Quick viewing(Text Mode)

Nicotinic Ach Receptors

Nicotinic Ach Receptors

DRIVINGDRIVING RESEARCHRESEARCH FURTHERFURTHER

This , was , 7 ε . High 1 Torpedo , whereas εδ 1 β predominates α- Together with 2 9 γδ 1) 1 α β 2 Electrophorus 1) α transmembrane segments,

An additional subunit, additional An 8 . δ , and eel,

-toxins, including α and γ subunit numbering), form a disulphide a form numbering), subunit , α β

Torpedo , α Torpedo Bgt), enabled the nAChR protein to be purified ‑ α the lipid bilayer to create a water-filled pore. subunit Each consists of the second transmembrane 4 segment (M2) lines the . The extracellular of N-terminal every domain subunit contains a signature sequence of this LGIC family: two cysteine ‘cys-loop’ that is the residues, separated by 13 amino acids 142, (Cys 128, Molecular Architecture of the nAChR Molecular Architecture of the nAChR (Figure 1) span nAChR the comprising subunits five the of Each and there followed detailed studies of the properties of nAChRs mediating these sites. synaptic nAChRs at transmission the in muscle endplate at sympathetic and by ganglia their could respective preferences polymethylene be for bistrimethylammonium C10 distinguished compounds, and notably C6 and . of the muscle nAChR, using biochemical approaches, biochemical using nAChR, muscle the of synapses nicotinic of abundance the by facilitated was akin to the muscle endplate in electric organs of the electric ray, affinity snake ( and subsequently resolved into 4 different subunits, designated subsequently identified in adult skeletal muscle. In the early 1980s, these subunits were cloned and the commenced. nAChRs of analysis molecular the of era The muscle endplate combination and nAChR stoichiometry ( has the extrajunctional the nAChR ( subunit (muscle-derived) and muscle, denervated or foetal in in nAChRs of density high The organs. electric structural resolution high facilitated has organ electric studies using electron microscopy. biochemical and biophysical approaches to studying a in resulted has this relationships, structure-function detailed molecular description of the nAChR. provided the first evidence that muscle and neuronal nAChRs are structurally different. function and structure the of elucidation 1970s, the In

, , ‑ C 1,2 Nicotinic , GABA A Neuromuscular 6 Tocris Bioscience Scientific Review Series In these studies he 5 and the development ACh Receptors ACh 3 support this research. Progress is 4 receptors, and invertebrate glutamate invertebrate and receptors, 3 physiological and pharmacological investigations, neurotransmitter (ACh), that take their products. natural these from names and ganglionic preparations lend themselves to and , leading pharmacologically to the distinct recognition (and functionally of unrelated) families of structurally two receptors for the and intracellular response by interaction with a molecular a with interaction by response intracellular responsive the of membrane the (‘’)in entity cell. Dale distinguished the actions of contraction is prevented .... by the ’. first notion that the This action of a was neurotransmitter or pharmacological is transduced into an of a ‘receptive substance’. showed that ‘nicotine causes tonic certain contraction muscles of fowl, frog and toad, of and that this nAChRs in vertebrate skeletal muscle have studied been for over a pivotal century; this in preparation Langley’s was formulation of the concept nAChRs to pathological conditions. Muscle nAChR being made in understanding the physiological roles physiological the understanding in made being molecular underlying the and brain the in nAChRs of and cellular mechanisms, and the contribution of of subtype-selective ligands generous to armamentarium of complement natural products the that target nAChRs, a range of neurological and psychiatric conditions. The generation of transgenic mice with deleted or mutated nAChR subunits in the central have more recently become brain the focus the of in intense nAChRs research that efforts. realisation This the by fuelled was and spinal cord are potential therapeutic targets for histamine-, and 5-HT-gated characterised been have muscle skeletal in chloride nAChRs channels. in detail whereas mammalian neuronal nAChRs prototype of the cys-loop family of ligand-gated ion channels (LGIC) that also includes GABA 5-HT glycine, Introduction The nicotinic (nAChR) is the Bath. Her research focuses on understanding the roles of nicotinic acetylcholine receptors in the mammalian brain was Barik Jacques stimulation. receptor nicotinic chronic and acute by initiated events cellular and molecular the and France in Paris. addiction research at the Collège de Bath group and is continuing in a PhD student in the Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK of Bath, Bath BA2 7AY, University and Biochemistry, Department of Biology Susan Wonnacott is Professor of Neuroscience in the Department of Biology and Biochemistry at the University of Susan Wonnacott and Jacques Barik and Susan Wonnacott Tocris Bioscience Scientific Review Series

Figure 1 | General structure of nAChRs1 Non-competitive Antagonist K+

Positive Allosteric Agonist / Competitive Complementary Modulator Antagonist : γ/(δ)

Channel Blocker Primary E D binding site: α Y B Y111 117 W149 W55/(57)

Y151 F Nic D180/(182) Y190 C C192 C193Y198 Y W86 a) N b) 93 A ACh binding protein

Cys-loop C C 2+ + Ca , Na C

M1 M2 M3 M4

M2 lines c) the channel a) Schematic of a nAChR with one subunit removed to reveal the ion channel lumen. Notional sites of action of interacting 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. bond to create a loop that has been implicated in californica and Bulinus truncatus.12,13 Each subunit the transduction of agonist binding into channel of this pentameric secreted protein is homologous opening.10 The principal agonist binding site resides to the N-terminal domain of a nAChR subunit, with in the N-terminal domain of α subunits, close to a pair conservation of all the residues implicated in ACh of adjacent (‘vicinal’) cysteine residues (Cys 192, binding to muscle nAChRs. These proteins provide a 193, Torpedo numbering) that define an α subunit. high resolution view of the extracellular portion of the Mutagenesis and photoaffinity labelling experiments receptor, notably of the binding sites at the interface have highlighted the importance of 4 aromatic between adjacent subunits, and the interaction of residues (Tyr 93, Trp 149, Tyr 190, Tyr 198, Torpedo with these sites.10 numbering), consistent with 3 polypeptide loops of Upon agonist binding, nAChRs undergo an allosteric the α subunit (loops A-C) contributing to the primary 11 transition from the closed, resting conformation to agonist binding site (see Figure 1). The adjacent an open state that allows an influx of Na+, and to a subunit (γ/ε or δ) also contributes to the binding site lesser extent Ca2+, and an efflux of K+ under normal (complementary site: ‘loops’ D-F, now recognised to physiological conditions. In the closed state the ion be mostly β strands). One consequence of this is that channel is occluded by a ‘hydrophobic girdle’ that the αγ/ε and αδ binding sites are not identical with 1 constitutes a barrier to ion permeation. Agonist respect to ligand affinity. However, occupancy of binding in the extracellular domain promotes a both binding sites is required to open the channel. conformational change that results in a rotational Knowledge of ligand binding to nAChRs has been movement of the M2 helices lining the pore. Twisting greatly augmented by the crystal structure of an ACh of the girdle widens the pore by ~3 Å, sufficient for binding protein first identified in the snail Lymaea ion permeation.9 At the muscle endplate, the ensuing stagnalis and subsequently also cloned from Aplysia depolarisation elicits muscle contraction. Despite the

 | Nicotinic Receptors presence of agonist, the nAChR channel closes within seconds to minutes, to enter a desensitised state. In Figure 2 | Relationship between the major this condition, the nAChR is refractory to activation. conformational states of a nAChR Multiple desensitised states have been proposed to agonist exist.14 In the active (open) conformation, the nAChR RESTING ACTIVE Channel closed Channel open Agonist binds binds agonists with low affinity (Figure 2; e.g. Kd for with low affinity 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. DESENSITISED Fast onset

Sites on the Muscle nAChR for Ligand Agonist binds Interactions (Figure 1) with high affinity In addition to agonists binding to the agonist binding sites in the extracellular domain, competitive DESENSITISED antagonists also bind at or close to these sites, Slow onset preventing access to agonists. Their antagonism can be overcome by increasing the agonist concentration (unless the antagonist binds irreversibly, as is the agonist binding sites, and include channel blocking case for α-Bgt), hence competitive antagonism is drugs that occlude the channel. Their inhibition is not referred to as ‘surmountable’. The concentration surmountable with increasing agonist concentration. of competitive antagonist necessary for nAChR In addition to compounds that interact specifically blockade will depend on the experimental conditions. with residues in the mouth or lumen of the pore, any Non-competitive antagonists bind to sites distinct from small positively charged species may be predicted

Table 1 | Selected compounds that interact with mammalian muscle nAChRs

Drug Comment Potencya Agonists

(±)-Anatoxin A A bicyclic from blue-geen algae that is a potent, enantio-selective ACh-like agonist lacking EC50 = 50 nM significant activity at muscarinic receptors or AChE.88,237

(-)-Nicotine The natural is ~6 times more potent than its unnatural enantiomer at muscle ED20 = 20 µM nAChRs.238 Competitive Antagonists Benzoquinonium A classical neuromuscular blocking agent,239 also used for invertebrate preparations.240 More recently reported to act as an allosteric potentiating ligand and open of muscle and neuronal nAChR subtypes.241

α-Bungarotoxin Polypeptide snake toxin from Bungarus multicinctus; most potent of the ‘long’ α‑neurotoxins. Binds Kd = 0.01-10 nM (α-Bgt) pseudo-irreversibly, reflecting very slow dissociation kinetics. Interacts potently with α subunit 1,242 sequence around Tyr190-X-Cys192-Cys193.

α- MI One of several α- from Conus sp. that specifically block muscle nAChRs. MI exhibits a Kd ~ 0.1-1 nM 10000-fold preference for the α/δ versus the α/γ agonist binding site interface of mammalian muscle nAChRs.139,243

Decamethonium Often used as a competitive antagonist but it produces a depolarising neuromuscular block akin to ED95 = 0.12 µmol/kg; nicotine and other agonists. It is more accurately classified as a .7,244 0.03 mg/kg

245,246 Pancuronium Used clinically as a non-depolarizing muscle relaxant. IC50 ~ 5 nM 1,246,247 d-Tubocurarine As a photoaffinity label, it discriminates αδ and αγ agonist binding sites. Kd1 = 30 nM,

Kd2 = 8 µM

IC50 ~ 50-100 nM Channel Blockers

Chlorpromazine This neuroleptic also blocks the nAChR channel, interacting with hydrophobic residues in the IC50 >300 nM Torpedo M2 channel lining.248,249,250

251,252,253 Histrionicotoxin First isolated from frogs of the dendrobatid genera. Ki ~ 0.1-1 µM (Bold Text Denotes Compounds Available From Tocris)

a EC50, 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

www.tocris.com |  Tocris Bioscience Scientific Review Series to channel block, and many agonists including ACh necessitated the presence of specific receptors. In do this at high concentrations.15 The efficiency of the early 1980’s it was apparent that [125I]-α-Bgt and channel blockade is ‘state dependent’; access to the [3H]-nicotine labelled distinct sites that differ in their channel requires the channel to be open. Hence the pharmacological profiles and anatomical distributions speed of block will be influenced by the state of the in rodent brain.16,17 This raised the (then) novel and receptor: resting, open or desensitised (Figure 2). controversial prospect of nAChR heterogeneity in the A selection of compounds acting at such sites on brain. Since the first publication of a cloned neuronal the muscle or Torpedo nAChR is listed in Table 1. nAChR subunit (α3) in 1986,18 eleven neuronal Allosteric modulators can act at a number of sites to nAChR subunits have been identified in mammals influence agonist interactions or channel function. (α2-α7, α9, α10, β2-β4),4,19 with an additional subunit, α8, cloned from avian species.20 Neuronal nAChR α Subunits are defined by the presence of a pair of Heterogeneity of Subtypes (Figure 3) vicinal cysteines equivalent to those that characterise In addition to their presence in skeletal muscle, the muscle α1 subunit. This led to the supposition that nAChRs in autonomic neurones were implicit, and all α subunits could constitute the primary agonist recognised to be pharmacologically distinct, since binding subunit in neuronal nAChRs. However, the α5 the studies of Paton and Zaimis.7 The existence subunit is not capable of fulfilling this role as it lacks of nAChRs in the brain was controversial, but the critical from loop C (Tyr190, Torpedo α1 realisation that the habit is labelling, Figure 1).21 β Subunits lack the N-terminal underpinned by the psychoactive actions of nicotine 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. α99 α99 α99 α99 α1010 α1010 α9 α99 α99 αα99 α99 α10 Neuronal, homomeric α7 α 7 α 8 α 7 pCa2+/Na+ α7 α8 α7 αα77 αα77 α88 α88 α88 α88 α8 ~10 α77 α77 α88 α88 α77 α77 α1 β1 Muscle, ε/γ δ α1 α1 pCa2+/Na+ γ 0.2 δ β1 ε α2 β2 α44 α44 xX = β2, α4, α5,… α4 α3 β2 xX Neuronal, α6 heteromeric β2 β2 α5 pCa2+/Na+ α66 α44 α66 α66 ~1-1.5 β3 β2 β3 β2 β3 β2 β2 β2 β4 β4 α33 α33 α33 α33 α33 α33 β2 Y β4 Y β4 Y

