case of α7 nAChRs).42

The α7 nAChR is particularly prominent in the hippocampus, where it is found on GABAergic interneurones of stratum oriens and stratum radiatum, and on pyramidal neurones. Presynaptic α7 nAChRs are present on glutamate terminals and facilitate transmitter release in various brain regions, including hippocampus, cortex and ventral tegmental area.43,44,45 Nicotine acting at α7 nAChRs can enhance hippocampal LTP,46 and α7 nAChRs are associated with attentional processes and working memory.47,48 As a consequence, α7 nAChRs are a therapeutic target for treating cognitive impairment, notably in Alzheimer’s disease and schizophrenia, and this has prompted the generation of α7 nAChR-selective ligands,49 some of which are listed in Table 2.

α4β2 nAChRs have high affinity for nicotine (and account for >90% of [3H]-nicotine binding to brain tissues). A stoichiometry of (α4)2(β2)3 has been proposed, generating two agonist binding sites consistent with the model of the muscle nAChR (Figure 3).50,51 Manipulation of the stoichiometry of α4β2 nAChRs expressed in Xenopus oocytes indicates that (α4)3(β2)2 nAChRs are also viable, displaying lower affinity for ACh and higher Ca2+ permeability;52,53 whether native nAChRs with this subunit stoichiometry exist is not known.

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Table 2 | Potencies of selected compounds that interact preferentially with α7 nAChR

Drug CommentPotency

Binding FunctionAgonist Ki a EC50

b

AR-R17779 A synthetic, structurally rigid spirooxazolidone with 100-fold great potency for binding to α7 nAChRs than α4β2 nAChRs. No activation of α4β2, α3β4, α3β2, 5-HT3 receptors.254,255 Central effects at 1-2 mg/kg s.c.256

0.2 µM 10-20 µM

Choline The substrate and breakdown product of ACh is a weak α7-selective agonist, 10 times less potent than ACh.257,258,260 A very weak or partial agonist at α3β4* nAChRs in PC12 cells, noncompetitive inhibitor of α3β4* nAChRs in bovine chromaffin cells, blocks α3β4* and α4β2* nAChRs with IC50 of 15 and 370 µM. IC50 for desensitisation of α7 nAChRs ~40 µM.259,261,296

~2 mM 0.4-1.6 mM

Compound A A potent and selective α7 nAChR agonist (referred to a Compound B in earlier abstracts).262,263 In conjunction with the allosteric potentiator PNU 120596, 10 nM Compound A activates α-Bgt-sensitive Ca2+ signals in PC12 cells.265 Effective in vivo at doses of 3-10 mg/kg.264

40 nM 14 nM- 0.95 µM

GTS-21(aka DMXB)

A partial agonist at α7 nAChRs, eliciting 12-30% of maximum response to nicotine or ACh. Also interacts with α4β2 nAChRs (Ki = 84 nM versus [3H]-cytisine): very low partial agonist activity but significant antagonism of α4β2 nAChRs.266,267,268,269,296 Effective in vivo in cognitive tasks and normalises sensory gating.270

0.2-0.5 µM 6-26 µM

PNU 282987 A synthetic α7-selective agonist, with weak activity at 5-HT3 receptors (Ki = 0.9 µM). When administered at 1 mg/kg i.v. it restored amphetamine-induced sensory gating deficit and augmented hippocampal theta oscillation in anaesthetised rats.271,272,273

26 nM 128 nM

SSR180711 A synthetic α7-selective partial agonist (Emax = 36-50% of maximum response to ACh). Elicits central effects after i.p. or oral administration.274,275

~20 nM 1-4 µM

Competitive antagonists Kia IC50

c

α-Bungarotoxin Blocks α7, α8, α9* nAChRs; faster dissociation kinetics than at muscle nAChRs, but requires long preincubation for maximum effect. Preincubation time may be reduced by increasing concentration (e.g. 100 nM for 20 min).265,269,276 Not suitable for in vivo studies (unless locally applied).

0.5-1 nM (1-100 nM)

a-Conotoxin ImI From Conus imperialis, this peptide toxin selectively inhibits rat α7 nAChRs. Weaker antagonist of α9 (IC50 1.8 µM) and muscle nAChRs (50 µM), with no action at heteromeric nAChRs. Open channel blocker of 5-HT3 receptor.139,277,278 May be species selective, as reported to block bovine α3β4* nAChRs (IC50 2.5 µM).279 Also blocks certain invertebrate nAChRs.139

ND 86-220 nM

Methyllycaconitine(MLA)

Isolated from Delphinium sp., this hexacyclic norditerpenoid antagonist discriminates between muscle and α7 nAChRs, unlike α-Bgt.155,260,269,282 Crosses blood brain barrier following systemic administration, but access to brain is reduced after chronic nicotine treatment.280,281 Effective i.c.v. in rat (10 µg).256 The relatively high potency at α6β2* nAChRs compromises the use of MLA to define α7 nAChRs in catecholaminergic brain regions where α6 is expressed.157

1 nM 10-200 nM

MG 624 A 4-oxystilbene derivative that shows a 30-fold preference for blocking chick α7 nAChR, compared with α4β2 nAChRs.160,161 Related F3 derivative targets non-α7 nAChRs in rat chromaffin cells.162

100 nM 100 nM

Strychnine This glycine receptor antagonist is also a competitive antagonist at α7, α8, α9/α10 nAChRs; non-competitive block of muscle nAChRs and voltage-dependent block of heteromeric neuronal nAChRs (IC50 = 7-30 µM).27,66,283,284,285,286

6 µM 1 µM

Allosteric modulators EC50b

5-Hydroxyindole A metabolite of 5-HT that potentiates α7 nAChR responses to ACh: increases potency and efficacy of ACh without affecting desensitisation. Effective at 100 µM in brain preparations.287,288,289

2.5 mM

Ivermectin This semi-synthetic anthelminthic agent is an allosteric potentiator of α7 nAChRs; pre-application (16 seconds) necessary, effective at 30 µM. More potent interactions with mammalian GABAA and glycine receptors.290,291,292

ND

PNU 120596 Positive allosteric potentiator of α7 nAChR responses, prolongs agonist-evoked currents. No effect on responses from α4β2, α3β4 or α9α10 nAChRs. In vivo, CNS effects in response to 1mg/kg in rats. Limited solubility.265,293

200 nM

(Bold Text Denotes Compounds Available From Tocris)aKi, Inhibition constant, from competition binding assays for [125I]-α-Bgt or [3H]-MLA binding to brain membranes or heterologously expressed human or rat α7 nAChR;bEC50, effective concentration producing 50% of maximum activation; from electrophysiological recording of whole cell currents or intracellular Ca2+ responses, from native receptors in hippocampal neurones or cell lines or heterologously expressed human or rat α7 nAChR;cIC50, concentration of competitive antagonist producing 50% inhibition of functional responses; dependent on experimental conditions, especially agonist concentration and whether antagonist is co- or pre-applied (and, in the case of α-Bgt, preincubation time, due to slow association kinetics);n.d. = not determined

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Nicotinic Receptors

Transgenic knockout of either of these subunits eliminates nicotine self administration, whereas virally targeted re-expression of the β2 subunit in mesolimbic areas of β2 knockout mice recovers this behaviour, implicating a role for α4β2 nAChRs in nicotine addiction.54,55 α4β2 nAChRs are highly expressed in the thalamus. As a consequence of their putative role in thalamo-cortical circuitry, gain of function mutations in the M2 domain of either the α4 or β2 subunit give rise to some forms of autosomal dominant nocturnal frontal lobe epilepsy.56

α3 and β4 subunits have a much more restricted distribution in the CNS, for example in medial habenula and locus coeruleus they are often, but not always, co-expressed.19

α6 and β3 subunits are largely restricted to catecholaminergic neurones and contribute to nAChRs of complex subunit composition, e.g. α6β2β3 and α4α6β2β3 nAChRs on dopaminergic terminals.57 The β3 subunit is suggested to be necessary for the correct assembly, stability and/or targeting of α6* nAChRs.58 The α2 subunit has the most limited expression pattern of any nAChR subunit in the rodent CNS, being largely restricted to the interpeduncular nucleus.59 As a consequence its contribution to native nAChR has been rather little studied. However, its distribution in the primate brain appears to be more extensive.60

• Mechanosensory hair cells express (exclusively) α9 and α10 subunits that coassemble to generate predominantly heteromeric nAChRs that mediate effects of the efferent olivocochlear system on auditory processing.26,61

