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Home , Anabaseine, Anabasine, Chlorisondamine, Cholinesterase, Cholinesterase inhibitor, Cytisine

NEURONAL NICOTINIC RECEPTORS

Dr Christopher G V Sharples and preparations lend themselves to physiological and pharmacological investigations, and there followed a Professor Susan Wonnacott period of intense study of the properties of nAChR- mediating transmission at these sites. nAChRs at the Department of Biology and , muscle endplate and in sympathetic ganglia could be University of Bath, Bath BA2 7AY, UK distinguished by their respective preferences for C10 and C6 polymethylene bistrimethylammonium Susan Wonnacott is Professor of compounds, notably and Neuroscience and Christopher Sharples is a ,5 providing the first hint of diversity post-doctoral research officer within the among nAChRs. Department of Biology and Biochemistry at Biochemical approaches to elucidate the structure the University of Bath. Their research and function of the nAChR protein in the 1970’s were focuses on understanding the molecular and facilitated by the abundance of nicotinic cellular events underlying the effects of akin to the muscle endplate, in electric organs of the acute and chronic nicotinic electric ray,Torpedo , and eel, Electrophorus . High stimulation. This is with the goal of affinity snakea -, principallyaa - ( - Bgt), enabled the nAChR protein to be purified, and elucidating the structure, function and subsequently resolved into 4 different subunits regulation of neuronal nicotinic receptors. designateda ,bg , and d .6 An additional subunit, e , was subsequently identified in adult muscle. In the early 1980’s, these subunits were cloned and sequenced, The nicotinic receptor (nAChR) arguably and the era of the molecular analysis of the nAChR has the longest history of experimental study of any commenced. The muscle endplate nAChR has the receptor, and is the prototype -gated ion subunit combination and stoichiometry (a 1)bed 1 , channel (LGIC). nAChRs in muscle and, to a lesser 2 whereas the extrajunctional nAChR (a 1)2bgd 1 extent, in autonomic neurones have been predominates in foetal or denervated muscle. characterised in detail. In contrast, nAChRs in the CNS have only lately become the focus of intense Neuronal nAChRs research efforts. The family of neuronal nAChRs in the brain and spinal cord is increasingly recognised to The biochemical and molecular approaches to have therapeutic potential in a wide range of studying the muscle nAChR eclipsed interest in conditions, yet the physiological functions of these neuronal nAChRs, as no model systems existed for nAChRs remain enigmatic and their subunit these receptors. The credentials for ganglionic composition, assembly, trafficking and regulation, and nAChRs were firmly established from the significance of their heterogeneity, are still to be electrophysiological and pharmacological approaches fully elucidated. Progress in these endeavours has but the notion of nAChRs in the brain was viewed with been hampered by a dearth of subtype-selective scepticism. Whilea -Bgt, the defining ligand of muscle tools. Historically, Nature has provided the majority of nAChRs, specifically bound to brain tissue, it did not nAChR-selective compounds, but the nicotinic antagonise central functions.7 However, pharmacopoeia is now expanding with a number of the smoking habit was increasingly accepted novel synthetic ligands that have been developed in to be an to , implying that nicotine the last few years. exerts a powerful central effect. In 1980, [3 H]-nicotine was reported to specifically label sites in the rat brain Historical perspective1 that have a unique nicotinic .83 [ H]- The use of nicotine as an experimental tool was Nicotine binding was not blocked bya -Bgt, and the pivotal in Langley’s formulation of the concept of a anatomical distributions of binding sites for these two nicotinic ligands were quite distinct, raising the (then) ‘receptive substance’.2 In these studies he showed that ‘nicotine causes tonic contraction of certain novel and controversial prospect of nAChR muscles of fowl, and toad, and that this heterogeneity in the brain. The first publication of a contraction is prevented… by ’. Subsequently, cloned neuronal nAChR subunit (a 3) appeared in Loewi and Dale developed the theory of 1986.9,10 The nAChR credentials of thea -Bgt binding neurohumoural transmission, and recognised site in neurones were conclusively provided by acetylcholine (ACh) as an endogenous transmitter.3 cloning of thea 7 nAChR subunit gene in 1990.11 The Dale distinguished the actions of and current tally of neuronal nAChR subunits in mammals nicotine, leading to the recognition of two is eleven (aaaa 2- 7, 9, 10,bb 2- 4), with an additional pharmacologically distinct (and now known to be subunit,a 8, identified in avian species.12,13 structurally and functionally unrelated) families of receptors for ACh, that take their names from these A distinct but related gene family ofa andb subunits natural products.4 Neuromuscular and ganglionic has been revealed in .14 The C. elegans