Y = β2, β4, α5,…

 | Nicotinic Receptors tryptophan residue characteristic of loop D (Figure 1); α3β2* and α7 nAChRs (where * indicates the hence these subunits can act like γ and δ muscle possible inclusion of unspecified subunits; see subunits to provide the complementary binding site Figure 3).35 Additional subunits (including α4 and in an αβ pair. The absence of this residue in the β3 α10) have been reported in dorsal root ganglia;36,37 subunit makes it the true homologue of the muscle β1 nAChRs in these sensory neurones are of interest subunit that does not contribute to an agonist binding as therapeutic targets for modulating nociceptive site. Indeed, the sequence similarity between α5 and signals. β3 subunits (see Figure 3) is consistent with both • There is a heterogeneous distribution of α2-α7 having this role.22 and β2-β4 subunits in the mammalian CNS:19 In contrast, the α7, α8 and α9 subunits are α4, β2 and α7 are the most wide-spread subunits distinguished by their ability to form robust homomeric with α4β2* and α7 nAChRs having a somewhat receptors in expression systems in the absence complementary distribution. In contrast to their of a β subunit. Hence these subunits provide both roles at the and in primary and complementary faces of the agonist sympathetic ganglia, there are rather few reports binding site,19 resulting in five putative binding sites of neuronal nAChRs mediating per receptor monomer (Figure 3). It is not known if synaptic transmission in the CNS. There is more than 2 sites must be occupied by the agonist to abundant evidence in the brain for presynaptic open the channel, but occupancy of a single site by nAChRs that modulate the release of many the antagonist (MLA) is predicted different neurotransmitters38 and this has led to to be sufficient to inhibit α7 nAChR function.23 In the unproven supposition that the majority of avian tissues α7α8 also occur and an nAChRs are located presynaptically. However, association between α7 and β2 subunits has been nAChRs also exist on somatodendritic regions, suggested to occur in mammalian brain.24 A splice in perisynaptic or extrasynaptic locations.39,40,41 variant of the α7 nAChR subunit that incorporates a The current perspective is that presynaptic and novel 87 base pair cassette in the N-terminal domain extrasynaptic nAChRs serve to modulate short has also been reported to be expressed in rat and longer term neuronal activity in response intracardiac neurones, and possibly other tissues.25 to non-synaptic (‘paracrine’) levels of ACh (or It differs in having slower desensitisation kinetics and , in the case of α7 nAChRs).42 more reversible blockade by α-Bgt. The related α10 subunit is only incorporated into a functional nAChR The α7 nAChR is particularly prominent in the when co-expressed with the α9 subunit.26,27 hippocampus, where it is found on GABAergic interneurones of stratum oriens and stratum A distinct but related gene family of α and β subunits radiatum, and on pyramidal neurones. Presynaptic has been uncovered in invertebrates. The C. elegans α7 nAChRs are present on glutamate terminals genome sequence incorporates 29 candidate nAChR and facilitate transmitter release in various brain subunits, Drosophila melanogaster and Anopholes regions, including hippocampus, cortex and gambiae each have 10 nAChR subunit genes, while ventral tegmental area.43,44,45 Nicotine acting at α7 the honey bee Apis mellifera has 11 such genes.28,29 nAChRs can enhance hippocampal LTP,46 and α7 Twelve nAChR subunits have been found in the nAChRs are associated with attentional processes mollusc Lymnaea stagnalis.30 and working memory.47,48 As a consequence, α7 Distribution and Physiological nAChRs are a therapeutic target for treating cognitive Significance of nAChR Subtypes impairment, notably in Alzheimer’s disease and In situ hybridisation has shown that nAChR subunits , and this has prompted the generation have distinct and often widespread distributions in the of α7 nAChR-selective ligands,49 some of which are vertebrate nervous system. The subunit composition listed in Table 2. of native nAChRs has proved a more challenging quest. The following methodologies have contributed α4β2 nAChRs have high affinity for nicotine (and to the current understanding of subunit composition: account for >90% of [3H]-nicotine binding to brain subtype-selective radioligand binding (Table 5); tissues). A stoichiometry of (α4)2(β2)3 has been pharmacological characterisation; single cell PCR proposed, generating two agonist binding sites and electrophysiology; immunoprecipitation with consistent with the model of the muscle nAChR subunit-specific antibodies; knock out mice. (Figure 3).50,51 Manipulation of the stoichiometry • Autonomic neurones (including sympathetic of α4β2 nAChRs expressed in Xenopus oocytes ganglia, parasympathetic innervation, sensory indicates that (α4)3(β2)2 nAChRs are also viable, ganglia, chromaffin, neuroblastoma and PC12 displaying lower affinity for ACh and higher Ca2+ cells) typically express α3, α5, α7, β2 and β4 permeability;52,53 whether native nAChRs with subunits,31,32,33,34 with the likely assembly of α3β4*, this subunit stoichiometry exist is not known.

www.tocris.com |  Tocris Bioscience Scientific Review Series

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

Potency Drug Comment Binding Function

a b Agonist Ki EC50 AR-R17779 A synthetic, structurally rigid spirooxazolidone with 100-fold great potency for binding to α7 0.2 µM 10-20 µM 254,255 nAChRs than α4β2 nAChRs. No activation of α4β2, α3β4, α3β2, 5‑HT3 receptors. Central effects at 1-2 mg/kg s.c.256 Choline The substrate and breakdown product of ACh is a weak α7-selective agonist, 10 times less ~2 mM 0.4-1.6 mM 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 Compound A A potent and selective α7 nAChR agonist (referred to a Compound B in earlier 40 nM 14 nM- abstracts).262,263 In conjunction with the allosteric potentiator PNU 120596, 10 nM 0.95 µM Compound A activates α-Bgt-sensitive Ca2+ signals in PC12 cells.265 Effective in vivo at doses of 3-10 mg/kg.264 GTS-21 A partial agonist at α7 nAChRs, eliciting 12-30% of maximum response to nicotine or ACh. 0.2-0.5 µM 6-26 µM 3 (aka DMXB) Also interacts with α4β2 nAChRs (Ki = 84 nM versus [ H]-): 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

PNU 282987 A synthetic α7-selective agonist, with weak activity at 5-HT3 receptors (Ki = 0.9 µM). When 26 nM 128 nM administered at 1 mg/kg i.v. it restored -induced sensory gating deficit and augmented hippocampal theta oscillation in anaesthetised rats.271,272,273

SSR180711 A synthetic α7-selective partial agonist (Emax = 36-50% of maximum response to ACh). ~20 nM 1-4 µM Elicits central effects after i.p. or oral administration.274,275

a c Competitive antagonists Ki IC50 α-Bungarotoxin Blocks α7, α8, α9* nAChRs; faster dissociation kinetics than at muscle nAChRs, but 0.5-1 nM (1-100 nM) 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). a-Conotoxin ImI From Conus imperialis, this peptide toxin selectively inhibits rat α7 nAChRs. Weaker ND 86-220 nM

antagonist of α9 (IC50 1.8 µM) and muscle nAChRs (50 µM), with no action at heteromeric 139,277,278 nAChRs. Open channel blocker of 5-HT3 receptor. May be species selective, as 279 reported to block bovine α3β4* nAChRs (IC50 2.5 µM). Also blocks certain invertebrate nAChRs.139 Methyllycaconitine Isolated from Delphinium sp., this hexacyclic norditerpenoid antagonist discriminates 1 nM 10-200 nM (MLA) 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 MG 624 A 4-oxystilbene derivative that shows a 30-fold preference for blocking chick α7 nAChR, 100 nM 100 nM compared with α4β2 nAChRs.160,161 Related F3 derivative targets non-α7 nAChRs in rat chromaffin cells.162 This glycine is also a competitive antagonist at α7, α8, α9/α10 nAChRs; 6 µM 1 µM non-competitive block of muscle nAChRs and voltage-dependent block of heteromeric 27,66,283,284,285,286 neuronal nAChRs (IC50 = 7-30 µM). b Allosteric modulators EC50 5-Hydroxyindole A metabolite of 5-HT that potentiates α7 nAChR responses to ACh: increases potency and of 2.5 mM ACh without affecting desensitisation. Effective at 100 µM in brain preparations.287,288,289 This semi-synthetic anthelminthic agent is an allosteric potentiator of α7 nAChRs; pre-application ND

(16 seconds) necessary, effective at 30 µM. More potent interactions with mammalian GABAA and glycine receptors.290,291,292 PNU 120596 Positive allosteric potentiator of α7 nAChR responses, prolongs agonist-evoked currents. No effect on 200 nM responses from α4β2, α3β4 or α9α10 nAChRs. In vivo, CNS effects in response to 1mg/kg in rats. Limited solubility.265,293

(Bold Text Denotes Compounds Available From Tocris) a 125 3 Ki, Inhibition constant, from competition binding assays for [ I]-α-Bgt or [ H]-MLA binding to brain membranes or heterologously expressed human or rat α7 nAChR; b 2+ EC50, effective concentration producing 50% of maximum activation; from electrophysiological recording of whole cell currents or intracellular Ca responses, from native receptors in hippocampal neurones or cell lines or heterologously expressed human or rat α7 nAChR; c IC50, 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

 | Nicotinic Receptors

Transgenic knockout of either of these subunits targets has stimulated the generation of synthetic eliminates nicotine self administration, whereas ligands to add to this pharmacopoeia. However, there virally targeted re-expression of the β2 subunit in remains a lack of subtype-selective tools, in particular mesolimbic areas of β2 knockout mice recovers antagonists. Only the α7 nAChR has a significant this behaviour, implicating a role for α4β2 nAChRs and growing list of selective agonists, antagonists in nicotine addiction.54,55 α4β2 nAChRs are highly and allosteric modulators, these are described in expressed in the thalamus. As a consequence of Table 2. Selected pan-acting or less discriminating their putative role in thalamo-cortical circuitry, gain of agonists and antagonists are summarised in Tables function mutations in the M2 domain of either the α4 3 and 4 respectively, and some are briefly discussed or β2 subunit give rise to some forms of autosomal below. More comprehensive accounts of the families dominant nocturnal frontal lobe epilepsy.56 of synthetic nicotinic ligands have been published recently.4,65 α3 and β4 subunits have a much more restricted distribution in the CNS, for example in medial Agonists (Table 3) habenula and locus coeruleus they are often, but not Structurally diverse naturally occurring nicotinic always, co-expressed.19 agonists include: (-)-nicotine, (‑)‑cytisine, (+)‑anatoxin A, (+)-, and α6 and β3 subunits are largely restricted to . Synthetic agonists range from catecholaminergic neurones and contribute to the classical “ganglionic agonist” dimethyl- nAChRs of complex subunit composition, e.g. phenylpiperazine (DMPP) developed in the 1960s, α6β2β3 and α4α6β2β3 nAChRs on to novel agonists created more recently in order to terminals.57 The β3 subunit is suggested to be provide greater subtype selectivity and therapeutic necessary for the correct assembly, stability and/ efficacy.4 Typically, agonists bind with highest affinity or targeting of α6* nAChRs.58 The α2 subunit has at the α4β2 nAChR, with 2-3 orders of magnitude the most limited expression pattern of any nAChR lower affinity at α7 nAChRs and with intermediate subunit in the rodent CNS, being largely restricted affinity to α3* nAChRs (Table 3). With respect to to the interpeduncular nucleus.59 As a consequence functional potency, a similar relationship is observed, its contribution to native nAChR has been rather little except that differences in EC values between studied. However, its distribution in the primate brain 50 subtypes are less marked, especially between α4β2 appears to be more extensive.60 and α3* nAChRs. Agonists are ~2 fold more potent at • Mechanosensory hair cells express (exclusively) α8 nAChRs66,104 and ~10 fold more potent in binding α9 and α10 subunits that coassemble to generate to α9α10 nAChRs,27 compared with α7 nAChRs.

predominantly heteromeric nAChRs that mediate Binding affinities (Ki values) are typically 2-3 orders effects of the efferent olivocochlear system on of magnitude lower, in terms of concentration, 26,61 auditory processing. than EC50 values for nAChR activation (Table 3). With the exception of some recently described α7 • Expression of nAChR subunits has also nAChR-selective agonists described in Table 2, few been detected in diverse non-neuronal cells. agonists have sufficient nAChR subtype selectivity These comprise astrocytes, macrophages, to exclusively activate one particular subtype in a keratinocytes, endothelial cells of the vascular mixed population. system, muscle cells, lymphocytes, intestinal epithelial cells and various cell-types of the • ACh is the endogenous agonist for all nAChR lungs.62,63 mRNAs encoding most nAChR subtypes. It is a popular choice for activating subunits (but not α6) have been detected in such nAChRs in electrophysiological experiments but cells. The identity and functional significance its utility is compromised by its lack of selectivity of assembled nAChRs in non-neuronal cells for nAChRs versus muscarinic AChRs, and remain poorly understood, although α7 nAChRs its susceptibility to hydrolysis. A muscarinic on macrophages have excited interest in the antagonist (typically , ~1 µM) and an possibility that they might be involved in anti- inhibitor must be included inflammatory responses.64 with ACh in biological preparations; some of these agents may also interact with nAChRs (see Nicotinic Ligands for Neuronal below). nAChR • Carbamoylcholine () is formed Due to the critical roles of muscle and ganglionic by the modification of ACh to a , nAChRs, nature has elaborated a diverse array of resulting in a hydrolysis-resistant analogue. This plant and animal toxins that target these receptors has reduced affinity at α4β2 and α7 nAChRs and their counterparts in the CNS. More recently, the (Table 3) but is potent at muscarinic sites and perceived validity of neuronal nAChRs as therapeutic is commonly used as a .