• Expression of nAChR subunits has also been detected in diverse non-neuronal cells. These comprise astrocytes, macrophages, keratinocytes, endothelial cells of the vascular system, muscle cells, lymphocytes, intestinal epithelial cells and various cell-types of the lungs.62,63 mRNAs encoding most nAChR subunits (but not α6) have been detected in such cells. The identity and functional significance of assembled nAChRs in non-neuronal cells remain poorly understood, although α7 nAChRs on macrophages have excited interest in the possibility that they might be involved in anti-inflammatory responses.64

Nicotinic Ligands for Neuronal nAChR Due to the critical roles of muscle and ganglionic nAChRs, nature has elaborated a diverse array of plant and animal toxins that target these receptors and their counterparts in the CNS. More recently, the perceived validity of neuronal nAChRs as therapeutic

targets has stimulated the generation of synthetic ligands to add to this pharmacopoeia. However, there remains a lack of subtype-selective tools, in particular antagonists. Only the α7 nAChR has a significant and growing list of selective agonists, antagonists and allosteric modulators, these are described in Table 2. Selected pan-acting or less discriminating agonists and antagonists are summarised in Tables 3 and 4 respectively, and some are briefly discussed below. More comprehensive accounts of the families of synthetic nicotinic ligands have been published recently.4,65

Agonists (Table �)Structurally diverse naturally occurring nicotinic agonists include: (-)-nicotine, (-)-cytisine, (+)-anatoxin A, (+)-epibatidine, anabasine and anabaseine. Synthetic agonists range from the classical “ganglionic agonist” dimethyl-phenylpiperazine (DMPP) developed in the 1960s, to novel agonists created more recently in order to provide greater subtype selectivity and therapeutic efficacy.4 Typically, agonists bind with highest affinity at the α4β2 nAChR, with 2-3 orders of magnitude lower affinity at α7 nAChRs and with intermediate affinity to α3* nAChRs (Table 3). With respect to functional potency, a similar relationship is observed, except that differences in EC50 values between subtypes are less marked, especially between α4β2 and α3* nAChRs. Agonists are ~2 fold more potent at α8 nAChRs66,104 and ~10 fold more potent in binding to α9α10 nAChRs,27 compared with α7 nAChRs. Binding affinities (Ki values) are typically 2-3 orders of magnitude lower, in terms of concentration, than EC50 values for nAChR activation (Table 3). With the exception of some recently described α7 nAChR-selective agonists described in Table 2, few agonists have sufficient nAChR subtype selectivity to exclusively activate one particular subtype in a mixed population. • ACh is the endogenous agonist for all nAChR

subtypes. It is a popular choice for activating nAChRs in electrophysiological experiments but its utility is compromised by its lack of selectivity for nAChRs versus muscarinic AChRs, and its susceptibility to hydrolysis. A muscarinic antagonist (typically atropine, ~1 µM) and an acetylcholinesterase inhibitor must be included with ACh in biological preparations; some of these agents may also interact with nAChRs (see below).

• Carbamoylcholine (carbachol) is formed by the modification of ACh to a carbamate, resulting in a hydrolysis-resistant analogue. This has reduced affinity at α4β2 and α7 nAChRs (Table 3) but is potent at muscarinic sites and is commonly used as a muscarinic agonist.

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Tocris Bioscience Scientific Review Series

Table 3 | Binding affinities and functional potencies of selected agonists at neuronal nAChRsa

AgonistnAChR subtype

α4β2* α7 α3β4 α4β2* α7 α3β4Binding affinity Ki (nM) Functional potency EC50 (µM)

A-85380 0.017-0.14103,107,108,110

17-290107,108,110 14-78103,107,110,298 0.7108 8.9108 0.8108

5-Iodo-A-85380 0.01-0.2103,110,111 250-6145110,111 50-280103,110,111 0.013111 – 5111

Acetylcholine 33-4467,103,339 4000-18000067,269,285,339

620-85067,103 0.5-68104,131,294,295 28-18080,104,131,269,285,296

35-20367,104,131,297

(+)-Anabasine 260-520125,339 58-340125,339 – – 16.8125 –

Anabaseine 32125 58-759125,269 – 4.2125 6.7125 –

(±)-Anatoxin A 1.9-3.5294,299 91-380269,299,300 53298 0.048-0.1390,294 0.58-3.990,300 –

Carbamoylcholine 35-1000

67,69,103,294,33912000-580000269,285,339

3839-4700103,298 17-34294,295 580285 –

(-)-Cotinine 0.04-0.06; >100000069,301

– – – (175*)80 –

(-)-Cytisine 0.012-1.5

67,69,103,110,294,339260-1500067,110,269,28

5,300,33954-22067,103,110,298

0.019-2.6131,294,295 5.6-71131,296,300 14-7267.131.297

Dimethylphenyl piperazinium (DMPP)

8.7-190 69,298,294,339 470-7600300,339 820103 1.9-18131,294,295 26-6480,131,300 14-19131,297

(±)-Epibatidine 0.042-0.15

67,103,107,29420-24067,107,269 0.21-0.98

67,103,107,2980.0045-0.0085104,295

1.2-1.3104,266 0.024-0.07104

Lobeline 1.5-1669,302,303 11600-13100269,303 – (0.73-21*)295,303 (8.5*)80 –

Methylcarbamyl-choline (MCC)

1.7-2867,69 4400067 2,70067 2.6294 – 4467

(-)-Nicotine 0.6-10

67,69,103,125,294,339400-15,00067,125,269,285,339

290-47667,103,107,298

0.35-5104,131,295 49-113131,266,269 8.1-11067,104,131,297

RJR 2403 26304 36000304 – 0.73-1685,304 24085 –

TC 2559 5.5-2298,344 >1000098 >100000344 0.18345 >100345 >30345

(±)-UB165 0.27305 2760305 6.5305 ** 6.9114 0.27-0.31114,305

Varenicline 0.17100 620100 85100 2.3-5.2100,306 18306 13-55100,306

(Bold Text Denotes Compounds Available From Tocris)*IC50 (drug inhibits rather than activating nAChR responses)**weak partial agonistareferences are indicated with each entry; preparations used are as follows:

Reference: Species and Preparation 67. Rat/HEK or tsA cells, or rat brain69. Rat α4β2/brain [3H]-nicotine binding80. Human α7/Xenopus oocytes85. Human/Xenopus oocytes90. Chicken/heterologous or Rat/native98. Human/mammalian cells/binding100. Human/HEK/neuroblastoma 103. Rat/HEK104. Human (or chicken)/Xenopus oocytes107. Rat brain/IMR32 cells/binding108. Rat brain/binding; Human/Xenopus oocytes or cell lines110. Rat brain/adrenal glands/binding111. Rat brain/rat α3β4 cells/binding/rat striatal dopamine release/rat α3β4

cells Ca2+ increases114. Human/Xenopus oocytes125. Rat/binding to brain/Xenopus oocytes131. Human/Xenopus oocytes266. Human α7/Xenopus oocytes269. Human α7/HEK cells

285. Chicken brain α7/binding294. Mouse thalamus (α4β2*)/[3H]-nicotine binding/Rb+ efflux295. Human α4β2/SH-EP1 cells296. Rat α7/Xenopus oocytes297. Human α3β4/HEK cells298. Rat α3β4/HEK cells299. Rat brain/binding300. Rat brain α7/binding; Chicken α7/Xenopus oocytes301. Rat brain/[3H]-epibatidine binding302. Rat brain α4β2/binding/Xenopus oocytes303. Rat brain binding/rat thalamus Rb+ efflux304. Rat brain/binding/rat thalamus Rb+ efflux305. Rat brain/rat α3β4 cells/binding/rat α3β4 cells Ca2+ increases306. Rat/Xenopus oocytes339. Rat/mouse brain/[3H]-nicotine and [125I]-α-Bgt binding 344. Rat cortex/binding345. Human/mammalian cell lines/Ca2+ respnses

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Nicotinic Receptors

N-Methylation of the carbamate nitrogen to yield N-methylcarbamylcholine (MCC) recovers high (nanomolar) binding affinity at α4β2 nAChRs, comparable to ACh.67 N-Methylation also confers substantial selectivity for nAChRs over muscarinic AChRs.68,69 The permanently charged quaternary nitrogen atom renders MCC membrane impermeant; this has been exploited to discriminate cell surface nAChRs from the total population that includes a large proportion of intracellular nAChRs.70