Tocris Cookson Ltd., UK Tocris Cookson Inc., USA Tel: + 44 (0)117 982 6551 Tel: (800) 421-3701 Fax: + 44 (0)117 982 6552 www.tocris.com Fax: (800) 483-1993 e-mail: [email protected] [email protected] e-mail: [email protected] genome sequence incorporates 17 candidate nAChR nAChRs are present (albeit in relatively low subunits; nAChRs in nematodes are the target for the abundance) throughout the central and peripheral anthelmintic . In , nAChRs are the nervous systems. In contrast to their pivotal role in principal excitatory receptors in the CNS, and autonomic neurotransmission and in the initiation of constitute a major target (e.g. for , nAChRs in the CNS are ). Here we will focus on considered to exert a more modulatory influence.16,17 nAChRs; the reader is referred to a recent review14 Neuronal nAChRs are gaining credence as significant for a comprehensive account of nAChRs. players in the from their relationship with a number of disease states in which they are Heterogeneity of neuronal nAChR subunits and perceived as novel targets.18 These include subtypes Alzheimer’s and Parkinson’s diseases, , nAChRs are pentameric LGICs and members of the Tourette’s Syndrome and attention deficit disorder. In superfamily of receptors that addition, neuronal nAChRs are also targets for includes the GABA , glycine and 5-HT receptors.15 analgesia, anxiolysis, neuroprotection and smoking A3 cessation. Figure 1. nAChR structure and heterogeneity

A. Structure of a neuronal nAChR subunit. N-Terminal

Glycosylation

Cys Cys C-Terminal

M1 M2 M3 M4

P Phosphorylation

B. Structural overview of a nAChR with one subunit removed revealing channel lumen. Shown are binding sites for (including loop model), competitive antagonists, non-competitive antagonists and positive allosteric modulators.38

A F Agonist/Competitive Non-competitive antagonist antagonist interacting with B E K+ extracellular domain CD Positive a b subunit subunit AGONIST BINDING SITE LOOP MODEL

Channel blocking non-competitive antagonist

Ca2+/Na+

C. Major nAChR subtypes present in the CNS and peripheral nervous system (PNS). Blue circles represent putative agonist binding sites at subunit interfaces.