www.tocris.com |  Tocris Bioscience Scientific Review Series

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

nAChR subtype Agonist α4β2* α7 α3β4 α4β2* α7 α3β4

Binding affinity Ki (nM) Functional potency EC50 (µM) A-85380 0.017- 17-290107,108,110 14-78103,107,110,298 0.7108 8.9108 0.8108 0.14103,107,108,110 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 4000-180000 620-85067,103 0.5-68104,131,294,295 28-180 35-20367,104,131,297 67,269,285,339 80,104,131,269,285,296

(+)-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 12000- 3839-4700103,298 17-34294,295 580285 – 67,69,103,294,339 580000269,285,339 (-)- 0.04-0.06; – – – (175*)80 – >100000069,301 (-)-Cytisine 0.012-1.5 260-1500067,110,269,28 54- 0.019-2.6131,294,295 5.6-71131,296,300 14-7267.131.297 67,69,103,110,294,339 5,300,339 22067,103,110,298 Dimethylphenyl 8.7-190 69,298,294,339 470-7600300,339 820103 1.9-18131,294,295 26-6480,131,300 14-19131,297 piperazinium (DMPP) (±)-Epibatidine 0.042-0.15 20-24067,107,269 0.21-0.98 0.0045- 1.2-1.3104,266 0.024-0.07104 67,103,107,294 67,103,107,298 0.0085104,295 1.5-1669,302,303 11600-13100269,303 – (0.73-21*)295,303 (8.5*)80 –

Methylcarbamyl- 1.7-2867,69 4400067 2,70067 2.6294 – 4467 choline (MCC) (-)-Nicotine 0.6-10 400- 290- 0.35-5104,131,295 49-113131,266,269 8.1-11067,104,131,297 67,69,103,125,294,339 15,00067,125,269,285,339 47667,103,107,298 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 agonist areferences are indicated with each entry; preparations used are as follows:

Reference: Species and Preparation 67. Rat/HEK or tsA cells, or rat brain 285. Chicken brain α7/binding 69. Rat α4β2/brain [3H]-nicotine binding 294. Mouse thalamus (α4β2*)/[3H]-nicotine binding/Rb+ efflux 80. Human α7/Xenopus oocytes 295. Human α4β2/SH-EP1 cells 85. Human/Xenopus oocytes 296. Rat α7/Xenopus oocytes 90. Chicken/heterologous or Rat/native 297. Human α3β4/HEK cells 98. Human/mammalian cells/binding 298. Rat α3β4/HEK cells 100. Human/HEK/neuroblastoma 299. Rat brain/binding 103. Rat/HEK 300. Rat brain α7/binding; Chicken α7/Xenopus oocytes 104. Human (or chicken)/Xenopus oocytes 301. Rat brain/[3H]-epibatidine binding 107. Rat brain/IMR32 cells/binding 302. Rat brain α4β2/binding/Xenopus oocytes 108. Rat brain/binding; Human/Xenopus oocytes or cell lines 303. Rat brain binding/rat thalamus Rb+ efflux 110. Rat brain/adrenal glands/binding 304. Rat brain/binding/rat thalamus Rb+ efflux 111. Rat brain/rat α3β4 cells/binding/rat striatal release/rat α3β4 305. Rat brain/rat α3β4 cells/binding/rat α3β4 cells Ca2+ increases cells Ca2+ increases 306. Rat/Xenopus oocytes 114. Human/Xenopus oocytes 339. Rat/mouse brain/[3H]-nicotine and [125I]-α-Bgt binding 125. Rat/binding to brain/Xenopus oocytes 344. Rat cortex/binding 131. Human/Xenopus oocytes 345. Human/mammalian cell lines/Ca2+ respnses 266. Human α7/Xenopus oocytes 269. Human α7/HEK cells

 | Nicotinic Receptors

N‑Methylation of the carbamate nitrogen to activity in in vitro functional assays and in a yield N-methylcarbamylcholine (MCC) recovers number of behavioural tests.80,81 Because of its

high (nanomolar) binding affinity at α4β2 extended half life (T½ = 5-6 hours, compared with nAChRs, comparable to ACh.67 N-Methylation <1 hour for nicotine in rat brain)77 cotinine attains also confers substantial selectivity for nAChRs high concentrations in vivo and can desensitise over muscarinic AChRs.68,69 The permanently nAChR responses,80,82,83 although ganglionic charged quaternary nitrogen atom renders MCC nicotinic responses appear to be less affected.81 membrane impermeant; this has been exploited • Trans-metanicotine (RJR-2403 or TC‑2403) to discriminate cell surface nAChRs from the generated by opening the ring of total population that includes a large proportion nicotine, also occurs naturally as a minor tobacco of intracellular nAChRs.70 alkaloid. It shows some functional selectivity for • (-)-Nicotine, a tobacco alkaloid, is the prototypic α4β2 nAChRs compared with α3* nAChRs,84,85 nAChR agonist that was used historically to and is effective in vivo when administered at classify the receptors.5 All nAChR subtypes are doses of 1-7 µmol/kg (0.2-1.2 mg/kg) s.c.86 It has activated by nicotine, with the exception of α9 recently been tested, with encouraging results, in and α9α10 nAChRs; nicotine is an antagonist an in vitro model of ulcerative colitis.87

at these subtypes (IC50 values are 31 µM and 4 µM respectively).26,71 Neuronal heteromeric nAChRs exhibit a marked preference for the TC 2559 and RJR 2403, Subtype- natural enantiomer over (+)-nicotine whereas α β α7 nAChRs appear to be less stereoselective. Selective 4 2 Ligands Nicotine crosses the blood brain barrier readily MeHN and its and N 72 TC 2559 are well documented. For a comprehensive Cat. No. 2737 .2C4H4O4 discussion of nicotine doses used in vivo in OEt various vertebrate and invertebrate organisms, see ref. 343. Behavioural responses typically RJR 2403 NHMe Cat. No. 1053 .C4H4O4 show a bell-shaped dose-response profile with N maximum responses in rats elicited by doses of 0.4 mg/kg s.c. or less. For chronic (continuous) TC 2559 and RJR 2403 (TC-2403) are subtype-selective administration, nicotine may be given via osmotic ligands for α4β2 nicotinic acetylcholine receptors that display good CNS-PNS selectivity ratios. TC 2559 displays minipumps or indwelling s.c. cannulae and a daily selectivity for (α4)2(β2)3 receptor stoichiometry and acts as a delivery of 2-4 mg/kg/day in rats reproduces the partial agonist, whilst RJR 2403 is a full agonist. plasma concentrations that are found in human smokers.73,74 Nicotine in flavoured drinking water In vivo Both compounds are active in vivo; TC 2559 and RJR 2403 (e.g. 2% saccharin, to disguise the aversive taste significantly improve passive avoidance retention following of nicotine) provides a means of self-delivery scopolamide-induced cognitive deficits in rats. of nicotine chronically. This route is often used with mice (e.g. 200 µg/ml in drinking water)75 RJR 2403 TC 2559 because surgical implantation of osmotic pumps α4β2 0.026 0.18 in mice is less convenient due to their size. Mice α4β4 – 12.5 have a much faster turnover of nicotine (T½ <10 α2β4 – 14.0 min) than rats (T ~55 min) and consequently ½ α3β4 >1000 >30 require higher doses.76,77,78 However, high doses of nicotine (>1 mg/kg s.c. in rats) produce α1β1γδ >1000 – adverse effects, most likely due to ganglionic α3β2 15 >100

or neuromuscular actions, although higher α7 36 (Ki) >100

concentrations elicit convulsions. These doses EC50 values (in µM). Data taken from Bencherif et al (1996) and Chen et al refer to the free base concentration of nicotine; (2003). 3-times higher concentrations of the tartrate salt Bencherif et al (1996) RJR-2403: a with CNS selectivity I. In vitro characterization. J.Pharmacol.Exp.Ther. 279 1413. Lippiello et al (1996) are required to achieve these concentrations of RJR-2403: a nicotinic agonist with CNS selectivity II. In vivo characterization. nicotine base; published doses should indicate J.Pharmacol.Exp.Ther. 279 1422. Damaj et al (1999) Antinociceptive and pharmacological effects of metanicotine, a selective nicotinic agonist. the free base concentrations. 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. • Cotinine is the principal metabolite of nicotine. 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 Its ability to displace nicotinic radioligands from midbrain slices. Neuropharmacol. 45 334. Zwart et al (2006) 5-I A-85380 and binding to rat brain is inconsistent,69,79 but recently TC-2559 differentially activate heterologously expressed α42 nicotinic receptors. cotinine has been reported to have weak nicotinic Eur.J.Pharmacol. 539 10.

www.tocris.com |  Tocris Bioscience Scientific Review Series

• TC 2559 is the 5-ethoxy derivative of trans- affinity binding (Ki ~1 nM), but its differential metanicotine. This modification of the parent interactions with other nAChR subtypes has compound results in greatly improved selectivity enabled it to be used to distinguish subpopulations for α4β2 nAChRs, coupled with relatively low of nicotinic binding sites labelled by [3H]- efficacy (~30%).4,344,345 Interestingly, TC 2559 epibatidine that differ in having high or low affinity is suggested to discriminate between α4β2* for cytisine.95 At α4β2* nAChRs cytisine is a partial and α6β2* nAChRs, as it provokes currents in agonist; its functional efficacy is dependent on midbrain dopamine neurones that are insensitive the identity of the β subunit present. Thus cytisine to α-conotoxin-MII.345 In vivo, TC 2559 (1-10 mg/ displays full efficacy at nAChRs containing the β4 kg) generalises to nicotine in a discriminative subunit expressed in Xenopus oocytes, while stimulus test that is considered to be mediated greatly reduced efficacy is observed at by α4β2 nAChRs.98 TC 2559 significantly β2‑containing nAChRs.96 Halogenation at the attenuated -induced cognitive 3‑position of the ring increases both deficits and reduced working memory errors in potency and efficacy.94 Cytisine is less potent a radial arm maze, at doses of 1-6 µmol/kg. In than nicotine in behavioural studies, and shows contrast to nicotine, no locomotor or hypothermia only partial generalisation to nicotine in a drug effects were observed, consistent with reduced discrimination test; doses of 1-3 mg/kg are peripheral side effects.344 effective in vivo.97,98 • Anatoxin A is a potent, semi-rigid, stereoselective • (ChantixTM (USA); ChampixTM (EU)) agonist originally isolated from freshwater blue is a cytisine congener developed to exploit the green algae, Anabaena flos aqua.88 Activity properties of cytisine (selectivity and partial resides in the natural (+)-enantiomer. At muscle agonism with respect to α4β2* nAChRs) as an nAChRs, anatoxin A is about 8 times more potent aid to .99 Although it appears than ACh89 but it activates neuronal nAChR selective for α4β2* nAChRs in binding assays, its subtypes at sub-micromolar concentrations agonist potencies at different nAChR subtypes, (Table 3), with EC values that are 20-100 times 50 determined by electrophysiological recordings lower than those for ACh.90,91 Despite being a from heterologous expression systems, show less secondary amine (that should cross the blood discrimination306 (Table 3). Varenicline is effective brain barrier readily), there are few reports of the in releasing dopamine in vitro and in vivo (efficacy in vivo effects of anatoxin A, although two reports ~45% of maximum response to nicotine) but it suggest that its responses are qualitatively is also capable of attenuating nicotine-evoked different from those of nicotine.92,93 dopamine release, a reflection of its partial agonist properties.100 Indeed, varenicline is more (±)-Anatoxin A, Potent Nicotinic potent at inhibiting nicotine-evoked responses in Xenopus oocytes (IC = 6 nM) than it is at Agonist 50 eliciting responses (EC50 = 3 µM), interpreted Me (±)-Anatoxin A O NH as a reflection of the higher affinity for agonists Cat. No. 0789 .C H O 4 4 4 shown by the desensitised state of nAChRs (α4β2 nAChRs in particular) (see Figure 2). Varenicline (±)-Anatoxin A is a nicotinic receptor agonist (Ki values is effective in vivo at doses of 0.01-3.0 mg/kg are 3.5 and 380 nM for α4β2 and α7 nicotinic receptors 100 3 (given s.c. or p.o.) and has a half life of 4 and respectively). The agonist stimulates [ H]-dopamine release 17 hours in rats and humans respectively, with from rat striatal synaptosomes (EC50 = 136 nM) with a higher 101 potency than (-)-nicotine. (±)-Anatoxin A displays powerful little metabolism. behavioural effects in the rat; it decreases locomotor activity in nicotine-tolerant and non-tolerant rats and decreases • Epibatidine, originally obtained from skin extracts rates of operant responding in a drug discrimination of the Amazonian frog Epidobates Tricolor, is procedure. one of the most potent nicotinic agonists,102 Wonnacott et al (1991) Nicotinic of anatoxin analogs. II. Side binding to multiple heteromeric nAChRs with chain structure-activity relationships at neuronal nicotinic ligand binding sites. 103 J.Pharmacol.Exp.Ther. 259 387. Stolerman et al (1992) Behavioural effects of sub-nanomolar affinities. Like anatoxin A, anatoxin, a potent nicotinic agonist in rats. Neuropharmacology 31 311. Thomas epibatidine also has a bicyclic moiety that confers et al (1993) (+)-Anatoxin is a potent agonist at neuronal nicotinic acetylcholine receptors. J.Neurochem. 60 2308. Sharples et al (2000) UB-165 implicates some rigidity to its structure, but in this case it is α4β2 nAChR in striatal dopamine release. J.Neurosci. 20 2783. a smaller azabicycloheptane ring, coupled to a chloropyridyl moiety. In contrast to anatoxin A, • (-)-Cytisine occurs in a number of plants of the the enantiomers of epibatidine show equivalent leguminosae family including laburnum. Its rigid biological activities.102,104,105 The functional structure has provided a template for modelling potency of epibatidine is exceptionally high, with nicotinic ligands.94 At α4β2 nAChRs it is sub-micromolar EC50 values for heteromeric comparable to nicotine, with respect to its high