• (-)-Nicotine, a tobacco alkaloid, is the prototypic nAChR agonist that was used historically to classify the receptors.5 All nAChR subtypes are activated by nicotine, with the exception of α9 and α9α10 nAChRs; nicotine is an antagonist at these subtypes (IC50 values are 31 µM and 4 µM respectively).26,71 Neuronal heteromeric nAChRs exhibit a marked preference for the natural enantiomer over (+)-nicotine whereas α7 nAChRs appear to be less stereoselective. Nicotine crosses the blood brain barrier readily and its pharmacokinetics and metabolism are well documented.72 For a comprehensive discussion of nicotine doses used in vivo in various vertebrate and invertebrate organisms, see ref. 343. Behavioural responses typically show a bell-shaped dose-response profile with maximum responses in rats elicited by doses of 0.4 mg/kg s.c. or less. For chronic (continuous) administration, nicotine may be given via osmotic minipumps or indwelling s.c. cannulae and a daily delivery of 2-4 mg/kg/day in rats reproduces the plasma concentrations that are found in human smokers.73,74 Nicotine in flavoured drinking water (e.g. 2% saccharin, to disguise the aversive taste of nicotine) provides a means of self-delivery of nicotine chronically. This route is often used with mice (e.g. 200 µg/ml in drinking water)75 because surgical implantation of osmotic pumps in mice is less convenient due to their size. Mice have a much faster turnover of nicotine (T½ <10 min) than rats (T½ ~55 min) and consequently require higher doses.76,77,78 However, high doses of nicotine (>1 mg/kg s.c. in rats) produce adverse effects, most likely due to ganglionic or neuromuscular actions, although higher concentrations elicit convulsions. These doses refer to the free base concentration of nicotine; 3-times higher concentrations of the tartrate salt are required to achieve these concentrations of nicotine base; published doses should indicate the free base concentrations.

• Cotinine is the principal metabolite of nicotine. Its ability to displace nicotinic radioligands from binding to rat brain is inconsistent,69,79 but recently cotinine has been reported to have weak nicotinic

activity in in vitro functional assays and in a number of behavioural tests.80,81 Because of its extended half life (T½ = 5-6 hours, compared with <1 hour for nicotine in rat brain)77 cotinine attains high concentrations in vivo and can desensitise nAChR responses,80,82,83 although ganglionic nicotinic responses appear to be less affected.81

• Trans-metanicotine (RJR-2403 or TC-2403) generated by opening the pyrrolidine ring of nicotine, also occurs naturally as a minor tobacco alkaloid. It shows some functional selectivity for α4β2 nAChRs compared with α3* nAChRs,84,85 and is effective in vivo when administered at doses of 1-7 µmol/kg (0.2-1.2 mg/kg) s.c.86 It has recently been tested, with encouraging results, in an in vitro model of ulcerative colitis.87

TC 2559 and RJR 2403, Subtype-Selective α4β2 Ligands

TC 2559 N

OEt

MeHN

.2C4H4O4Cat. No. 2737

RJR 2403

N

NHMe

.C4H4O4Cat. No. 1053

TC 2559 and RJR 2403 (TC-2403) are subtype-selective ligands for α4β2 nicotinic acetylcholine receptors that display good CNS-PNS selectivity ratios. TC 2559 displays selectivity for (α4)2(β2)3 receptor stoichiometry and acts as a partial agonist, whilst RJR 2403 is a full agonist.

In vivoBoth compounds are active in vivo; TC 2559 and RJR 2403 significantly improve passive avoidance retention following scopolamide-induced cognitive deficits in rats.

RJR 2403 TC 2559α4β2 0.026 0.18α4β4 – 12.5α2β4 – 14.0α3β4 >1000 >30α1β1γδ >1000 –α3β2 15 >100α7 36 (Ki) >100

EC50 values (in µM). Data taken from Bencherif et al (1996) and Chen et al (2003).

Bencherif et al (1996) RJR-2403: a nicotinic agonist with CNS selectivity I. In vitro characterization. J.Pharmacol.Exp.Ther. 279 1413. Lippiello et al (1996) RJR-2403: a nicotinic agonist with CNS selectivity II. In vivo characterization. J.Pharmacol.Exp.Ther. 279 1422. Damaj et al (1999) Antinociceptive and pharmacological effects of metanicotine, a selective nicotinic agonist. J.Pharmacol.Exp.Ther. 291 390. Bencherif et al (2000) TC-2559: a novel orally active ligand selective at neuronal acetylcholine receptors. Eur.J.Pharmacol. 409 45. Chen et al (2003) The nicotinic α4β2 receptor selective agonist, TC-2559, increases dopamine neuronal activity in the ventral tegmental area of rat midbrain slices. Neuropharmacol. 45 334. Zwart et al (2006) 5-I A-85380 and TC-2559 differentially activate heterologously expressed α42 nicotinic receptors. Eur.J.Pharmacol. 539 10.

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• TC 2559 is the 5-ethoxy derivative of trans-metanicotine. This modification of the parent compound results in greatly improved selectivity for α4β2 nAChRs, coupled with relatively low efficacy (~30%).4,344,345 Interestingly, TC 2559 is suggested to discriminate between α4β2* and α6β2* nAChRs, as it provokes currents in midbrain dopamine neurones that are insensitive to α-conotoxin-MII.345 In vivo, TC 2559 (1-10 mg/kg) generalises to nicotine in a discriminative stimulus test that is considered to be mediated by α4β2 nAChRs.98 TC 2559 significantly attenuated scopolamine-induced cognitive deficits and reduced working memory errors in a radial arm maze, at doses of 1-6 µmol/kg. In contrast to nicotine, no locomotor or hypothermia effects were observed, consistent with reduced peripheral side effects.344

• Anatoxin A is a potent, semi-rigid, stereoselective agonist originally isolated from freshwater blue green algae, Anabaena flos aqua.88 Activity resides in the natural (+)-enantiomer. At muscle nAChRs, anatoxin A is about 8 times more potent than ACh89 but it activates neuronal nAChR subtypes at sub-micromolar concentrations (Table 3), with EC50 values that are 20-100 times lower than those for ACh.90,91 Despite being a secondary amine (that should cross the blood brain barrier readily), there are few reports of the in vivo effects of anatoxin A, although two reports suggest that its responses are qualitatively different from those of nicotine.92,93

(±)-Anatoxin A, Potent Nicotinic Agonist

(±)-Anatoxin A NHMe

O

.C4H4O4Cat. No. 0789

(±)-Anatoxin A is a nicotinic receptor agonist (Ki values are 3.5 and 380 nM for α4β2 and α7 nicotinic receptors respectively). The agonist stimulates [3H]-dopamine release from rat striatal synaptosomes (EC50 = 136 nM) with a higher potency than (-)-nicotine. (±)-Anatoxin A displays powerful behavioural effects in the rat; it decreases locomotor activity in nicotine-tolerant and non-tolerant rats and decreases rates of operant responding in a drug discrimination procedure.Wonnacott et al (1991) Nicotinic pharmacology of anatoxin analogs. II. Side chain structure-activity relationships at neuronal nicotinic ligand binding sites. J.Pharmacol.Exp.Ther. 259 387. Stolerman et al (1992) Behavioural effects of anatoxin, a potent nicotinic agonist in rats. Neuropharmacology 31 311. Thomas et al (1993) (+)-Anatoxin is a potent agonist at neuronal nicotinic acetylcholine receptors. J.Neurochem. 60 2308. Sharples et al (2000) UB-165 implicates α4β2 nAChR in striatal dopamine release. J.Neurosci. 20 2783.

• (-)-Cytisine occurs in a number of plants of the leguminosae family including laburnum. Its rigid structure has provided a template for modelling nicotinic ligands.94 At α4β2 nAChRs it is comparable to nicotine, with respect to its high

affinity binding (Ki ~1 nM), but its differential interactions with other nAChR subtypes has enabled it to be used to distinguish subpopulations of nicotinic binding sites labelled by [3H]-epibatidine that differ in having high or low affinity for cytisine.95 At α4β2* nAChRs cytisine is a partial agonist; its functional efficacy is dependent on the identity of the β subunit present. Thus cytisine displays full efficacy at nAChRs containing the β4 subunit expressed in Xenopus oocytes, while greatly reduced efficacy is observed at β2-containing nAChRs.96 Halogenation at the 3-position of the pyridine ring increases both potency and efficacy.94 Cytisine is less potent than nicotine in behavioural studies, and shows only partial generalisation to nicotine in a drug discrimination test; doses of 1-3 mg/kg are effective in vivo.97,98

• Varenicline (ChantixTM (USA); ChampixTM (EU)) is a cytisine congener developed to exploit the properties of cytisine (selectivity and partial agonism with respect to α4β2* nAChRs) as an aid to smoking cessation.99 Although it appears selective for α4β2* nAChRs in binding assays, its agonist potencies at different nAChR subtypes, determined by electrophysiological recordings from heterologous expression systems, show less discrimination306 (Table 3). Varenicline is effective in releasing dopamine in vitro and in vivo (efficacy ~45% of maximum response to nicotine) but it is also capable of attenuating nicotine-evoked dopamine release, a reflection of its partial agonist properties.100 Indeed, varenicline is more potent at inhibiting nicotine-evoked responses in Xenopus oocytes (IC50 = 6 nM) than it is at eliciting responses (EC50 = 3 µM), interpreted as a reflection of the higher affinity for agonists shown by the desensitised state of nAChRs (α4β2 nAChRs in particular) (see Figure 2). Varenicline is effective in vivo at doses of 0.01-3.0 mg/kg (given s.c. or p.o.)100 and has a half life of 4 and 17 hours in rats and humans respectively, with little metabolism.101