b2 a7 b4 a4 a4 a7 a7 a3 a3

b2 b2 a7 a7 a5 bx

CNS CNS/PNS PNS

2 Neuronal nAChRs are composed of pentameric (M1-M4) that transverse the plasma membrane combinations ofab and subunits, giving the potential (Figure 1A); evidence indicates that M2 isa -helical for a great variety of native neuronal nAChRs.12 and lines the cation pore.1,37 M3 and M4 are Heterologous expression of nAChRs in Xenopus separated by a large, variable, intracellular loop which oocytes and mammalian cell lines has established contains putative phosphorylation sites for Ser/Thr certain rules of assembly that limit the subtypes of kinases. native nAChRs that may exist. Pairwise combinations Five subunits assemble to form a pentameric nAChR. ofab 2,aa 3 or 4 subunits with 2 and b 4 subunits can Cryo-electron microscopy at 4.6Å resolution of form functional nAChRs, whereas thea 5 andb 3 Torpedo nAChRs in a resting, non-activated state has subunits are generally unable to participate revealed a membrane spanning protein in which the successfully in pairwise combinations, but they have bulk of the structure is extracellular.39 It been expressed in with at least two other accommodates a cation conducting pore which types of subunits.12,19,20,21,22 Thea 6 subunit has been narrows to a putative gate within the membrane shown to form a functional nAChR in combination bilayer. Two agonist binding sites are located at the withb 4, but higher levels of expression were interfaces of theTorpedo aa /gd and / subunits observed whena 6 was co-expressed with at least (analogous toa /b subunit interfaces in neuronal two other types of subunit, includingb 3.23,24 In nAChRs, Figure 1C), approximately 20-40 Å from the contrast, theaa 7, 8 and a 9 subunits are .37,40 Most features deduced from this distinguished by their ability to form robust homomeric moderately resolvedTorpedo nAChR structure have receptors in expression systems, althoughaa 7 8 been confirmed with the determination of a high heteromers also occur in avian tissues.11,25,26,27 The resolution (2.7Å) crystal structure of an ACh binding relateda 10 subunit is only incorporated into a protein from the snailLymnaea stagnalis .41 This functional nAChR when co-expressed witha 9.13 protein is a structural and functional analogue of the extracellular domain of an nAChR pentamer, with In native systems, knowledge of the subunit closest similarity to the neuronala 7 nAChR. composition of nAChRs is generally lacking and only a few major subtypes have been identified. These Upon agonist binding, nAChRs undergo an allosteric include thea 4b 2* nAChR, which is relatively transition from the closed, resting conformation to an abundant in the CNS and constitutes over 90% of open state which conducts the cations sodium, high affinity [3 H]- binding in the rat brain (see potassium and calcium. In the active (open) below).28 Based on the expression ofa 4b 2 nAChRs conformation, the nAChR binds agonists with low affinity (Figure 2). The continued presence of agonist inXenopus oocytes, the stoichiometry (a 4)23 (b 2) has 29,30 been proposed. However, more complex subunit Figure 2. Relationship between major states that a combinations may existin vivo and an asterisk is nAChR may occupy used to denote the possible presence of additional types of subunit.12 Indeed in chick a small proportion Fast (msec) ofaa 4b 2* nAChRs may contain 5 subunits and in rat R A striatum an nAChR composed ofaa 4,b 2, 5 and a 6 RESTING ACTIVE Channel Channel 31,32 subunits has been proposed. closed Nicotine binds open with LOW The other major subtype of neuronal nAChR is AFFINITY comprised ofa 7 subunits which are generally thought (mM -m M) to form homomeric nAChRs in the CNS and Slow peripheral nervous system.33 In the chick brain and (sec-min) retina, thea 7 subunit also forms heteromeric nAChRs witha 8, and may combine with other subunits such D There are multiple asa 5 in chick sympathetic neurones.25,27,34 In the DESENSITISED desensitised states Channel peripheral nervous system, a variety of heteromeric closed Nicotine binds with a3* nAChRs may exist. The predominant subtype HIGH AFFINITY containsa 3 andb 4 subunits, which may assemble I (m M - nM) together witha 5 and/orb 2.35,36 This brief overview INACTIVE does not preclude the possibility that nAChRs comprised of different or more complex subunit combinations may also exist in the vertebrate nervous leads to ion channel closure and receptor system.aa 9 and 10 are both expressed in desensitisation. In this condition, the nAChR is mechanosensory hair cells while neither has been refractory to activation although it displays higher detected in the adult brain.13 affinity for agonist binding. A multiplicity of desensitised states is proposed to exist.15 The rates nAChR structure: sites for ligand interaction of desensitisation and recovery differ between nAChR Individual nAChR subunits consist of a number of subtypes: for example, thea 7 nAChR displays very rapid desensitisation.11,19 Prolonged agonist exposure distinct functional domains (Figure 1A).37 The large extracellular N-terminal domain contains putative may produce an inactivated state, from which glycosylation sites, a disulphide-linked loop recovery is very slow; theab 4 2 neuronal nAChR is between residues homologous to 128 and 142 of the prone to inactivation on chronic nicotine treatment.42 a1 subunit, and the interface for agonist binding Transitions between resting, open and desensitised states are reversible and different ligands may (loops A, B and C ina subunits and loops D, E and F stabilise different receptor states: agonists initially 37 in all subunits, Figure 1B). In addition,a subunits stabilise the activated (open) state whereas are defined by the presence of a cysteine pair at competitive antagonists preferentially stabilise the positions homologous toa 1 192-193 in loop C. The nAChR in a closed state, either the resting or loops that comprise the agonist binding site contain desensitised configuration (Figure 2). conserved residues, many of which possess aromatic side chains ( and ) which are The nAChR presents a number of sites which may be proposed to make cationic-p interactions with targeted by ligands (Figure 1B). Agonists and agonists.38 competitive antagonists compete for the agonist binding site in the extracellular domain. As this site The polypeptide chain of nAChR subunits contains exists at the interface between adjoining subunits, it four hydrophobic, putative transmembrane segments 3 offers the prospect of nAChR subtype-selective are well documented.50 The principal interactions. Non-competitive antagonists (NCAs) and metabolite of nicotine, , is devoid of significant allosteric modulators act at sites distinct from the nicotinic binding activity.46 A minor tobacco is agonist binding site. , which is structurally related to nicotine: the pyrimidine ring of nicotine is replaced by a larger, Agonists (Table 1) unmethylated ring. Anabasine binds with 52 Nicotinic agonists interact with the nAChR agonist mid-nanomolar affinities toab42 and a 7 nAChRs. binding site to initiate an allosteric change within the Trans-meta nicotine (RJR2403) is generated by pentameric complex which leads to opening of the opening the ring of nicotine, and occurs cation conducting pore. The agonist interaction with naturally as a minor tobacco alkaloid. It is claimed to the activated state occurs with low affinity. Hence the show functional selectivity forab42 nAChRs EC50 values for agonist activation of nAChRs are compared witha 3* nAChRs, which is an important distinct from the higher affinity interaction with the consideration in developing targeted at CNS desensitised state which is measured in binding dysfunctions.51 Thus it was found to be as potent as assays, as a consequence of the prolonged nicotine in activatingab42 nAChRs in rat thalamic incubation conditions necessary to reach equilibrium synaptosomes but did not significantly activatea 3* (Figure 2, Table 1). nAChRs are the molecular targets for the offensive or defensive mechanisms of a wide anda 7 nAChRs expressed in the PC12 cell line. It variety of and animals, hence Nature has was ten-fold less potent and considerably less provided an array of potent ligands. These include the efficacious than nicotine in inducing guinea pig agonists (-)-nicotine, (-)-cytisine, (+)-anatoxin A, (+)- contractions, an effect probably mediated bya 3* , and anabasine. Synthetic nAChRs. agonists range from the classical “ganglionic agonist” Figure 3. Chemical structures of some nAChR dimethylphenylpiperazine (DMPP) developed in the agonists 1960’s, to novel agonists created more recently in H Me order to provide greater subtype selectivity and O therapeutic . Typically, agonists bind with NH highest affinity at theab 4 2 nAChR and with 2-3 HN orders of magnitude lower affinity ata 7 nAChRs N