10 | Nicotinic Receptors

and α8 neuronal nAChR subtypes. Only α7 and This analogue shows improved functional

muscle nAChRs exhibited higher EC50 values, selectivity for β2-containing nAChRs over other in the low micromolar range.4,104,105 In vivo, nAChR subtypes.110,111 It has good credentials in epibatidine displays potent non- analgesic vivo for use as a SPECT ligand,112 and has been activity.102 The therapeutic window for epibatidine reported to be self-administered by rats.113 is very narrow, attributed to side effects that • UB-165 is a novel hybrid molecule, comprising reflect its lack of nAChR subtype selectivity, but the azabicyclononene bicycle of anatoxin A it has generated interest in neuronal nAChRs as and the chloropyridyl moiety of epibatidine.114 potential targets for antinociceptive drugs.106 UB-165 exhibits intermediate potency, with • A-85380 came out of a synthetic programme that stereoselectivity comparable to that of anatoxin aimed to recapitulate the potency of epibatidine A. In contrast to the parent molecules, it is a at α4β2* nAChRs while minimising interactions partial agonist at α4β2* nAChRs.114,115 with other nAChR subtypes.107 Like epibatidine, A-85380 binds to heteromeric nAChRs with sub- • Dimethylphenylpiperazine (DMPP) has a long nanomolar affinities but its interaction with α7 or history as a ganglionic agonist. It shows little Torpedo nAChRs requires >1000 times higher selectivity between neuronal nAChR subtypes concentrations.108 A-85380 has been used to with respect to binding affinity or potency of 4 distinguish populations of [125I]-epibatidine binding activation, hence it is relatively more potent sites in mouse brain that are either sensitive or at α3β4* nAChRs than nicotine, and has been resistant to inhibition by this ligand; the A-85380- used in preparations in which such ganglionic- resistant population is also heterogeneous, type nAChRs prevail. For example, DMPP was with respect to the involvement of α3 nAChR recently used to show that nAChRs exert an subunits.109 Functionally, A-85380 is a potent anti-inflammatory influence in monocytes and 116 full agonist in several functional assays that macrophages. DMPP is a partial agonist correlate with different nAChR subtypes. In vivo at various heterologously expressed nAChR 117,118 A-85380 (0.01-0.1 mg/kg s.c.) fully generalises subtypes. It is membrane impermeant, to nicotine in an operant drug discrimination task due to the quaternary nitrogen atom, and has in rats, a behavioural response attributed to α4β2 been used to selectively block cell surface 119 nAChRs.98 A-85380 has analgesic properties, nAChRs. The related compound 1-acetyl-4- methylpiperazine is a weaker nicotinic agonist with ED50 values of 0.1-0.2 µmol/kg i.p. in the rat; side effects (transient prostration and ataxia) that exhibits a behavioural profile indicative of occur at doses of 0.5 µmol/kg and above.107 different pharmacodynamics compared with nicotine.120 The methiodide salt is 100 times more • 5-Iodo-A-85380 was created by introduction of potent but does not access the brain. an iodine atom in the pyridine ring of A-85380 to generate an iodinated radioligand (see Table 5). • Lobeline, an alkaloid from the Indian tobacco inflate, is an atypical nicotinic ligand and shows anomolous behaviour in vivo. Lobeline 5-Iodo-A-85380, Potent, Subtype- binds with high affinity to α4β2* nAChRs, and has both agonist and antagonist activities.121,122 Selective Nicotinic Agonist In addition, it inhibits vesicular monoamine (IC50 NH 5-Iodo-A-85380 ~1 μM) and dopamine transporters (IC50 = 40- Cat. No. 1518 O 123 N 100 μM) and μ-opioid receptors (IC50 = 1 μM). .2HCl In vivo, lobeline shows some nicotine-like

I effects but also differs from nicotine in several 5-Iodo-A-85380 is a highly potent and subtype-selective behavioural assays, for example lobeline at ligand for the α4β2 and α6β2 nicotinic acetylcholine doses of 0.3 and 0.9 mg/kg improves learning on receptors. The agonist activates α-conotoxin-MII-sensitive a radial arm maze task in rats, whereas nicotine and -insensitive components of [3H]-dopamine release from (0.1, 0.3 mg/kg) does not.124 rat striatal synaptosomes, corresponding to α6β2 and α4β2 receptors (EC values are 12.7 and ~35 nM respectively). 50 • Anabasine is a tobacco alkaloid with rather non- The compound is ~ 5000-, 25000- and 140000-fold selective over α3β4, α7 and muscle nAChR receptors respectively. selective nicotinic activity. Anabaseine, a toxin that occurs naturally in nemertines (a marine worm), Koren et al (1998) 2-, 5-, and 6-Halo-3-(2(S)-azetidinylmethoxy): synthesis, affinity for nicotinic acetylcholine receptors, and molecular modeling. differs from anabasine only in bond order, but this J.Med.Chem. 41 3690. Mukhin et al (2000) 5-Iodo-A-85380, an α4β2 subtype- difference confers a functional selectivity for α7 selective ligand for nicotinic acetylcholine receptors. Mol.Pharmacol. 57 642. 125 Mogg et al (2004) Functional responses and subunit composition of presynaptic nAChRs. This structure led to the development nicotinic receptor subtypes explored using the novel agonist 5-iodo-A-85380. of a series of derivatives of which GTS-21 is the Neuropharmacology 47 848. best known (see Table 2).

www.tocris.com | 11 Tocris Bioscience Scientific Review Series

• is an anthelmintic that acts as a full acts as a weak agonist at avian α7

agonist at nematode muscle nAChRs, producing nAChRs (EC50 = 270 µM) but showed no activity muscle paralysis.28 Levamisole-sensitive muscle at α4β2 nAChRs.129. nAChRs in C. elegans contain the α subunits UNC-38, UNC-63 and LEV-8, and non-α subunits • is a 5-HT3 receptor antagonist that UNC-29 and LEV-1. In cultured C. elegans muscle displays potent partial agonist at α7 nAChRs cells, levamisole activates single channels with a (EC50 ~1 µM). Low micromolar concentrations of conductance similar to that of channels activated this compound are also reported to inhibit α3β4 4. by ACh. It is 5 times more potent than ACh, nAChRs. activating currents at concentrations greater than Antagonists (Table 4) 100 nM.126 The structurally unrelated anthelmintic Competitive antagonists agonists and activate the same Competitive antagonists interact reversibly with receptors. Levamisole is devoid of agonist activity the nAChR at, or close to, the agonist binding site, at mammalian nAChRs but has been reported to stabilising the receptor in a conformation with the act as an (see below). channel closed and preventing access for agonists. • Imidacloprid is a drug that acts Inhibition by reversible competitive antagonists is at nAChRs in the insect CNS; these receptors surmountable with increasing agonist concentration, constitute a major pesticide target.127 Data from shifting the concentration response relationship heterologous expression systems indicate that to the right (e.g. dihydro-β-erythroidine (DHβE) nAChRs containing Drosophila α2 subunits are versus nicotine-evoked [3H]-dopamine release130).

susceptible to activation by imidacloprid (EC50 Consequently the degree of functional blockade ~2 µM),128 whereas those containing Dα1 subunits achieved by a given concentration of competitive are not. Mutagenesis studies highlight loops B antagonist will be influenced by the agonist and C in the primary binding site of Dα2 subunit concentration. Most competitive antagonists originate (see Figure 1) as important for neonicotinoid from a wide variety of natural sources. Unfortunately selectivity (and possibly resistance).127 there is a very limited range of subtype-selective

Table 4 | Specificity of selected antagonists for neuronal nAChRsa

Effective concentration rangeb Antagonist Mode of action Specificity Reference In vitro In vivo d-Tubocurarine Competitive (and Non-selective; 10 µM Muscle block 4, 131, 339 other interactions) Also blocks predominates

5-HT3 > GABAA Dihydro-β-erythroidine Competitive β2>β4 1-10 µM 1-6 mg/kg 4, 137, 138, (DHβE) 166, 295, 307 a-Conotoxin MII Competitive? α3β2*/α6β2* 10-120 nM Does not access brain; 139, 141, 145, ‘Dock and lock’ local injection: 147, 308, 309 5 nmol/VTA 0.25-25 pmol/locus coeruleus a-Conotoxin AuIB Competitive? α3β4* 1-10 µM Does not access brain; 139, 140, 148, local injection: 309 1-25 pmol/locus coeruleus Non-competitive Neuronal 1-10 µM 1-4 mg/kg s.c. or i.p. 4, 149, 166, αβ > α7 310, 311 Hexamethonium Non-competitive / Neuronal 1-100 µM Does not access brain 149, 165, 166, weakly competitive 1-3 mg/kg s.c. for 339 ganglionic blockade Non-competitive / Neuronal (persistent 10-100 µM 10 mg/kg s.c. 168, 169, 171, long lasting (weeks or block of CNS 10 µg i.c.v. 312 months) nAChRs, transient block of ganglionic nAChRs) TMPH Non-competitive: use- αβ>α7 or muscle; 0.1-10 µM 1-5 mg/kg s.c. 173, 174 dependent, voltage- influenced by addition independent of α5, α6, β3

(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

12 | Nicotinic Receptors agents and not all are commercially available. Table nature means that they do not cross the blood 4 lists some antagonists that have been used to brain barrier but examples of local delivery of inhibit neuronal nAChRs; those with specificity for α7 these toxins into the brain are given in Table 4. nAChRs are included in Table 2. Certain α-conotoxins can adhere to plastic: use of sialinised glass or plastic and/or addition of • d-Tubocurarine (d-TC), produced by the South BSA can reduce this problem. American shrub Chondodendron tomentosum, is a classical non-selective nAChR antagonist. • α-Conotoxin MII was originally shown to be a d-TC does not discriminate appreciably between potent and selective antagonist of α3β2 nAChRs nAChR subtypes and it is able to fully antagonise expressed in Xenopus oocytes; α3β2 nAChRs functional responses mediated by muscle are fully blocked by 100 nM toxin. At other and neuronal nAChRs at a concentration of heterologously expressed nAChRs, the potency approximately 10 μM.131 It should be noted that the of α-conotoxin MII was 2-4 orders of magnitude mechanism of inhibition by d-TC can be complex, lower.141,142 It has subsequently been found to be also involving non-competitive interactions.117 an equally effective antagonist of α6β2* nAChRs,

In addition, d-TC is a potent 5-HT3 receptor reflecting the sequence similarities between α3 143 antagonist; it binds with Ki ~0.1 µM, and 1-10 µM and α6 subunits (Figure 3). α-Conotoxin MII

d-TC inhibits 5-HT3 receptor-mediated currents has been especially useful in elucidating the in hippocampal interneurones and transfected contribution of α3/α6β2* nAChRs to dopaminergic HEK-293 cells.132,133,134 Higher concentrations pathways in rodents and primates; it is typically 57,144,145,146 of d-TC also block GABAA receptor-mediated used at concentrations of 10-120 nM. currents.135 Recently α-conotoxin PIA from Conus purpurascens has been described; it is the first • Dihydro-β-erythroidine (DHβE), an alkaloid ligand to exhibit a marked preference for α6β2* originating from Erythrina seeds, is a purely over α3β2 nAChRs.147 The mode of action of α- competitive antagonist for neuronal nAChRs. conotoxin MII and related α-conotoxins is complex. Sub-micromolar concentrations of DHβE Studies of binding kinetics, crystal structures block human and rat α4β2 and α3β2 nAChRs and docking simulations have indicated a 2 step but it is 10-50 fold less potent at α3β4 and α7 ‘dock and lock’ model, with initial binding to the nAChRs expressed in Xenopus oocytes.4,131,136 In complementary β nAChR subunit followed by a hippocampal neurones, 100 nM DHβE blocked stabilising interaction with the primary α subunit.4 type II currents, attributed to α4β2*, whereas type I currents mediated by α7 nAChRs were • α-Conotoxin AuIB from Conus aulicus is a much insensitive to DHβE concentrations below less potent toxin but the only one reported to have 10 µM.91 Therefore DHβE can be regarded as a selectivity for α3β4* nAChRs; 1-10 μM α‑conotoxin non-α7 nAChR antagonist with a preference for AuIB is required for complete blockade of α3β4 β2-containing subtypes; it is typically employed nAChRs.148 α-Conotoxin AuIB partially inhibits at a concentration of 1-10 µM. DHβE is also nicotine-evoked [3H]-noradrenaline and [3H]-ACh effective in vivo (see Table 4), for example, it has release from rat brain preparations, implicating been used to implicate non-α7 nAChRs in nicotine α3β4* nAChRs in these responses, consistent reward and in the enhancement of contextual with weak sensitivity to DHβE.148,149 137,138 fear conditioning by nicotine. • α-Conotoxin ImI (Table 2) is a selective antagonist of α7 nAChRs, fully blocking responses • α-Conotoxins, present in the venoms of various at 1 µM.150,151 It has weaker interactions with species of Conus snails, provide a growing family muscle and α9 nAChRs (see Table 2). More of subtype-selective nAChR antagonists.139,140 potent α‑conotoxins with greater selectivity for α7 α-Conotoxins are small peptide toxins (12-18 nAChRs over other subtypes are under amino acids) with 4 cysteine residues forming investigation.152 One caveat with regard to using two disulphide bonds, between the first and third, the α-conotoxins is that their specificity may be and second and fourth cysteines. They can be highly species specific, limiting extrapolation generated by peptide synthesis with sequential between species. For example, although α- de-protection of the cysteine pairs.141 The different conotoxin ImI inhibits rat α7 nAChRs, it is reported specificities of related α-conotoxins that retain the to block a non-α7 nAChR (but not α7 nAChRs) in cysteine residues in the same positions reflect bovine chromaffin cells.153 the side chains of the non-conserved amino acids. Three well characterised α-conotoxins • α-Bungarotoxin (α-Bgt; Tables 1, 2) is the most with different specificities for neuronal nAChRs well-established subtype-selective nicotinic are α‑conotoxins MII, AuIB and ImI. These have antagonist. Isolated from the venom of the been exploited in in vitro studies. Their peptidergic Taiwanese banded krait, Bungarus Multicinctus,

www.tocris.com | 13 Tocris Bioscience Scientific Review Series

α‑Bgt. However, like α-Bgt, MLA also blocks α9 Methyllycaconitine, α7 nAChR and α9α10 nAChRs with low nanomolar affinity. Antagonist Of more practical concern for brain studies, OMe MLA interacts with α6β2* nAChRs with only Methyllycaconitine MeO H OMe ~30-fold lower affinity (Ki ~30 nM) than with α7 40,157 Cat. No. 1029 Me H nAChRs. In addition, non-α7 interactions of N OH MLA have been indicated in avian preparations,