• Epibatidine, originally obtained from skin extracts of the Amazonian frog Epidobates Tricolor, is one of the most potent nicotinic agonists,102 binding to multiple heteromeric nAChRs with sub-nanomolar affinities.103 Like anatoxin A, epibatidine also has a bicyclic moiety that confers some rigidity to its structure, but in this case it is a smaller azabicycloheptane ring, coupled to a chloropyridyl moiety. In contrast to anatoxin A, the enantiomers of epibatidine show equivalent biological activities.102,104,105 The functional potency of epibatidine is exceptionally high, with sub-micromolar EC50 values for heteromeric

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Nicotinic Receptors

and α8 neuronal nAChR subtypes. Only α7 and muscle nAChRs exhibited higher EC50 values, in the low micromolar range.4,104,105 In vivo, epibatidine displays potent non-opioid analgesic activity.102 The therapeutic window for epibatidine is very narrow, attributed to side effects that reflect its lack of nAChR subtype selectivity, but it has generated interest in neuronal nAChRs as potential targets for antinociceptive drugs.106

• A-85380 came out of a synthetic programme that aimed to recapitulate the potency of epibatidine at α4β2* nAChRs while minimising interactions with other nAChR subtypes.107 Like epibatidine, A-85380 binds to heteromeric nAChRs with sub-nanomolar affinities but its interaction with α7 or Torpedo nAChRs requires >1000 times higher concentrations.108 A-85380 has been used to distinguish populations of [125I]-epibatidine binding sites in mouse brain that are either sensitive or resistant to inhibition by this ligand; the A-85380-resistant population is also heterogeneous, with respect to the involvement of α3 nAChR subunits.109 Functionally, A-85380 is a potent full agonist in several functional assays that correlate with different nAChR subtypes. In vivo A-85380 (0.01-0.1 mg/kg s.c.) fully generalises to nicotine in an operant drug discrimination task in rats, a behavioural response attributed to α4β2 nAChRs.98 A-85380 has analgesic properties, with ED50 values of 0.1-0.2 µmol/kg i.p. in the rat; side effects (transient prostration and ataxia) occur at doses of 0.5 µmol/kg and above.107

• 5-Iodo-A-85380 was created by introduction of an iodine atom in the pyridine ring of A-85380 to generate an iodinated radioligand (see Table 5).

This analogue shows improved functional selectivity for β2-containing nAChRs over other nAChR subtypes.110,111 It has good credentials in vivo for use as a SPECT ligand,112 and has been reported to be self-administered by rats.113

• UB-165 is a novel hybrid molecule, comprising the azabicyclononene bicycle of anatoxin A and the chloropyridyl moiety of epibatidine.114 UB-165 exhibits intermediate potency, with stereoselectivity comparable to that of anatoxin A. In contrast to the parent molecules, it is a partial agonist at α4β2* nAChRs.114,115

• Dimethylphenylpiperazine (DMPP) has a long history as a ganglionic agonist. It shows little selectivity between neuronal nAChR subtypes with respect to binding affinity or potency of activation,4 hence it is relatively more potent at α3β4* nAChRs than nicotine, and has been used in preparations in which such ganglionic-type nAChRs prevail. For example, DMPP was recently used to show that nAChRs exert an anti-inflammatory influence in monocytes and macrophages.116 DMPP is a partial agonist at various heterologously expressed nAChR subtypes.117,118 It is membrane impermeant, due to the quaternary nitrogen atom, and has been used to selectively block cell surface nAChRs.119 The related compound 1-acetyl-4-methylpiperazine is a weaker nicotinic agonist that exhibits a behavioural profile indicative of different pharmacodynamics compared with nicotine.120 The methiodide salt is 100 times more potent but does not access the brain.

• Lobeline, an alkaloid from the Indian tobacco Lobelia inflate, is an atypical nicotinic ligand and shows anomolous behaviour in vivo. Lobeline binds with high affinity to α4β2* nAChRs, and has both agonist and antagonist activities.121,122 In addition, it inhibits vesicular monoamine (IC50 ~1 μM) and dopamine transporters (IC50 = 40-100 μM) and μ-opioid receptors (IC50 = 1 μM).123 In vivo, lobeline shows some nicotine-like effects but also differs from nicotine in several behavioural assays, for example lobeline at doses of 0.3 and 0.9 mg/kg improves learning on a radial arm maze task in rats, whereas nicotine (0.1, 0.3 mg/kg) does not.124

• Anabasine is a tobacco alkaloid with rather non-selective nicotinic activity. Anabaseine, a toxin that occurs naturally in nemertines (a marine worm), differs from anabasine only in bond order, but this difference confers a functional selectivity for α7 nAChRs.125 This structure led to the development of a series of derivatives of which GTS-21 is the best known (see Table 2).

5-Iodo-A-85380, Potent, Subtype-Selective Nicotinic Agonist

5-Iodo-A-85380 NH

O

I

N

.2HCl

Cat. No. 1518

5-Iodo-A-85380 is a highly potent and subtype-selective ligand for the α4β2 and α6β2 nicotinic acetylcholine receptors. The agonist activates α-conotoxin-MII-sensitive and -insensitive components of [3H]-dopamine release from rat striatal synaptosomes, corresponding to α6β2 and α4β2 receptors (EC50 values are 12.7 and ~35 nM respectively). The compound is ~ 5000-, 25000- and 140000-fold selective over α3β4, α7 and muscle nAChR receptors respectively.Koren et al (1998) 2-, 5-, and 6-Halo-3-(2(S)-azetidinylmethoxy)pyridines: synthesis, affinity for nicotinic acetylcholine receptors, and molecular modeling. J.Med.Chem. 41 3690. Mukhin et al (2000) 5-Iodo-A-85380, an α4β2 subtype-selective ligand for nicotinic acetylcholine receptors. Mol.Pharmacol. 57 642. Mogg et al (2004) Functional responses and subunit composition of presynaptic nicotinic receptor subtypes explored using the novel agonist 5-iodo-A-85380. Neuropharmacology 47 848.

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• Levamisole is an anthelmintic that acts as a full agonist at nematode muscle nAChRs, producing muscle paralysis.28 Levamisole-sensitive muscle nAChRs in C. elegans contain the α subunits UNC-38, UNC-63 and LEV-8, and non-α subunits UNC-29 and LEV-1. In cultured C. elegans muscle cells, levamisole activates single channels with a conductance similar to that of channels activated by ACh. It is 5 times more potent than ACh, activating currents at concentrations greater than 100 nM.126 The structurally unrelated anthelmintic agonists pyrantel and morantel activate the same receptors. Levamisole is devoid of agonist activity at mammalian nAChRs but has been reported to act as an allosteric modulator (see below).

• Imidacloprid is a neonicotinoid drug that acts at nAChRs in the insect CNS; these receptors constitute a major pesticide target.127 Data from heterologous expression systems indicate that nAChRs containing Drosophila α2 subunits are susceptible to activation by imidacloprid (EC50 ~2 µM),128 whereas those containing Dα1 subunits are not. Mutagenesis studies highlight loops B and C in the primary binding site of Dα2 subunit (see Figure 1) as important for neonicotinoid selectivity (and possibly resistance).127

Imidacloprid acts as a weak agonist at avian α7 nAChRs (EC50 = 270 µM) but showed no activity at α4β2 nAChRs.129.

• Tropisetron is a 5-HT3 receptor antagonist that displays potent partial agonist at α7 nAChRs (EC50 ~1 µM). Low micromolar concentrations of this compound are also reported to inhibit α3β4 nAChRs.4.