OH where functional responses sensitive to MLA, but OMe O 158,159 not α-Bgt, have been observed. Therefore, .C6H8O7 O this antagonist is selective, rather than specific, N O O for α7 over other nAChR subtypes. In particular,

Me its interactions with α6β2* nAChR subtypes could Methyllycaconitine is a potent competitive antagonist of confound interpretation of results from in vivo

α7-containing neuronal nicotinic receptors (Ki = 1.4 nM) that studies, in which the local concentration of MLA also interacts with α4β2 and α6β2 receptors at nanomolar is not known. concentrations. Unlike α-Bgt, MLA can distinguish between α7 and muscle nAChRs. The antagonist attenuates • MG 624 (Table 2) is a 4-oxystilbene derivative -induced neurotoxicity in the mouse that is a selective and quite potent antagonist striatum in vivo. of chicken α7 nAChRs. The IC50 for blocking α7 Ward et al (1990) Methyllycaconitine: a selective probe for neuronal nAChR responses (100 nM) is ~30 fold lower α‑bungarotoxin binding sites. FEBS Lett. 270 45. Wonnacott et al (1993) than that for muscle-type or α4β2 nAChRs.160,161 Methyllycaconitine: a new probe that discriminates between nicotinic acetylcholine receptor subclasses. Methods in Neurosci. 12 263. Dobelis However, the related oxystilbene derivative F3 et al (1999) Effects of delphinium on neuromuscular transmission. (that displayed similar properties to MG 624 in J.Pharmacol.Exp.Ther. 291 538. Escubedo et al (2005) Methyllycaconitine 160 prevents methamphetamine-induced effects in mouse striatum: involvement of chick preparations) was found to block non-α7 α7 nicotinic receptors. J.Pharmacol.Exp.Ther. 315 658. nAChR responses in rat chromaffin cells, albeit 162 with somewhat lower potency (IC50 ~350 nM). it is an 8kDa peptide that binds to muscle and This raises the possibility that MG 624 may also Torpedo nAChRs, and to α7-α9* nAChRs with display species differences or a broader range an affinity (Ki) of ~1 nM. However, its association of nAChR interactions than first thought. Indeed, binding kinetics are very slow and typically a pre- MG 624 significantly attenuated nicotine-evoked incubation of up to one hour with a low nanomolar [3H]-dopamine release in rat cortical slices, in concentration (10 nM) of toxin is necessary to contrast to α-conotoxin ImI, α-Bgt or MLA.115 achieve a complete blockade. This is commonly circumvented by increasing the concentration Non-Competitive Antagonists and decreasing the preincubation time. This Non-competitive antagonists, as indicated above, do strategy is possible because, even at micromolar not compete for binding to the agonist binding sites, concentrations, α-Bgt does not appear to interact but interact with distinct sites that can include the with α/β heteromeric nAChRs. However, it was lumen of the nAChR channel.

recently reported to block a subset of GABAA • Mecamylamine is the archetypal non- receptors containing an adjacent pair of β3 competitive antagonist for neuronal nAChRs. It

subunits (Kd ~50 nM; t½ ~10 min); whether GABAA was developed as a for the receptors with this subunit composition exist in treatment of hypertension. Mecamylamine inhibits 154 nature is presently unclear. α-Bgt binding to most neuronal nAChRs with IC50 values typically nAChRs exhibits very slow dissociation kinetics, in the range 0.1-1 µM, and 10 µM mecamylamine such that functional blockade is not reversed is a standard concentration used to achieve a by washout within the timescale of a typical complete block in vitro. α7 nAChRs are somewhat experiment (e.g. 1 hour). less sensitive than α/β heteromers, requiring >10 μM for full blockade.4,131 High concentrations • Methyllycaconitine (MLA, Table 2) is a of mecamylamine (100 µM) can transiently inhibit norditerpenoid alkaloid produced by Delphinium NMDA receptors.163 Mecamylamine crosses sp. It is a potent competitive antagonist, selective the blood brain barrier freely and is typically for α7 nAChRs and, unlike α-Bgt, it discriminates administered at a concentration of around 1mg/kg between α7 and muscle nAChRs.155 MLA binds to in rodents to block CNS nAChRs in behavioural α7 nAChRs with a K of approximately 1 nM, and i studies. Unexpectedly, lower concentrations of picomolar concentrations are reported to block mecamylamine have been reported to improve α7 nAChR-mediated currents recorded from cognitive performance.164 hippocampal neurones or Xenopus oocytes.23,156 Inhibition of α7 nAChRs by MLA is rapid and • Hexamethonium, like mecamylamine, was reversible, making it a useful complement to also first recognised as a ganglionic nAChR

14 | Nicotinic Receptors

blocking agent but the hydrophilic nature of nAChRs.175 These agents cannot be considered this polymethylene bistrimethylammonium to be specific for nAChRs, but the interactions can compound prevents it from crossing the blood- be of pharmacological or physiological interest or brain barrier.7 Comparative studies of the effects they may raise practical concerns. The examples of mecamylamine and hexamethonium in vivo mentioned here do not constitute an exhaustive list have been used to establish if a particular but are chosen to represent some of the more well behaviour is centrally or peripherally mediated, known or topical classes. as peripherally administered hexamethonium is • MK-801 () is an anticonvulsant unable to block CNS nAChRs in vivo.165 In vitro agent developed as a channel blocker of the hexamethonium is used at 1-100 µM to block NMDA receptor. It is able to perform the same neuronal nAChRs.149,166 function on neuronal nAChRs, where it is an

• Chlorisondamine, a bisquaternary nicotinic open channel blocker at α4β2 (IC50 = 15 μM) 176,177,178 antagonist, was also originally used as a and α7 nAChRs (IC50 = 15 μM). Studies ganglionic blocker.167 When administered in vivo on Torpedo nAChRs indicate that MK-801 may it is unique in producing a persistent blockade also interact with a non-luminal site, in addition of nAChRs within the CNS that lasts for weeks to the channel.179 The dissociative anaesthetics or even months, in contrast to a transient (PCP) and that interact

ganglionic blockade. For example, nicotine- with NMDA receptors also block nAChRs (IC50 evoked dopamine release was abolished from rat ~2-10 μM).180,181,182 The tryptophan metabolite striatal synaptosomes prepared several weeks , a competitive antagonist of after administration of chlorisondamine in vivo the glycine site of NMDA receptors, is also (10 mg/kg s.c).168 It is hypothesised that this long a non-competitive antagonist of α7 nAChRs 175,183 lasting inhibition may arise from an intracellular (IC50 = 7 μM). accumulation of the drug,169 and a specific • Buproprion (ZybanTM) was originally developed interaction with the α2 nAChR subunit has been as an antidepressant but is now marketed as proposed.170 However, given the paucity of α2 an aid to smoking cessation. Its principle site nAChR subunit expression in the rodent brain, of action is the dopamine and noradrenaline this is unlikely to explain the widespread central transporters, but at low micromolar concentrations antagonism by chlorisondamine. Its persistent it also inhibits nAChRs.184,185 It non-competitively action is particularly useful for antagonising brain inhibits rat α3β2, α4β2 and α7 nAChRs expressed nAChRs during chronic studies involving repeated in Xenopus oocytes or native nAChRs in cell or continuous nicotine administration.171 lines and nicotine-evoked [3H]-dopamine release • 2,2,6,6-Tetramethylpiperidin-4-yl heptanoate from rat striatal preparations.186,187,188,189,190 (TMPH) is a synthetic derivative of the parent The possibility that this interaction contributes bis-tetramethylpiperidine compound BTMPS. to the efficacy of as a smoking The latter produces a nearly irreversible cessation agent is debated. Low micromolar non-competitive block of neuronal nAChRs, concentrations of other antidepressants whereas its inhibition of muscle nAChRs is including , and 172 191,192 readily reversible. Similarly, low micromolar also inhibit nAChRs (IC50 = 1‑12 µM). The concentrations of TMPH produced a long-lasting neuroleptic and the anti-epileptic inhibition of heteromeric nAChRs comprised of α3 drug lamotrigine also interact with nAChR or α4 with β2 or β4 subunits, whereas blockade channels.193,194 of α7 and muscle nAChRs was readily reversible, • L-type voltage-operated Ca2+ channel allowing the different classes of nAChRs to be blockers inhibit nAChRs in chromaffin cells distinguished. However, incorporation of an or neuroblastoma cell lines with IC values additional subunit (either α5, α6 or β3) into 50 in the low micromolar range. These drugs heteromeric nAChRs also decreased inhibition, include furnidipine, verapamil and diltiazem and attributed to a residue within the M2 channel lining the dihydropyridines: nimodipine, nifedipine, domain (Figure 1).173 TMPH was also effective in nitrendipine and furnidipine.195,196,197 The N/P/ vivo, showing a differential inhibition of nicotine- Q‑type ω‑conotoxin MVIIC induced analgesia and nicotine discrimination, and the N-type blocker ω‑conotoxin GVIA were compared with hypothermia and locomotor without effect, although another study reported effects.174 that they too can block nAChRs expressed in Other Modulators Producing Non- Xenopus oocytes, with rat α3β4 nAChRs being Competitive Inhibition of Neuronal nAChRs more susceptible than α7 nAChRs.198 The block Many compounds that have other primary targets of α3β4 nAChRs by ω‑conotoxins was shown also act as non-competitive antagonists of to be reversible, whereas these inhibitors exert

www.tocris.com | 15 Tocris Bioscience Scientific Review Series

a longer lasting inhibition of voltage-operated calcium channels. This difference has been PNU 120596 and PNU 282987, exploited to discriminate between these two Selective α7 nAChR Ligands targets.199

• Steroids, including corticosterone, , MeO OMe estradiol, hydrocortisone and aldosterone, have PNU 120596 Cat. No. 2498 O N HN Cl been shown to inhibit neuronal nAChRs expressed N O in the SH-SY5Y cell line, as well as α4β2 nAChRs H

in HEK-293 cells or Xenopus oocytes, with IC50 N values ranging from 0.1-10 μM.200,201,202 PNU 282987 O Cat. No. 2303 N • β-Amyloid peptide Aβ1-42, the endogenous H agent that accumulates in Alzheimer’s disease, Cl is reported to interact with nAChRs, principally PNU 120596 is a positive allosteric modulator of α7 neuronal 203 nicotinic acetylcholine receptors (EC50 = 216 nM) that the α7 subtype, in a variety of ways. Aβ has displays with no detectable effect on α4β2, α3β4 and α9α10 been reported to bind competitively (IC50 ~5 receptors. pM) to α7 nAChRs204 but to inhibit nAChRs non- 205,206,207 PNU 282987 is a highly selective α7 nAChR agonist competitively (1-100 nM). Picomolar (K = 26 nM) that displays negligible blockade of α1β1γδ and concentrations of Aβ have been found to activate i α3β4 nAChRs (IC50 ≥ 60 µM). The agonist was found to be α7 and non-α7 nAChRs in Xenopus oocytes inactive against a panel of 32 receptors at 1 µM, except 208,209 or synaptosomes, but these results are 5‑HT3 receptors (Ki = 930 nM). 205,207 controversial. α7 nAChRs have also been In vivo proposed to mediate the modulation of NMDA Both compounds improve auditory gating deficits induced receptor trafficking by Aβ.210 The nature of the by D-amphetamine in anesthetised rats; a model proposed interactions of Aβ with nAChRs is particularly to reflect a circuit level disturbance associated with intriguing in view of the changes in nAChRs in schizophrenia. Alzheimer’s disease and the ability of nicotinic Bodnar et al (2005) Discovery and structure-activity relationship of quinuclidine benzamides as agonists of α7 nicotinic acetylcholine receptors. J.Med.Chem. agonists to enhance cognition and/or offer 48 905. Hajós et al (2005) The selective α7 nicotinic acetylcholine receptor neuroprotection, but more work is required to 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 clarify the relationship. 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 Positive Allosteric Modulators nicotinic acetylcholine receptor: in vitro and in vivo characterization. J.Neurosci. In addition to divalent cations such as Ca2+ and 25 4396. Zn2+, several structurally diverse compounds can potentiate responses to nicotinic agonists by acting at a site distinct from the agonist binding site. Such effect as an AChE inhibitor, its ability to enhance effects are well established for other receptor classes the effects of residual ACh could contribute to TM (e.g. and glycine, acting at GABAA the efficacy of galanthamine (Reminyl ) as a and NMDA receptors respectively), but have only treatment for cognitive decline in Alzheimer’s relatively recently been characterised for neuronal disease. nAChRs.4,175 The site of action of these positive allosteric • and galanthamine were among modulators is proposed to be at, or close to, Lys the first acetylcholinesterase inhibitors to be 125 of the extracellular N-terminal domain of the recognised. These compounds potentiate nAChR neuronal nAChR α subunit.211 Other allosteric responses evoked by sub-maximal concentrations potentiators acting at the same site include the of nicotinic agonists, thus shifting the dose , the neuromuscular blocking agent response curve for agonists to the left. Alone, benzoquinonium (Table 1) and the neurotransmitter physostigmine or galanthamine can activate 5-HT; sub-micromolar concentrations of 5-HT mimic single-channel currents in muscle and neuronal the potentiating effects of galanthamine in PC12 cells but the probability of channel opening is cells.175,211 Interestingly 5-hydroxyindole, a metabolite too low to generate macroscopic (whole cell) of 5-HT, is also a positive allosteric modulator but currents.211 Galanthamine and physostigmine shows selectivity for α7 nAChRs (Table 2).215 The site produce a modest potentiation (~30%) of ACh- of action of 5-hydroxyindole has not been established. evoked currents or nicotine-evoked increases Recently another more potent and more efficacious in intracellular Ca2+ over a narrow concentration synthetic positive allosteric modulator of α7 nAChRs range (1-10 µM); higher concentrations (>10 µM) has been described; PNU 120596 (see Table 2).217 inhibit responses by acting as an open channel Such compounds could have therapeutic utility in blocker.175,212,213,214 In addition to galanthamine’s maximising the effects of endogenous agonists.

16 | Nicotinic Receptors

nAChRs in wound healing. The related SLURP-2 Sazetidine A, Novel α4β2 protein is proposed to interact with α3* nAChRs Receptor Desensitiser on keratinocytes to prevent apoptosis. Thus the different effects observed for SLURP-1 and -2 Sazetidine A NH O are attributed to their differential binding to the Cat. No. 2736 N nAChR subtypes expressed in mucocutaneous

epithelial cells.224 .2HCl Radioligands (Table 5) HO Nicotinic radioligands have been valuable tools for Sazetidine A is a subtype-selective α4β2 nicotinic the identification, quantitation, pharmacological acetylcholine receptor desensitiser that binds with high characterisation and localisation of nAChRs. Table 5 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 summarises the features of the major nicotinic 3 receptor subtypes respectively). The compound does not radioligands currently in use. [ H]-Nicotine binding to activate these receptors or inhibit activation when added with brain tissue was first described in detail by Romano nicotine, but causes potent inhibition of nicotine-stimulated and Goldstein.16 In the majority of reports [3H]-nicotine α4β2 activation following a 10 minute preincubation. identifies a single population of sites in the CNS Sazetidine A potently drives the α4β2 receptors into a with a K of 1-10 nM. Although by definition nicotine desensitised state without receptor activation, an inhibitory d action termed silent desensitisation. interacts with all nAChR subtypes, the radiolabelled version labels predominantly α4β2* nAChRs; Xiao et al (2006) Sazetidine-A, a novel ligand that desensitizes α4β2 nicotinic 3 acetylcholine receptors without activating them. Mol.Pharmacol. 70 1454. [ H]‑nicotine binding is almost totally absent in α4 or β2 null mutant mice.225,226 This apparent anomaly • Ivermectin, an anthelminthic drug, also reflects the binding affinity (typically <20 nM) required potentiates α7 nAChRs,216 whereas the to retain bound ligand during the removal of ‘free’ anthelmintic levamisole has been reported to (unbound) ligand (typically by filtration through glass show dual potentiation and inhibition of ACh- fibre filters, a process that takes ~15 sec). Only α4β2* evoked responses recorded from Xenopus nAChRs bind nicotine with high enough affinity to oocytes expressing human α3* nAChRs.218 This retain it during this step. In contrast, [3H]-epibatidine dual behaviour is reminiscent of the modulatory binds with sub-nanomolar affinity to multiple nAChR effects of galanthamine and physostigmine subtypes.229 Subpopulations have been defined by described above. their differential sensitivity to A-85380, cytisine or α‑conotoxin MII.225,227,228,230 • β-estradiol has been observed to potentiate the activation of human, but not rat, α4β2 nAChRs A second issue that can be confusing is the disparity

expressed in Xenopus oocytes; this action was between binding affinity (Kd) and functional potency

distinct from the inhibitory effects of steroids (EC50) of nAChR agonists that are also radioligands;

and mediated by interaction with the C-terminal Kd correlates with Ki for the unlabelled version tail of the α4 subunit.219,220 In view of the high derived from competition binding assays (Table 3) concentrations required for this effect, its and is typically 1-3 orders of magnitude lower than 175 biological significance is uncertain. EC50. This difference reflects the conditions of the equilibrium binding assay in which the incubation • Recently, novel members of the Ly-6/u-PAR time will be sufficient to stabilise the desensitised superfamily have been described and shown state of the nAChR that binds agonist with higher to act as endogenous modulators of nAChR affinity (Figure 2). Indeed, analysis of the kinetics of function. Lynx-1 is a GPI-anchored protein found [3H]-nicotine binding to brain membranes revealed a in brain and other tissues including lung. It co- two-state model, consistent with interconversion from localises with both α7 and α4β2 nAChRs, altering a low affinity binding component (K 150 nM) to a high single channel conductance and enhancing d affinity conformation (K 1 nM).231 Similarly, the affinity desensitisation.221,222 SLURP-1 is a 9 kDa d with which unlabelled agonists displace radioligand secreted protein lacking a GPI anchor that is binding in competition assays (K; Table 3) will reflect produced by keratinocytes. Like Lynx-1, it also i their affinity for a desensitised form of the nAChR. has a high degree of structural homology with 3‑fingered snake neurotoxins like α-Bgt. Picomolar The antagonist radioligands α-Bgt and MLA exhibit concentrations of SLURP-1 were without binding affinities of ~1 nM that are in line with effect when applied alone to Xenopus oocytes their functional potency at α7 nAChRs. However, expressing human α7 nAChRs but increased the competitive antagonism is influenced by agonist amplitude of ACh-evoked currents several fold.223 concentration and binding kinetics, as discussed The association of the SLURP-1 gene with an above. Therefore higher concentrations would inflammatory skin disorder suggests a role for typically be used for nAChR blockade (see Table 2).

www.tocris.com | 17 Tocris Bioscience Scientific Review Series

Table 5 | Selected radioligands that label neuronal nAChRs

a nAChR Radioligand Comment Kd Ref subtype (nM) [3H]-ACh AChE inhibitor (100 µM DFP) and muscarinic AChR antagonist (1.5 µM 12.3r 225, 313, atropine) required in assay mix; labels same site(s) as [3H]-nicotine; used at 314, 315, 2 nM for autoradiography 339 [3H]-Cytisine Labels same site(s) as [3H]-nicotine in rodents; 0.15-1.1r 69, 95, comparable autoradiographic labelling with [3H]-nicotine in monkey; 0.2h 225, 316, binding largely abolished in β2 null mutant mice; unaffected in β4 and α7 null 317, 318, α4β2* mutant mice; used at 5 nM for autoradiography 319, 320 [3H]-Nicotine Add mercaptoacetic acid for storage; purification of [3H]-nicotine by 0.9-2.0r 16, 69, chromatography, or addition of 200 mM Tris to assay buffer, and filtration 10m 225, 315, through glass fibre filters soaked overnight in 0.3% polyethylene imine (PEI) 2.8h 321, 322, reduces non-specific binding; comparable labelling of heterologously 339

expressed α4β2 nAChRs; Bmax increased in smokers and in animals after chronic nicotine; used at 3.5-5 nM for autoradiography α6β2* [125I]-α-Conotoxin Mostly used for quantitative autoradiography; membrane binding assay 0.6-0.8r 109, 157, MII possible with addition of BSA and a dilution step to reduce non-specific binding; 0.3-1.9m 323, 324, corresponds to a minor population of sites labelled by [3H]-epibatidine; 0.9p 325, 326, binding abolished in α6 (but not α3) and β2 null-mutant mice; ~50% reduction in 327 α4 and β3 null-mutant mice; labelling of basal ganglia greatly reduced in Parkinsonian model; used at 0.5 nM for autoradiography β2* 5-[125I]-A85380 Incorporation of an iodine atom into A85380, to generate a ligand for PET and 0.010r 110, SPECT, produced a more potent and more selective nicotinic agonist; labelling 0.012h 328, absent in β2 null mutant mice; used at 0.2 nM for autoradiography; 329, [123I]‑labelled version used for SPECT in smokers versus non-smokers 330 [3H]-Epibatidine A potent ligand with low non-specific binding under standard incubation 0.015-0.05r 95, conditions; potency can result in ligand depletion at very low concentrations, (0.4 low 225, ameliorated by increasing assay volume; labels multiple sites in brain (at least affinity site) 227, six) distinguished by differential competition by more selective ligands (e.g. 331, m cytisine, α-conotoxinMII) or analysis in null mutant mice; Kd for different αβ 0.014 332 combinations expressed in HEK293 cells range from 21-94 pM; used at 0.5 nM (7.2 low for autoradiography affinity site) Non-α7 [125I]-Epibatidine (+/-)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane (IPH), analogue of 0.9r 0.05- 227, epibatidine incorporating an iodine atom in place of the chlorine atom in the 0.07m 325, chloropyridyl ring of natural epibatidine, displays identical properties to parent 328, compound; labels multiple sites in brain with high affinity, predominantly α4β2 333 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 [3H]-MLA Non-specific binding can be decreased by inclusion of 0.1% BSA and/or 1.9r 27, 232, m 200 mM Tris in assay buffer; labels α9α10/5-HT3 chimeric receptors 2.2 260, 334,

(Kd 7.5 nM); also used to label invertebrate nAChRs (Kd~1 nM); used at 5 nM 335 (rat) / 10 nM (mouse) for autoradiography [125I]-MLA Comparable properties with [3H]-MLA; i.v. administration shows rapid clearance 1.8r 336, 337 α7 and poor brain penetration; increased binding in primate model of Parkinson’s 33-46p disease [125I]-α-Bgt The classical ligand for defining α7 nAChRs; slow association kinetics require 0.1-1.2r 95, 315, long incubation time; labelling abolished in α7 null mutant mice; used at 0.12m 338, 339, 0.7‑1.5 nM for autoradiography 340, 341 a 125 Kd , Binding affinity derived from saturation binding experiments carried out on brain membranes or heterologously expressed nAChRs. Binding affinities of [ I]- 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, 5´ position increased both potency and selectivity.110 together with the generation of novel synthetic Iodinated ligands are typically labelled to higher ligands in the quest for greater nAChR subtype specific radioactivity than their tritiated counterparts selectivity, can lead to new nicotinic radioligands, and hence provide greater sensitivity and resolution and [125I]-5-iodo-A85380 is an example to come out (especially for autoradiography).232 They have the of this process. Introduction of a halogen atom can disadvantage of a short half life (60 days) and the change the properties of the ligand. Fortuitously in higher energy γ-radiation emitted requires more the case of A85380, addition of an iodine atom in the stringent handling conditions.