Antagonists (Table �)Competitive antagonistsCompetitive antagonists interact reversibly with the nAChR at, or close to, the agonist binding site, stabilising the receptor in a conformation with the channel closed and preventing access for agonists. Inhibition by reversible competitive antagonists is surmountable with increasing agonist concentration, shifting the concentration response relationship to the right (e.g. dihydro-β-erythroidine (DHβE) versus nicotine-evoked [3H]-dopamine release130). Consequently the degree of functional blockade achieved by a given concentration of competitive antagonist will be influenced by the agonist concentration. Most competitive antagonists originate from a wide variety of natural sources. Unfortunately there is a very limited range of subtype-selective

Table 4 | Specificity of selected antagonists for neuronal nAChRsa

Antagonist Mode of action SpecificityEffective concentration rangeb

ReferenceIn vitro In vivo

d-Tubocurarine Competitive (and other interactions)

Non-selective; Also blocks 5-HT3 > GABAA

10 µM Muscle block predominates

4, 131, 339

Dihydro-β-erythroidine (DHβE)

Competitive β2>β4 1-10 µM 1-6 mg/kg 4, 137, 138, 166, 295, 307

a-Conotoxin MII Competitive? ‘Dock and lock’

α3β2*/α6β2* 10-120 nM Does not access brain;local injection: 5 nmol/VTA0.25-25 pmol/locus coeruleus

139, 141, 145, 147, 308, 309

a-Conotoxin AuIB Competitive? α3β4* 1-10 µM Does not access brain;local injection: 1-25 pmol/locus coeruleus

139, 140, 148, 309

Mecamylamine Non-competitive Neuronal αβ > α7

1-10 µM 1-4 mg/kg s.c. or i.p. 4, 149, 166, 310, 311

Hexamethonium Non-competitive / weakly competitive

Neuronal 1-100 µM Does not access brain1-3 mg/kg s.c. for ganglionic blockade

149, 165, 166, 339

Chlorisondamine Non-competitive / long lasting (weeks or months)

Neuronal (persistent block of CNS nAChRs, transient block of ganglionic nAChRs)

10-100 µM 10 mg/kg s.c.10 µg i.c.v.

168, 169, 171,312

TMPH Non-competitive: use-dependent, voltage-independent

αβ>α7 or muscle; influenced by addition of α5, α6, β3

0.1-10 µM 1-5 mg/kg s.c. 173, 174

(Bold Text Denotes Compounds Available From Tocris)aFor muscle and α7 nAChR-selective antagonists see Tables 1 and 2 respectively.bRange of concentrations typically used to achieve substantial functional blockade in rodents

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Nicotinic Receptors

agents and not all are commercially available. Table 4 lists some antagonists that have been used to inhibit neuronal nAChRs; those with specificity for α7 nAChRs are included in Table 2.

• d-Tubocurarine (d-TC), produced by the South American shrub Chondodendron tomentosum, is a classical non-selective nAChR antagonist. d-TC does not discriminate appreciably between nAChR subtypes and it is able to fully antagonise functional responses mediated by muscle and neuronal nAChRs at a concentration of approximately 10 μM.131 It should be noted that the mechanism of inhibition by d-TC can be complex, also involving non-competitive interactions.117 In addition, d-TC is a potent 5-HT3 receptor antagonist; it binds with Ki ~0.1 µM, and 1-10 µM d-TC inhibits 5-HT3 receptor-mediated currents in hippocampal interneurones and transfected HEK-293 cells.132,133,134 Higher concentrations of d-TC also block GABAA receptor-mediated currents.135

• Dihydro-β-erythroidine (DHβE), an alkaloid originating from Erythrina seeds, is a purely competitive antagonist for neuronal nAChRs. Sub-micromolar concentrations of DHβE block human and rat α4β2 and α3β2 nAChRs but it is 10-50 fold less potent at α3β4 and α7 nAChRs expressed in Xenopus oocytes.4,131,136 In hippocampal neurones, 100 nM DHβE blocked type II currents, attributed to α4β2*, whereas type I currents mediated by α7 nAChRs were insensitive to DHβE concentrations below 10µM.91 Therefore DHβE can be regarded as a non-α7 nAChR antagonist with a preference for β2-containing subtypes; it is typically employed at a concentration of 1-10 µM. DHβE is also effective in vivo (see Table 4), for example, it has been used to implicate non-α7 nAChRs in nicotine reward and in the enhancement of contextual fear conditioning by nicotine.137,138

• α-Conotoxins, present in the venoms of various species of Conus snails, provide a growing family of subtype-selective nAChR antagonists.139,140 α-Conotoxins are small peptide toxins (12-18 amino acids) with 4 cysteine residues forming two disulphide bonds, between the first and third, and second and fourth cysteines. They can be generated by peptide synthesis with sequential de-protection of the cysteine pairs.141 The different specificities of related α-conotoxins that retain the cysteine residues in the same positions reflect the side chains of the non-conserved amino acids. Three well characterised α-conotoxins with different specificities for neuronal nAChRs are α-conotoxins MII, AuIB and ImI. These have been exploited in in vitro studies. Their peptidergic

nature means that they do not cross the blood brain barrier but examples of local delivery of these toxins into the brain are given in Table 4. Certain α-conotoxins can adhere to plastic: use of sialinised glass or plastic and/or addition of BSA can reduce this problem.

• α-Conotoxin MII was originally shown to be a potent and selective antagonist of α3β2 nAChRs expressed in Xenopus oocytes; α3β2 nAChRs are fully blocked by 100 nM toxin. At other heterologously expressed nAChRs, the potency of α-conotoxin MII was 2-4 orders of magnitude lower.141,142 It has subsequently been found to be an equally effective antagonist of α6β2* nAChRs, reflecting the sequence similarities between α3 and α6 subunits (Figure 3).143 α-Conotoxin MII has been especially useful in elucidating the contribution of α3/α6β2* nAChRs to dopaminergic pathways in rodents and primates; it is typically used at concentrations of 10-120 nM.57,144,145,146 Recently α-conotoxin PIA from Conus purpurascens has been described; it is the first ligand to exhibit a marked preference for α6β2* over α3β2 nAChRs.147 The mode of action of α-conotoxin MII and related α-conotoxins is complex. Studies of binding kinetics, crystal structures and docking simulations have indicated a 2 step ‘dock and lock’ model, with initial binding to the complementary β nAChR subunit followed by a stabilising interaction with the primary α subunit.4

• α-Conotoxin AuIB from Conus aulicus is a much less potent toxin but the only one reported to have selectivity for α3β4* nAChRs; 1-10 μM α-conotoxin AuIB is required for complete blockade of α3β4 nAChRs.148 α-Conotoxin AuIB partially inhibits nicotine-evoked [3H]-noradrenaline and [3H]-ACh release from rat brain preparations, implicating α3β4* nAChRs in these responses, consistent with weak sensitivity to DHβE.148,149

• α-Conotoxin ImI (Table 2) is a selective antagonist of α7 nAChRs, fully blocking responses at 1µM.150,151 It has weaker interactions with muscle and α9 nAChRs (see Table 2). More potent α-conotoxins with greater selectivity for α7 nAChRs over other subtypes are under investigation.152 One caveat with regard to using the α-conotoxins is that their specificity may be highly species specific, limiting extrapolation between species. For example, although α-conotoxin ImI inhibits rat α7 nAChRs, it is reported to block a non-α7 nAChR (but not α7 nAChRs) in bovine chromaffin cells.153

• α-Bungarotoxin (α-Bgt; Tables 1, 2) is the most well-established subtype-selective nicotinic antagonist. Isolated from the venom of the Taiwanese banded krait, Bungarus Multicinctus,

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it is an 8kDa peptide that binds to muscle and Torpedo nAChRs, and to α7-α9* nAChRs with an affinity (Ki) of ~1 nM. However, its association binding kinetics are very slow and typically a pre-incubation of up to one hour with a low nanomolar concentration (10 nM) of toxin is necessary to achieve a complete blockade. This is commonly circumvented by increasing the concentration and decreasing the preincubation time. This strategy is possible because, even at micromolar concentrations, α-Bgt does not appear to interact with α/β heteromeric nAChRs. However, it was recently reported to block a subset of GABAA receptors containing an adjacent pair of β3 subunits (Kd ~50 nM; t½ ~10 min); whether GABAA receptors with this subunit composition exist in nature is presently unclear.154 α-Bgt binding to nAChRs exhibits very slow dissociation kinetics, such that functional blockade is not reversed by washout within the timescale of a typical experiment (e.g. 1 hour).

• Methyllycaconitine (MLA, Table 2) is a norditerpenoid alkaloid produced by Delphinium sp. It is a potent competitive antagonist, selective for α7 nAChRs and, unlike α-Bgt, it discriminates between α7 and muscle nAChRs.155 MLA binds to α7 nAChRs with a Ki of approximately 1 nM, and picomolar concentrations are reported to block α7 nAChR-mediated currents recorded from hippocampal neurones or Xenopus oocytes.23,156 Inhibition of α7 nAChRs by MLA is rapid and reversible, making it a useful complement to

α-Bgt. However, like α-Bgt, MLA also blocks α9 and α9α10 nAChRs with low nanomolar affinity. Of more practical concern for brain studies, MLA interacts with α6β2* nAChRs with only ~30-fold lower affinity (Ki ~30 nM) than with α7 nAChRs.40,157 In addition, non-α7 interactions of MLA have been indicated in avian preparations, where functional responses sensitive to MLA, but not α-Bgt, have been observed.158,159 Therefore, this antagonist is selective, rather than specific, for α7 over other nAChR subtypes. In particular, its interactions with α6β2* nAChR subtypes could confound interpretation of results from in vivo studies, in which the local concentration of MLA is not known.