18 | Nicotinic Receptors

Fluorescent or streptavidin labels covalently specific ligands will facilitate a better understanding attached to α-Bgt facilitate identification of muscle of the physiological roles and therapeutic potential and α7 nAChRs by light, confocal and electron of individual neuronal nAChR subtypes. The more microscopy.44,233 The development of other selective allosteric potentiating drugs that have fluorescently labelled probes would be desirable and recently been described (Table 2) will also assist progress in labelling α-conotoxin MII has recently in revealing the often small or quiescent nAChR been reported.234 responses. With the first nicotinic agonist in the clinic (varenicline)235 and several more at different Future Directions for Nicotinic stages of clinical trials,49,236 nicotinic drug discovery Ligands is expanding and the products of these endeavours Despite the growing nicotinic pharmacoepia,4 there will enhance nAChR research as it proceeds on its is still a lack of availability of subtype-selective upward trajectory. agonists and antagonists. The design of more

69. Anderson & Arneric (1994) Eur.J.Pharmacol. 253 261. 136. Harvey et al (1996) J.Neurochem. 67 1953. References 70. Whiteaker et al (1998) Mol.Pharmacol. 53 950. 137. Kenny & Markou (2006) Neuropsychopharmacology 31 1. Karlin (2002) Nat.Rev.Neurosci. 3 102. 71. Elgoyhen et al (1994) Cell 79 705. 1203. 2. Jones & Sattelle (2006) Invert.Neurosci. 6 123. 72. Benowitz & Jacob (1998) New York.Wiley 213. 138. Davis & Gould (2006) Psychopharmacology 184 345. 3. Champtiaux & Changeux (2004) Prog.Brain Res. 145 73. Sanderson et al (1993) Brain.Res. 617 349. 139. McIntosh et al (1999) Ann.Rev.Neurosci. 68 59. 235. 74. Rowell & Li (1997) J.Neurochem. 68 1982. 140. Nicke et al (2004) Eur.J.Biochem. 271 2305. 4. Jensen et al (2005) J.Med.Chem. 48 4705. 75. Brunzell et al (2006) Psychopharmacology 184 328. 141. Cartier et al (1996) J.Biol.Chem. 271 7522. 5. Langley (1907) J.Physiol. 36 347. 76. Plowchalk et al (1992) Toxicol.Applied.Pharmacol. 116 142. Harvey et al (1997) Mol.Pharmacol. 51 336. 6. Dale (1914) J.Pharmacol.Exp.Ther. 6 147. 177. 143. McIntosh et al (2004) Mol.Pharmacol. 65 944. 7. Paton & Zaimis (1949) Br.J.Pharmacol. 4 381. 77. Ghosheh et al (1999) Drug.Metab.Dispos. 27 1448. 144. Kulak et al (1997) J.Neurosci. 17 5263. 8. Raftery et al (1980) Science 208 1454. 78. Siu & Tyndale (2007) Mol.Pharmacol. 71 826. 145. Kaiser et al (1998) J.Neurochem. 70 1069. 9. Unwin (2003) FEBS Lett. 555 91. 79. Vainio & Tuominen (2001) Nicotine.Tobacco.Res. 3 177. 146. McCallum et al (2005) Mol.Pharmacol. 68 737. 10. Sine & Engel (2006) Nature 440 448. 80. Briggs & McKenna (1998) Neuropharmacology 37 1095. 147. Dowell et al (2003) J.Neurosci. 23 8445. 11. Grutter et al (2004) Curr.Top.Med.Chem. 4 645. 81. Buccafusco et al (2007) Neurosci.Lett. 413 68. 148. Luo et al (1998) J.Neurosci. 18 8571. 12. Smit et al (2003) Ann.N.Y.Acad.Sci. 998 81. 82. Vainio et al (1998) Pharmacol.Toxicol. 83 188. 149. Grady et al (2001) J.Neurochem. 76 258. 13. Hansen et al (2005) EMBO.J. 24 3635. 83. Sziraki et al (1999) Neurochem.Res.. 24 1471. 150. Johnson et al (1995) Mol.Pharmacol. 48 194. 14. Changeux & Edelstein (2005) Science 308 1424. 84. Bencherif et al (1996) J.Pharmacol.Exp.Ther. 279 1413. 151. Periera et al (1996) J.Pharmacol.Exp.Ther. 278 1472. 15. Ogden & Colquhoun (1985) Proc.R.Soc.Lond.B.Biol. 85. Papke et al (2000) J.Neurochem. 75 204. 152. Whiteaker et al (2007) Biochemistry 46 6628. Sci. 225 329. 86. Summers et al (1996) Neurochem.Res. 21 1181. 153. Broxton et al (1999) J.Neurochem. 72 1656. 16. Romano & Goldstein (1980) Science 210 647. 87. Spoetti et al (2007) Int.J.Colorectal.Dis. 22 303. 154. McCann et al (2006) Proc.Natl.Acad.Sci. USA 103 5149. 17. Clarke (1992) TiPS 13 407. 88. Wonnacott & Gallagher (2006) Marine Drugs 4 228. 155. Wonnacott et al (1993) Methods in Neuroscience 12 263. 18. Boulter et al (1986) Nature 319 368. 89. Swanson et al (1986) Mol.Pharmacol. 29 250. 156. Alkondon et al (1992) Mol.Pharmacol. 41 802. 19. Gotti et al (2006) TiPS 27 482. 90. Thomas et al (1993) J.Neurochem. 60 2308. 157. Mogg et al (2002) J.Pharmacol.Exp.Ther. 302 197. 20. Schoepfer et al (1990) Neuron 5 35. 91. Alkondon & Albuquerque (1993) J.Pharmacol.Exp.Ther. 158. Yum et al (1996) Neuroscience 72 545. 21. Wang et al (1996) J.Biol.Chem. 271 17656. 265 1455. 159. Yu & Role (1998) J.Physiol. 509 607. 22. Corringer et al (2000) Annu.Rev.Pharmacol.Toxicol. 40 92. Stolerman et al (1992) Neuropharmacology 31 311. 160. Gotti et al (1998) Br.J.Pharmacol. 124 1197. 431. 93. MacPhail et al (2007) Toxicology 234 83. 161. Maggi et al (1999) Br J Pharmacol. 126 285. 23. Palma et al (1996) J.Physiol. 491 151. 94. Cassels et al (2005) Drug Discovery Today 10 1657. 162. Di Angelantonio et al (2000) Br.J.Pharmacol. 129 1771. 24. Khiroug et al (2002) J. Physiol. 540 425. 95. Marks et al (2006) Mol.Pharmacol. 70 947. 163. Papke et al (2001) J.Pharmacol.Exp.Ther. 297 646. 25. Severance et al (2004) Mol. Pharmacol. 66 420. 96. Leutje & Patrick (1991) J.Neurosci. 11 837. 164. Levin & Caldwell (2006) Neurobiol.Learn.Mem. 86 117. 26. Elgoyhen et al (2001) Proc.Natl.Acad.Sci. USA 98 3501. 97. Chandler & Stolerman (1997) Psychopharmacology 129 165. DeNoble & Mele (2006) Psychopharmacology 184 266. 27. Baker et al (2004) Mol.Pharmacol. 65 453. 257. 166. Eaton et al (2003) Mol.Pharmacol. 64 1283. 28. Brown et al (2006) Int.J.parasitol. 36 617. 98. Smith et al (2007) Psychopharmacology 190 157. 167. Plummer et al (1955) J.Pharmacol.Exp.Ther. 115 172. 29. Jones et al (2007) Invert.Neurosci. 7 67. 99. Coe et al (2005) J.Med.Chem. 48 3474. 168. El-Bizri and Clarke (1994) Br.J.Pharmacol. 111 414. 30. Van Nierop et al (2006) J.Biol.Chem. 28 1680. 100. Rollema et al (2007) Neuropharmacology 52 985. 169. El-Bizri et al (1995), Br.J.Pharmacol. 116 2503. 31. Rogers et al (1992) J.Neurosci. 12 4611. 101. Obach et al (2006) Drug.Metabolism and Disposition 34 121. 170. Woods et al (2003) J.Proteome.Res. 2 207. 32. Wang et al (2002) Prog.Neurobiol. 68 341. 102. Badio & Daly (1994) Mol.Pharmacol. 45 563. 171. Costa et al (2001) Brain.Res. 888 336. 33. Di Angelantonio et al (2003) Eur.J.Neurosci. 17 2313. 103. Xiao & Kellar (2004) J.Pharmacol.Exp.Ther. 310 98. 172. Francis et al (1998) Biophys.J. 74 2306. 34. Mao et al (2006) Mol.Pharmacol. 70 1693. 104. Gerzanich et al (1995) Mol.Pharmacol. 48 774. 173. Papke et al (2005) Mol.Pharmacol. 67 1977. 35. Lukas et al (1999) Pharmacol.Rev. 51 397. 105. Alkondon & Albuquerque (1995) J.Pharmacol.Exp.Ther. 174. Damaj et al (2005) Eur.J.Pharmacol. 521 43. 36. Genzen et al (2001) J.Neurophysiol. 86 1773. 274 771. 175. Pereira et al (2002) Prog in Neurobiol. 53 479. 37. Haberberger et al (2004) Auton.Neurosci. 113 32. 106. Jain (2004) Curr.Opin.Investig.Drugs. 5 76. 176. Ramoa et al (1990) J.Pharmacol.Exp.Ther. 254 71. 38. Wonnacott (1997) Trends Neurosci. 20 92. 107. Rueter et al (2006) CNS.Drug.Rev. 12 100. 177. Briggs & McKenna (1996) Neuropharmacol. 35 407. 39. Albuquerque et al (1997) J.Recept.Signal.Transduct. 108. Sullivan et al (1996) Neuropharmacol. 35 725. 178. Buisson & Bertrand (1998) Mol.Pharmacol. 53 555. Res. 17 243. 109. Whiteaker et al (2002) J.Neurosci. 22 2522. 179. Arias et al (2001) Mol.Pharmacol. 59 1051. 40. Klink et al (2001) J.Neurosci. 21 1452. 110. Mukhin et al (2000) Mol.Pharmacol. 57 642. 180. Furuya et al (1999) Anesth.Analg. 88 174. 41. Fabian-Fine et al (2001) J.Neurosci. 21 7993. 111. Mogg et al. (2004) Neuropharmacology 47 848. 181. Fryer & Lukas (1999) J.Pharmacol.Exp.Ther. 288 88. 42. Dajas-Bailador & Wonnacott (2004) TiPS 25 317. 112. Vaupel et al (1998) Neuroreport. 9 2311. 182. Yamakura et al (2000) Anesthesiology 92 1144. 43. Mansvelder & McGehee (2000) Neuron 27 349. 113. Liu et al (2003) Neuroreport. 14 1503. 183. Hilmas et al (2001) J.Neurosci. 21 7463. 44. Jones & Wonnacott (2004) J.Neurosci. 24 11244. 114. Sharples et al (2000) J.Neurosci. 20 2783. 184. Damaj et al (2004), Mol.Pharmacol. 66 675. 45. Patti et al (2006) Neuropharmacology 50 706. 115. Cao et al (2005) Neuropharmacology 48 72. 185. Dwoskin et al (2006) CNS Drug Rev. 12 178. 46. Welsby et al (2006) Eur.J.Neurosci. 24 3109. 116. Blanchet et al (2006) Am.J.Physiol.Lung.Cell.Mol. 186. Fryer & Lukas (1999a) J.Neurochem. 72 1117. 47. Hoyle et al (2006) Psychopharmacology (berl) 189 211. Physiol. 291 L757. 187. Slemmer et al (2000) J.Pharmacol.Exp.Ther. 295 321. 48. Young et al (2007) Eur.Neuropsychopharmacol. 17 145. 117. Bertrand et al (1992) Neurosci.Lett. 146 87. 188. Miller et al (2002) J.Pharmacol.Exp.Ther. 302 1113. 49. Mazurov et al (2006) Curr.Med.Chem. 13 1567. 118. Wong et al (1995) Brain.Res.Mol.Brain.Res. 28 101. 189. Damaj et al (2004) Mol.Pharmacol. 66 675. 50. Cooper et al (1991) Nature 350 235. 119. Peng et al (1997) Mol.Pharmacol. 51 776. 190. Sidhpura et al (2007), Eur.J.Pharmacol. 567 102. 51. Anand et al (1991) J.Biol.Chem. 266 11192. 120. Garcha et al (1993) Psychopharmacology 110 347. 191. Garcia-Colunga et al (1997) Proc.Natl.Acad.Sci. USA 52. Moroni et al (2006) Mol.Pharmacol. 70 755. 121. Miller et al (2000) Neuropharmacology 39 2654. 94 2041. 53. Tapia et al (2007) Mol.Pharmacol. 71 769. 122. Dwoskin & Crookes (2001) J.Pharmacol.Exp.Ther. 298 192. Fryer & Lukas (1999) J.Neurochem. 72 1117. 54. Picciotto et al (2001) Pharmacol.Ther. 92 89. 395. 193. Arias (1998) Biochim.Biophys.Acta. 1376 173. 55. Maskos (2007) J.Neurochem. 100 596. 123. Miller et al (2007) Drug..Depend. (in press). 194. Valles et al (2007) Neuroreport. 18 45. 56. Raggenbass & Bertrand (2002) J.Neurobiol. 53 580. 124. Levin & Christopher (2003) Pharmacol.Biochem.Behav. 195. Lopez et al (1993) Eur.J.Pharmacol. 247 199. 57. Champtiaux et al (2003) J.Neurosci. 23 7820. 76 133. 196. Donnelly-Roberts et al (1995) Biochem.Biophys.Res. 58. Gotti et al (2005) Mol.Pharmacol. 68 1162. 125. Kem et al (1997) J.Pharmacol.Exp.Ther. 283 979. Comm. 213 657. 59. Wada et al (1989) J.Comp.Neurol. 284 314. 126. Rayes et al (2007) Mol.Pharmacol. 71 1407. 197. Villarroya et al (1997) Eur.J.Pharmacol. 320 249. 60. Han et al (2000) Eur.J.Neurosci. 12 3664. 127. Thany et al (2007) Trends Pharmacol. Sci. 28 14. 198. Herrero et al (1999) Br.J.Pharmacol. 127 1375. 61. Lustig (2006) Anat.Rec.A.Discov.Mol.Cell.Evol.Biol. 288 128. Shimomura et al (2004) Neurosci.Lett. 363 195. 199. Kulak et al (2001) J.Neurochem. 21 5494. 424. 129. Ihara et al (2003) Neuropharmacology 45 133. 200. Valera et al (1992) Proc.Natl.Acad.Sci. USA 89 9949. 62. Gotti & Clementi (2004) Prog.Neurobiol. 74 363. 130. Abin et al (2006) Eur.J.Pharmacol. 536 1. 201. Ke & Lukas (1996) J.Neurochem. 67 1100. 63. Gahring & Rogers (2006) AAPS J. 7 E885. 131. Chavez-Noriega et al (1997) J.Pharmacol.Exp.Ther. 280 202. Paradiso et al (2000) Mol.Pharmacol. 58 341. 64. Wang et al (2003) Nature 421 383. 346. 203. Liu & Wu (2006), Acta.Pharmacologica.Sinica 27 1277. 65. Romanelli & Gualtieri (2003) Med.Res.Rev. 23 393. 132. McMahon & Kauer (1997) J.Neurophysiol. 78 2493. 204. Wang et al (2000) J.Neurochem. 75 1155. 66. Gotti et al (1997) Eur.J.Neurosci. 9 1201 133. Brown et al (1998) J.Physiol. 507 653. 205. Liu et al (2001) Proc.Natl.Acad.Sci. USA 98 4734. 67. Jensen et al (2003) Mol.Pharmacol. 64 865. 134. Yan et al (2006) Mol.Pharmacol. 70 571. 206. Pettit et al (2001) J.Neurosci. 21 1. 68. Boksa et al (1989) Eur.J.Pharmacol. 173 93. 135. Wotring & Yoon (1995) Neuroscience 67 293. 207. Grassi et al (2003) J.Physiol. 547 147.

www.tocris.com | 19 208. Dineley et al (2002) J.Biol.Chem. 277 25056. 256. Van Kampen et al (2004) Psychopharmacology 172 300. Amar et al (1993) FEBS.Lett. 327 284. 209. Dougherty et al (2003) J.Neurosci. 23 6740. 375. 301. Vainio et al (2001) Nicotine.Tob.Res. 3 177. 210. Snyder et al (2005) Nature Neuroscience 8 1051. 257. Alkondon et al (1997) Eur J Neurosci. 9 2734. 302. Damaj et al (1997) J.Pharmacol.Exp.Ther. 282 410. 211. Schrattenholz et al (1996) Mol.Pharmacol. 49 1. 258. Papke et al (1996) Neurosci.Lett. 213 201. 303. Miller et al (2004) J.Pharmacol.Exp.Ther. 310 1035. 212. Dajas-Bailador et al (2003) Mol.Pharmacol. 64 1217. 259. Gonzalez-Rubio et al (2006) Eur.J.Pharmacol. 535 53. 304. Bencheriff et al (1996) J.Pharmacol.Exp.Ther. 279 1413. 213. Zwart et al (2000) J.Neurochem. 75 2492. 260. Davies et al (1999) Neuropharmacology 38 679. 305. Sharples et al (2002) J.Med.Chem. 45 3235. 214. Akk & Steinbach (2005) J.Neurosci. 23 1992. 261. Alkondon & Albuquerque (2006) J.Pharmacol.Exp. 306. Mihalak et al (2006) Mol.Pharmacol. 70 801. 215. Zwart et al (2002) Neuropharm. 43 374. Ther. 318 268. 307. Stolerman et al (1997) Psychopharmacology 129 390. 216. Krause et al (1997) Mol.Pharmacol. 53 283. 262. De Filippi et al (2002) Soc.Neurosci.Abs. 137 5. 308. Larsson et al (2004) Alcohol 34 239. 217. Hurst et al (2005) J.Neurosci. 25 4396. 263. O’Neil et al (2002) Soc.Neurosci.Abs. 137 6. 309. Fu et al (1999) Neurosci Lett. 266 113. 218. Levandoski et al (2003) Eur.J.Pharmacol. 471 9. 264. Cilia et al (2005) Psychopharmacol. 182 214. 310. Rezvani et al (2002) Psychopharmacology 164 369. 219. Paradiso et al (2000) Mol.Pharmacol. 58 341. 265. Dickinson et al (2007) J.Neurochem. 100 1089. 311. Rabenstein (2006) Psychopharmacology 189 395. 220. Curtis et al (2002) Mol.Pharmacol. 61 127. 266. Briggs et al (1995) Neuropharmacol. 34 583. 312. Reuben et al (1998) Br.J.Pharmacol. 125 1218. 221. Miwa et al (1999) Neuron 23 105. 267. Buisson et al (1996) J.Neurosci. 16 7880. 313. Schwartz et al (1982) Mol.Pharmacol. 22 56. 222. Ibanez-Tallon et al (2002) Neuron 33 893. 268. De Fiebre et al 1995, Mol Pharmacol. 47 164. 314. Martino-Barrows & Kellar (1987) Mol.Pharmacol. 31 169. 223. Chimienti et al (2003) Human.Mol.Genetics 12 3017. 269. Gopalakrishnan et al (1995) Eur.J.Pharmacol. 290 237. 315. Clarke et al (1985) J.Neurosci. 5 1307. 224. Arredondo et al (2006) J.Cell.Physiol. 208 238. 270. Oneill et al (2003) Psychopharmacology 169 332. 316. Pabreza et al (1991) Mol.Pharmacol. 39 9. 225. Zoli et al (1998) J.Neurosci. 18 4461. 271. Bodnar et al (2005) J.Med.Chem. 48 905. 317. Flores et al (1992) Mol.Pharmacol. 31 41. 226. Marubio et al (1999) Nature 398 805. 272. Hajos et al (2005) J.Pharmacol.Exp.Ther. 312 1213. 318. Happe et al (1994) Neuroscience 62 929. 227. Whiteaker et al (2000) Br.J.Pharmacol. 131 729. 273. Siok et al (2006) Eur.J.Neurosci. 23 570. 319. Hall et al (1993) Brain.Res. 600 127. 228. Whiteaker et al (2002) J.Neurosci. 22 2522. 274. Biton et al (2006) Neuropsychopharmacology 32 1. 320. Han et al (2003) J.Comp.Neurol. 461 49. 229. Xiao et al (2004) Bioorg Med Chem Lett. 14 1845. 275. Pichat et al (2006) Neuropsychopharmacology 32 17. 321. Lippiello et al (1987) Mol.Pharmacol. 31 392. 230. Marks et al (2006) Mol.Pharmacol. 70 947. 276. Peng et al (1994) Mol.Pharmacol. 45 546. 322. Breese et al (1997) J.Pharmacol.Exp.Ther. 282 7. 231. Lipiello et al (1987) Mol.Pharmacol. 31 392. 277. Johnson et al (1995) Mol.Pharmacol. 48 194. 323. Whiteaker et al (2000) Mol.Pharmacol. 57 913. 232. Whiteaker et al (1999) Eur.J.Neurosci. 11 2689. 278. Pereira et al (1996) J.Pharmacol.Exp.Ther. 278 1472. 324. Champtiaux et al (2002) J.Neurosci. 22 1208. 233. Kawai et al (2002) J.Neurosci. 22 7903. 279. Broxton et al (1999) J.Neurochem. 72 1656. 325. Cui et al (2003) J.Neurosci. 23 11045. 234. Vishwanath & McIntosh (2006) Bioconjug.Chem. 17 1612. 280. Turek et al 1995) J.Neurosci.Meth. 61 113. 326. Quik et al (2001) J.Neurosci. 21 5494. 235. Rollema et al (2007) Trends Pharmacol. Sci. (in press). 281. Lockman et al (2005) J.Neurochem. 94 37. 327. Salminen et al (2005) Neuropharmacol. 48 696. 236. Livett et al (2006) Toxicon. 48 810. 282. Virginio et al (2002) Eur.J.Pharmacol. 445 153. 328. Perry et al (2002) J.Neurochem. 82 468. 237. Swanson et al (1991) J.Pharmacol.Exp.Ther. 259 377. 283. Matsubayashi et al (1998) J.Pharmacol.Exp.Ther. 284 329. Ueda et al (2004) Ann.Nucl.Med. 18 337. 238. Rozental et al (1989) J.Pharmacol.Exp.Ther. 251 395. 904. 330. Staley et al (2006) J.Neurosci. 26 8707. 239. Bowman (1958) Br.J.Pharmacol.Chemother. 13 521. 284. Garcia-Colunga & Miledi (1999) Proc.Natl.Acad.Sci. 331. Houghtling et al (1995) Mol.Pharmacol. 48 280. 240. Curtis et al (2001) Aquat.Toxicol. 52 29. USA 96 4113. 332. Xiao & Kellar (2004) J.Pharmacol.Exp.Ther. 310 98. 241. Pereira et al (1993) J.Recept.Res 13 413. 285. Anand et al (1993) FEBS.Lett. 327 241. 333. Davila-Garcia et al (1997) J.Pharmacol.Exp.Ther. 282 242. Fuchs et al (2003) Ann.N.Y.Acad.Sci. 998 93. 286. Rothlin et al (1999) Mol.Pharmacol. 55 248. 445. 243. Luo & McIntosh (2004) Biochemistry 43 6656. 287. Zwart et al (2002) Neuropharm. 43 374. 334. Lind et al (2001) Insect.Biochem.Mol.Biol. 31 533. 244. Liu & Dilger (1993) Synapse 13 57. 288. Grilli et al (2006) Br.J.Pharmacol. 149 724. 335. Mugnaini et al (2002) Eur.J.Neurosci. 16 1633. 245. Wenningman & Dilger (2001) Mol Pharmacol. 60 790. 289. Mok & Kew (2006) J.Physiol. 574 699. 336. Navarro et al (2002) Synapse 44 117. 246. Paul et al (2002) Anesth.Analg 94 597. 290. Krause et al (1997) Mol.Pharmacol. 53 283. 337. Kulak et al (2006) Eur.J.Neurosci. 23 2604. 247. Chiara & Cohen (1997) J.Biol.Chem. 272 32940. 291. Krusek & Semkova (1994) Eur.J.Pharmacol 259 121. 338. Rasmussen & Perry (2006) Neurosci.Lett. 404 9. 248. Changeux et al (1986) J.Physiol. 378 497. 292. Shan et al (2001) J.Bio.Chem. 276 12556. 339. Marks et al (1986) Mol Pharmacol. 30 427. 249. Revah et al (1990) Proc.Natl.Acad.Sci. USA 87 4675. 293. Hurst et al (2005) J.Neurosci. 25 4396. 340. Breese et al (1997) J.Comp.Neurol. 387 385. 250. Xu et al (2006) J.Phys.Chem.B. 110 20640. 294. Marks et al (1996) J.Pharmacol.Exp.Ther. 277 1383. 341. Orr-Utreger et al (1997) J.Neurosci. 17 9165. 251. Spivak et al (1982) Mol.Pharmacol. 21 351. 295. Eaton et al (2003) Mol.Pharmacol. 64 1283. 342. Le Novere et al (2002) J Neurobiol. 53 447. 252. Daly (1995) Proc.Natl.Acad.Sci. USA 92 9. 296. Papke & Porter (2002) Br.J.Pharmacol 13 49. 343. Matta et al (2007) Psychopharmacol. (Berl) 190 269. 253. Karatholuvhu et al (2006) J.Am.Chem.Soc. 128 12656. 297. Nelson (2001) J.Gen.Physiol. 118 563. 344. Chen et al (2003) Neuropharmacology 45 334. 254. Mullen et al (2000) J.Med.Chem. 43 4045. 298. Xiao et al (1998) Mol.Pharmacol. 54 322. 255. Papke et al (2004) Bioorg.Med.Chem.Lett. 14 1849. 299. Wonnacott et al (1991) J.Pharmacol.Exp.Ther. 259 387. Nicotinic Receptor Compounds Available from Tocris

Agonists 2241 DMAB-anabaseine 0352 4-Acetyl-1,1-dimethylpiperazinium Antagonist at α4β2 receptors Nicotinic agonist 2843 Mecamylamine 0351 1-Acetyl-4-methylpiperazine Neuronal nicotinic receptor antagonist Nicotinic agonist 1029 Methyllycaconitine 1971 (+)-Anabasine α7 neuronal nicotinic receptor antagonist Neuronal nicotinic receptor agonist 1356 MG 624 0789 (±)-Anatoxin A α7 neuronal nicotinic receptor antagonist Nicotinic agonist 0693 Pancuronium 1390 (-)-Cytisine Nicotinic (neuromuscular) antagonist Potent, selective neuronal nicotinic agonist 2785 Strychnine hydrochloride 2241 DMAB-anabaseine Nicotinic receptor antagonist Partial agonist at α7-containing receptors 2438 TMPH 0684 (±)-Epibatidine Neuronal nicotinic receptor antagonist Very potent nicotinic agonist 2820 (+)- 1077 (-)-Lobeline Nicotinic receptor antagonist Nicotinic agonist Other 2303 PNU 282987 2809 Acetylcholine chloride Selective α7 nAChR agonist Endogenous neurotransmitter 1053 RJR 2403 2722 Catestatin CNS selective nicotinic agonist Inhibitor of nicotinic cholinergic-stimulated catecholamine secretion 2737 TC 2559 1518 5-Iodo-A-85380 Selective partial agonist at α4β2 receptor High affinity α4β2 subtype-selective ligand 1348 UB 165 1527 5-Iodo-A-85380, 5-trimethylstannyl N-BOC derivative Subunit selective nAChR agonist Precursor to Cat. No. 1518 Antagonists 1260 Ivermectin 0424 Benzoquinonium Allosteric modulator of α7 nicotinic receptors 2498 PNU 120596 2133 α-Bungarotoxin Positive allosteric modulator of α7 nAChR; active in vivo α7 subtype-selective nAChR antagonist 2736 Sazetidine A 1001 Chlorisondamine Subtype-selective α4β2 receptor desensitiser Nicotinic antagonist; slow offset 2349 Dihydro-β-erythroidine Antagonist for neuronal α4-containing nicotinic receptors

Tocris Reviews No. 28 ©2007 Tocris Cookson

UK: US: Phone: + 44 (0)117 916 3333 Phone: 800-421-3701 Fax: + 44 (0)117 916 3344 Fax: 800-483-1993 [email protected] [email protected] Tocris House, Hung Road, 16144 Westwoods Business Park, www.tocris.com Bristol, BS11 9XJ, UK Ellisville, Missouri 63021 USA

Top View