• MG 624 (Table 2) is a 4-oxystilbene derivative that is a selective and quite potent antagonist of chicken α7 nAChRs. The IC50 for blocking α7 nAChR responses (100 nM) is ~30 fold lower than that for muscle-type or α4β2 nAChRs.160,161

However, the related oxystilbene derivative F3 (that displayed similar properties to MG 624 in chick preparations)160 was found to block non-α7 nAChR responses in rat chromaffin cells, albeit with somewhat lower potency (IC50 ~350 nM).162 This raises the possibility that MG 624 may also display species differences or a broader range of nAChR interactions than first thought. Indeed, MG 624 significantly attenuated nicotine-evoked [3H]-dopamine release in rat cortical slices, in contrast to α-conotoxin ImI, α-Bgt or MLA.115

Non-Competitive AntagonistsNon-competitive antagonists, as indicated above, do not compete for binding to the agonist binding sites, but interact with distinct sites that can include the lumen of the nAChR channel. • Mecamylamine is the archetypal non-

competitive antagonist for neuronal nAChRs. It was developed as a ganglionic blocker for the treatment of hypertension. Mecamylamine inhibits most neuronal nAChRs with IC50 values typically in the range 0.1-1 µM, and 10 µM mecamylamine is a standard concentration used to achieve a complete block in vitro. α7 nAChRs are somewhat less sensitive than α/β heteromers, requiring >10 μM for full blockade.4,131 High concentrations of mecamylamine (100 µM) can transiently inhibit NMDA receptors.163 Mecamylamine crosses the blood brain barrier freely and is typically administered at a concentration of around 1mg/kg in rodents to block CNS nAChRs in behavioural studies. Unexpectedly, lower concentrations of mecamylamine have been reported to improve cognitive performance.164

• Hexamethonium, like mecamylamine, was also first recognised as a ganglionic nAChR

Methyllycaconitine, α7 nAChR Antagonist

Methyllycaconitine MeO OMe

OH

O

ON

Me

OMe

NMe

.C6H8O7

H

O O

OMe

OH

HCat. No. 1029

Methyllycaconitine is a potent competitive antagonist of α7-containing neuronal nicotinic receptors (Ki = 1.4 nM) that also interacts with α4β2 and α6β2 receptors at nanomolar concentrations. Unlike α-Bgt, MLA can distinguish between α7 and muscle nAChRs. The antagonist attenuates methamphetamine-induced neurotoxicity in the mouse striatum in vivo.Ward et al (1990) Methyllycaconitine: a selective probe for neuronal α-bungarotoxin binding sites. FEBS Lett. 270 45. Wonnacott et al (1993) Methyllycaconitine: a new probe that discriminates between nicotinic acetylcholine receptor subclasses. Methods in Neurosci. 12 263. Dobelis et al (1999) Effects of delphinium alkaloids on neuromuscular transmission. J.Pharmacol.Exp.Ther. 291 538. Escubedo et al (2005) Methyllycaconitine prevents methamphetamine-induced effects in mouse striatum: involvement of α7 nicotinic receptors. J.Pharmacol.Exp.Ther. 315 658.

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blocking agent but the hydrophilic nature of this polymethylene bistrimethylammonium compound prevents it from crossing the blood-brain barrier.7 Comparative studies of the effects of mecamylamine and hexamethonium in vivo have been used to establish if a particular behaviour is centrally or peripherally mediated, as peripherally administered hexamethonium is unable to block CNS nAChRs in vivo.165 In vitro hexamethonium is used at 1-100 µM to block neuronal nAChRs.149,166

• Chlorisondamine, a bisquaternary nicotinic antagonist, was also originally used as a ganglionic blocker.167 When administered in vivo it is unique in producing a persistent blockade of nAChRs within the CNS that lasts for weeks or even months, in contrast to a transient ganglionic blockade. For example, nicotine-evoked dopamine release was abolished from rat striatal synaptosomes prepared several weeks after administration of chlorisondamine in vivo (10 mg/kg s.c).168 It is hypothesised that this long lasting inhibition may arise from an intracellular accumulation of the drug,169 and a specific interaction with the α2 nAChR subunit has been proposed.170 However, given the paucity of α2 nAChR subunit expression in the rodent brain, this is unlikely to explain the widespread central antagonism by chlorisondamine. Its persistent action is particularly useful for antagonising brain nAChRs during chronic studies involving repeated or continuous nicotine administration.171

• 2,2,6,6-Tetramethylpiperidin-4-yl heptanoate (TMPH) is a synthetic derivative of the parent bis-tetramethylpiperidine compound BTMPS. The latter produces a nearly irreversible non-competitive block of neuronal nAChRs, whereas its inhibition of muscle nAChRs is readily reversible.172 Similarly, low micromolar concentrations of TMPH produced a long-lasting inhibition of heteromeric nAChRs comprised of α3 or α4 with β2 or β4 subunits, whereas blockade of α7 and muscle nAChRs was readily reversible, allowing the different classes of nAChRs to be distinguished. However, incorporation of an additional subunit (either α5, α6 or β3) into heteromeric nAChRs also decreased inhibition, attributed to a residue within the M2 channel lining domain (Figure 1).173 TMPH was also effective in vivo, showing a differential inhibition of nicotine-induced analgesia and nicotine discrimination, compared with hypothermia and locomotor effects.174

Other Modulators Producing Non-Competitive Inhibition of Neuronal nAChRsMany compounds that have other primary targets also act as non-competitive antagonists of

nAChRs.175 These agents cannot be considered to be specific for nAChRs, but the interactions can be of pharmacological or physiological interest or they may raise practical concerns. The examples mentioned here do not constitute an exhaustive list but are chosen to represent some of the more well known or topical classes.

• MK-801 (dizocilpine) is an anticonvulsant agent developed as a channel blocker of the NMDA receptor. It is able to perform the same function on neuronal nAChRs, where it is an open channel blocker at α4β2 (IC50 = 15 μM) and α7 nAChRs (IC50 = 15 μM).176,177,178 Studies on Torpedo nAChRs indicate that MK-801 may also interact with a non-luminal site, in addition to the channel.179 The dissociative anaesthetics phencyclidine (PCP) and ketamine that interact with NMDA receptors also block nAChRs (IC50 ~2-10 μM).180,181,182 The tryptophan metabolite kynurenic acid, a competitive antagonist of the glycine site of NMDA receptors, is also a non-competitive antagonist of α7 nAChRs (IC50 = 7 μM).175,183

• Buproprion (ZybanTM) was originally developed as an antidepressant but is now marketed as an aid to smoking cessation. Its principle site of action is the dopamine and noradrenaline transporters, but at low micromolar concentrations it also inhibits nAChRs.184,185 It non-competitively inhibits rat α3β2, α4β2 and α7 nAChRs expressed in Xenopus oocytes or native nAChRs in cell lines and nicotine-evoked [3H]-dopamine release from rat striatal preparations.186,187,188,189,190

The possibility that this interaction contributes to the efficacy of bupropion as a smoking cessation agent is debated. Low micromolar concentrations of other antidepressants including fluoxetine, sertraline and paroxetine also inhibit nAChRs (IC50 = 1-12 µM).191,192 The neuroleptic chlorpromazine and the anti-epileptic drug lamotrigine also interact with nAChR channels.193,194

• L-type voltage-operated Ca2+ channel blockers inhibit nAChRs in chromaffin cells or neuroblastoma cell lines with IC50 values in the low micromolar range. These drugs include furnidipine, verapamil and diltiazem and the dihydropyridines: nimodipine, nifedipine, nitrendipine and furnidipine.195,196,197 The N/P/Q-type calcium channel blocker ω-conotoxin MVIIC and the N-type blocker ω-conotoxin GVIA were without effect, although another study reported that they too can block nAChRs expressed in Xenopus oocytes, with rat α3β4 nAChRs being more susceptible than α7 nAChRs.198 The block of α3β4 nAChRs by ω-conotoxins was shown to be reversible, whereas these inhibitors exert

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a longer lasting inhibition of voltage-operated calcium channels. This difference has been exploited to discriminate between these two targets.199

• Steroids, including corticosterone, progesterone, estradiol, hydrocortisone and aldosterone, have been shown to inhibit neuronal nAChRs expressed in the SH-SY5Y cell line, as well as α4β2 nAChRs in HEK-293 cells or Xenopus oocytes, with IC50 values ranging from 0.1-10 μM.200,201,202

• β-Amyloid peptide Aβ1-42, the endogenous agent that accumulates in Alzheimer’s disease, is reported to interact with nAChRs, principally the α7 subtype, in a variety of ways.203 Aβ has been reported to bind competitively (IC50 ~5 pM) to α7 nAChRs204 but to inhibit nAChRs non-competitively (1-100 nM).205,206,207 Picomolar concentrations of Aβ have been found to activate α7 and non-α7 nAChRs in Xenopus oocytes or synaptosomes,208,209 but these results are controversial.205,207 α7 nAChRs have also been proposed to mediate the modulation of NMDA receptor trafficking by Aβ.210 The nature of the interactions of Aβ with nAChRs is particularly intriguing in view of the changes in nAChRs in Alzheimer’s disease and the ability of nicotinic agonists to enhance cognition and/or offer neuroprotection, but more work is required to clarify the relationship.

Positive Allosteric ModulatorsIn addition to divalent cations such as Ca2+ and Zn2+, several structurally diverse compounds can potentiate responses to nicotinic agonists by acting at a site distinct from the agonist binding site. Such effects are well established for other receptor classes (e.g. benzodiazepines and glycine, acting at GABAA and NMDA receptors respectively), but have only relatively recently been characterised for neuronal nAChRs.4,175

• Physostigmine and galanthamine were among the first acetylcholinesterase inhibitors to be recognised. These compounds potentiate nAChR responses evoked by sub-maximal concentrations of nicotinic agonists, thus shifting the dose response curve for agonists to the left. Alone, physostigmine or galanthamine can activate single-channel currents in muscle and neuronal cells but the probability of channel opening is too low to generate macroscopic (whole cell) currents.211 Galanthamine and physostigmine produce a modest potentiation (~30%) of ACh-evoked currents or nicotine-evoked increases in intracellular Ca2+ over a narrow concentration range (1-10 µM); higher concentrations (>10 µM) inhibit responses by acting as an open channel blocker.175,212,213,214 In addition to galanthamine’s

effect as an AChE inhibitor, its ability to enhance the effects of residual ACh could contribute to the efficacy of galanthamine (ReminylTM) as a treatment for cognitive decline in Alzheimer’s disease.

The site of action of these positive allosteric modulators is proposed to be at, or close to, Lys 125 of the extracellular N-terminal domain of the neuronal nAChR α subunit.211 Other allosteric potentiators acting at the same site include the opiate codeine, the neuromuscular blocking agent benzoquinonium (Table 1) and the neurotransmitter 5-HT; sub-micromolar concentrations of 5-HT mimic the potentiating effects of galanthamine in PC12 cells.175,211 Interestingly 5-hydroxyindole, a metabolite of 5-HT, is also a positive allosteric modulator but shows selectivity for α7 nAChRs (Table 2).215 The site of action of 5-hydroxyindole has not been established. Recently another more potent and more efficacious synthetic positive allosteric modulator of α7 nAChRs has been described; PNU 120596 (see Table 2).217 Such compounds could have therapeutic utility in maximising the effects of endogenous agonists.

PNU 120596 and PNU 282987, Selective α7 nAChR Ligands

PNU 120596 OMeMeO

ClHN

NH

O

NOCat. No. 2498

PNU 282987 N

NH

O

Cl

Cat. No. 2303

PNU 120596 is a positive allosteric modulator of α7 neuronal nicotinic acetylcholine receptors (EC50 = 216 nM) that displays with no detectable effect on α4β2, α3β4 and α9α10 receptors.

PNU 282987 is a highly selective α7 nAChR agonist (Ki = 26 nM) that displays negligible blockade of α1β1γδ and α3β4 nAChRs (IC50 ≥ 60 µM). The agonist was found to be inactive against a panel of 32 receptors at 1 µM, except 5-HT3 receptors (Ki = 930 nM).

In vivoBoth compounds improve auditory gating deficits induced by D-amphetamine in anesthetised rats; a model proposed to reflect a circuit level disturbance associated with schizophrenia. Bodnar et al (2005) Discovery and structure-activity relationship of quinuclidine benzamides as agonists of α7 nicotinic acetylcholine receptors. J.Med.Chem. 48 905. Hajós et al (2005) The selective α7 nicotinic acetylcholine receptor agonist PNU-282987 [N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] enhances GABAergic synaptic activity in brain slices and restores auditory gating deficits in anesthetized rats. J.Pharmacol.Exp.Ther. 312 1213. Hurst et al (2005) A novel positive allosteric modulator of the α7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J.Neurosci. 25 4396.

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• Ivermectin, an anthelminthic drug, also potentiates α7 nAChRs,216 whereas the anthelmintic levamisole has been reported to show dual potentiation and inhibition of ACh-evoked responses recorded from Xenopus oocytes expressing human α3* nAChRs.218 This dual behaviour is reminiscent of the modulatory effects of galanthamine and physostigmine described above.

• β-estradiol has been observed to potentiate the activation of human, but not rat, α4β2 nAChRs expressed in Xenopus oocytes; this action was distinct from the inhibitory effects of steroids and mediated by interaction with the C-terminal tail of the α4 subunit.219,220 In view of the high concentrations required for this effect, its biological significance is uncertain.175

• Recently, novel members of the Ly-6/u-PAR superfamily have been described and shown to act as endogenous modulators of nAChR function. Lynx-1 is a GPI-anchored protein found in brain and other tissues including lung. It co-localises with both α7 and α4β2 nAChRs, altering single channel conductance and enhancing desensitisation.221,222 SLURP-1 is a 9 kDa secreted protein lacking a GPI anchor that is produced by keratinocytes. Like Lynx-1, it also has a high degree of structural homology with 3-fingered snake neurotoxins like α-Bgt. Picomolar concentrations of SLURP-1 were without effect when applied alone to Xenopus oocytes expressing human α7 nAChRs but increased the amplitude of ACh-evoked currents several fold.223 The association of the SLURP-1 gene with an inflammatory skin disorder suggests a role for

nAChRs in wound healing. The related SLURP-2 protein is proposed to interact with α3* nAChRs on keratinocytes to prevent apoptosis. Thus the different effects observed for SLURP-1 and -2 are attributed to their differential binding to the nAChR subtypes expressed in mucocutaneous epithelial cells.224

Radioligands (Table �)Nicotinic radioligands have been valuable tools for the identification, quantitation, pharmacological characterisation and localisation of nAChRs. Table 5 summarises the features of the major nicotinic radioligands currently in use. [3H]-Nicotine binding to brain tissue was first described in detail by Romano and Goldstein.16 In the majority of reports [3H]-nicotine identifies a single population of sites in the CNS with a Kd of 1-10 nM. Although by definition nicotine interacts with all nAChR subtypes, the radiolabelled version labels predominantly α4β2* nAChRs; [3H]-nicotine binding is almost totally absent in α4 or β2 null mutant mice.225,226 This apparent anomaly reflects the binding affinity (typically <20 nM) required to retain bound ligand during the removal of ‘free’ (unbound) ligand (typically by filtration through glass fibre filters, a process that takes ~15 sec). Only α4β2* nAChRs bind nicotine with high enough affinity to retain it during this step. In contrast, [3H]-epibatidine binds with sub-nanomolar affinity to multiple nAChR subtypes.229 Subpopulations have been defined by their differential sensitivity to A-85380, cytisine or α-conotoxin MII.225,227,228,230

A second issue that can be confusing is the disparity between binding affinity (Kd) and functional potency (EC50) of nAChR agonists that are also radioligands; Kd correlates with Ki for the unlabelled version derived from competition binding assays (Table 3) and is typically 1-3 orders of magnitude lower than EC50. This difference reflects the conditions of the equilibrium binding assay in which the incubation time will be sufficient to stabilise the desensitised state of the nAChR that binds agonist with higher affinity (Figure 2). Indeed, analysis of the kinetics of [3H]-nicotine binding to brain membranes revealed a two-state model, consistent with interconversion from a low affinity binding component (Kd 150 nM) to a high affinity conformation (Kd 1 nM).231 Similarly, the affinity with which unlabelled agonists displace radioligand binding in competition assays (Ki; Table 3) will reflect their affinity for a desensitised form of the nAChR.

The antagonist radioligands α-Bgt and MLA exhibit binding affinities of ~1 nM that are in line with their functional potency at α7 nAChRs. However, competitive antagonism is influenced by agonist concentration and binding kinetics, as discussed above. Therefore higher concentrations would typically be used for nAChR blockade (see Table 2).

Sazetidine A, Novel α4β2 Receptor Desensitiser

Sazetidine A N

O

NH

HO

.2HCl

Cat. No. 2736

Sazetidine A is a subtype-selective α4β2 nicotinic acetylcholine receptor desensitiser that binds with high affinity to rat and human α4β2 receptors (Ki values are 0.41, 0.61 and 10000 nM for rat α4β2, human α4β2 and rat α3β4 receptor subtypes respectively). The compound does not activate these receptors or inhibit activation when added with nicotine, but causes potent inhibition of nicotine-stimulated α4β2 activation following a 10 minute preincubation. Sazetidine A potently drives the α4β2 receptors into a desensitised state without receptor activation, an inhibitory action termed silent desensitisation.Xiao et al (2006) Sazetidine-A, a novel ligand that desensitizes α4β2 nicotinic acetylcholine receptors without activating them. Mol.Pharmacol. 70 1454.

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Table 5 | Selected radioligands that label neuronal nAChRs

nAChR subtype

Radioligand Comment Kda

(nM)Ref

α4β2*

[3H]-ACh AChE inhibitor (100 µM DFP) and muscarinic AChR antagonist (1.5 µM atropine) required in assay mix; labels same site(s) as [3H]-nicotine; used at 2 nM for autoradiography

12.3r 225, 313,314, 315,339

[3H]-Cytisine Labels same site(s) as [3H]-nicotine in rodents;comparable autoradiographic labelling with [3H]-nicotine in monkey;binding largely abolished in β2 null mutant mice; unaffected in β4 and α7 null mutant mice; used at 5 nM for autoradiography

0.15-1.1r

0.2h69, 95, 225, 316, 317, 318, 319, 320

[3H]-Nicotine Add mercaptoacetic acid for storage; purification of [3H]-nicotine by chromatography, or addition of 200 mM Tris to assay buffer, and filtration through glass fibre filters soaked overnight in 0.3% polyethylene imine (PEI) reduces non-specific binding; comparable labelling of heterologously expressed α4β2 nAChRs; Bmax increased in smokers and in animals after chronic nicotine; used at 3.5-5 nM for autoradiography

0.9-2.0r

10m 2.8h

16, 69, 225, 315,321, 322,339

α6β2* [125I]-α-Conotoxin MII

Mostly used for quantitative autoradiography; membrane binding assay possible with addition of BSA and a dilution step to reduce non-specific binding;corresponds to a minor population of sites labelled by [3H]-epibatidine;binding abolished in α6 (but not α3) and β2 null-mutant mice; ~50% reduction in α4 and β3 null-mutant mice; labelling of basal ganglia greatly reduced in Parkinsonian model; used at 0.5 nM for autoradiography

0.6-0.8r

0.3-1.9m

0.9p

109, 157,323, 324,325, 326,327

β2* 5-[125I]-A85380 Incorporation of an iodine atom into A85380, to generate a ligand for PET and SPECT, produced a more potent and more selective nicotinic agonist; labelling absent in β2 null mutant mice; used at 0.2 nM for autoradiography; [123I]-labelled version used for SPECT in smokers versus non-smokers

0.010r

0.012h110,328,329,330

Non-α7

[3H]-Epibatidine A potent ligand with low non-specific binding under standard incubation conditions; potency can result in ligand depletion at very low concentrations, ameliorated by increasing assay volume; labels multiple sites in brain (at least six) distinguished by differential competition by more selective ligands (e.g. cytisine, α-conotoxinMII) or analysis in null mutant mice; Kd for different αβ combinations expressed in HEK293 cells range from 21-94 pM; used at 0.5 nM for autoradiography

0.015-0.05r

(0.4 low affinity site)

0.014m

(7.2 low affinity site)

95,225,227,331,332

[125I]-Epibatidine (+/-)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane (IPH), analogue of epibatidine incorporating an iodine atom in place of the chlorine atom in the chloropyridyl ring of natural epibatidine, displays identical properties to parent compound; labels multiple sites in brain with high affinity, predominantly α4β2 nAChRs but other sites distinguished by differential competition by more selective ligands (e.g. A85380, cytisine); used at 0.25-0.5 nM for autoradiography

0.9r 0.05-0.07m

227,325,328,333

α7

[3H]-MLA Non-specific binding can be decreased by inclusion of 0.1% BSA and/or 200 mM Tris in assay buffer; labels α9α10/5-HT3 chimeric receptors (Kd 7.5 nM); also used to label invertebrate nAChRs (Kd~1 nM); used at 5 nM (rat) / 10 nM (mouse) for autoradiography

1.9r 2.2m

27, 232,260, 334,335

[125I]-MLA Comparable properties with [3H]-MLA; i.v. administration shows rapid clearance and poor brain penetration; increased binding in primate model of Parkinson’s disease

1.8r

33-46p336, 337

[125I]-α-Bgt The classical ligand for defining α7 nAChRs; slow association kinetics require long incubation time; labelling abolished in α7 null mutant mice; used at 0.7-1.5 nM for autoradiography

0.1-1.2r

0.12m95, 315,338, 339,340, 341

a Kd , Binding affinity derived from saturation binding experiments carried out on brain membranes or heterologously expressed nAChRs. Binding affinities of [125I]-αConotoxin MII were also derived from quantitative autoradiography on brain slices.h = human; m = mouse; p = monkey; r = rat

Interest in developing ligands for PET or SPECT, together with the generation of novel synthetic ligands in the quest for greater nAChR subtype selectivity, can lead to new nicotinic radioligands, and [125I]-5-iodo-A85380 is an example to come out of this process. Introduction of a halogen atom can change the properties of the ligand. Fortuitously in the case of A85380, addition of an iodine atom in the

5´ position increased both potency and selectivity.110

Iodinated ligands are typically labelled to higher specific radioactivity than their tritiated counterparts and hence provide greater sensitivity and resolution (especially for autoradiography).232 They have the disadvantage of a short half life (60 days) and the higher energy γ-radiation emitted requires more stringent handling conditions.

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Nicotinic Receptors

Fluorescent or streptavidin labels covalently attached to α-Bgt facilitate identification of muscle and α7 nAChRs by light, confocal and electron microscopy.44,233 The development of other fluorescently labelled probes would be desirable and progress in labelling α-conotoxin MII has recently been reported.234

Future Directions for Nicotinic LigandsDespite the growing nicotinic pharmacoepia,4 there is still a lack of availability of subtype-selective agonists and antagonists. The design of more

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Nicotinic Receptor Compounds Available from TocrisAgonists0352 4-Acetyl-1,1-dimethylpiperazinium

Nicotinic agonist0351 1-Acetyl-4-methylpiperazine

Nicotinic agonist1971 (+)-Anabasine

Neuronal nicotinic receptor agonist0789 (±)-Anatoxin A

Nicotinic agonist1390 (-)-Cytisine

Potent, selective neuronal nicotinic agonist2241 DMAB-anabaseine

Partial agonist at α7-containing receptors0684 (±)-Epibatidine

Very potent nicotinic agonist1077 (-)-Lobeline

Nicotinic agonist2303 PNU 282987

Selective α7 nAChR agonist1053 RJR 2403

CNS selective nicotinic agonist2737 TC 2559

Selective partial agonist at α4β2 receptor1348 UB 165

Subunit selective nAChR agonistAntagonists0424 Benzoquinonium

Nicotinic antagonist2133 α-Bungarotoxin

α7 subtype-selective nAChR antagonist1001 Chlorisondamine

Nicotinic antagonist; slow offset2349 Dihydro-β-erythroidine

Antagonist for neuronal α4-containing nicotinic receptors

2241 DMAB-anabaseineAntagonist at α4β2 receptors

2843 MecamylamineNeuronal nicotinic receptor antagonist

1029 Methyllycaconitineα7 neuronal nicotinic receptor antagonist

1356 MG 624α7 neuronal nicotinic receptor antagonist

0693 PancuroniumNicotinic (neuromuscular) antagonist

2785 Strychnine hydrochlorideNicotinic receptor antagonist

2438 TMPHNeuronal nicotinic receptor antagonist

2820 (+)-Tubocurarine chlorideNicotinic receptor antagonist

Other2809 Acetylcholine chloride

Endogenous neurotransmitter2722 Catestatin

Inhibitor of nicotinic cholinergic-stimulated catecholamine secretion1518 5-Iodo-A-85380

High affinity α4β2 subtype-selective ligand1527 5-Iodo-A-85380, 5-trimethylstannyl N-BOC derivative

Precursor to Cat. No. 15181260 Ivermectin

Allosteric modulator of α7 nicotinic receptors2498 PNU 120596

Positive allosteric modulator of α7 nAChR; active in vivo2736 Sazetidine A

Subtype-selective α4β2 receptor desensitiser