(Table 1). They bind toa 3* nAChRs with intermediate H O (±)-Anatoxin A affinity. In terms of functional , a similar (-)-Cytisine (Cat. No. 0789) (Cat. No. 1390) relationship is observed, except that EC50 values at aa4b 2 and 3* nAChRs are more similar. Binding Me affinities (K values) are typically 2-3 orders of Cl H O NOH i N magnitude higher (i.e. lower concentration) than EC 50 N Ph Ph values for nAChR activation (Table 1).43 Few agonists have sufficient nAChR subtype selectivity for the (±)-Epibatidine (Cat. No. 1077) exclusive activation of a particular subtype. (Cat. No. 0684) The neurotransmitter ACh is the endogenous agonist NHMe for nAChRs. ACh binds with a high affinity at ab42 RJR 2403 (Cat. No. 1053) nAChRs (Ki ~ 10nM) but its utility as a nicotinic ligand N is limited by its lack of selectivity for nAChRs versus (Bold text denotes compounds available from Tocris) muscarinic AChRs (mAChRs) and its susceptibility to hydrolysis. Therefore a mAChR antagonist (typically (-)-Cytisine occurs in a number of plants of the ) and an (AChE) leguminosae family including laburnum. Its rigid inhibitor must be added with ACh in biological structure has provided a template for modelling preparations; some of these agents may also interact nicotinic ligands.53 It is comparable to nicotine with with nAChRs (see below). respect to its high affinity binding atab42 nAChRs Modification of ACh by conversion of its group (Ki ~ 1 nM) but has been used to distinguish sites to a yields the hydrolysis-resistant with low affinity for cytisine amongst the analogue carbamylcholine (). This has population labelled by [3 H]/[ 125 I]-epibatidine. 47,54,55 reduced affinity atab42 anda 7 nAChRs and is more However, in contrast to many nAChR agonists, potent as a .44,45 TheN -methyl including nicotine, the functional efficacy of derivative of carbachol,N -methylcarbamylcholine cytisine is dependent on the identity of the b (MCC) recovers a high affinity for binding to ab42 subunit present in the nAChR. (-)-Cytisine has full 46,47 efficacy at nAChRs containing theb 4 subunit nAChRs (Ki ~ 2 nM), comparable to ACh.N - methylation also confers substantial selectivity for expressed inXenopus oocytes, while atb 2- nAChRs over mAChRs, in binding and functional containing nAChRs greatly reduced efficacy is assays.46,48 MCC has a permanently charged observed.56 Thus (-)-cytisine is a at quaternary nitrogen atom which renders the molecule a4bab 2 and 3 2 nAChRs: this emphasises the membrane impermeant and hence useful for the importance of theb subunit in determining agonist study of cell surface nAChRs.47 interactions with neuronal nAChRs. The prototypic agonist for nAChRs is the tobacco (+)-Anatoxin A is an azabicyclononene alkaloid alkaloid, (-)-nicotine. Nicotine binds to the ab42 originally isolated from freshwater blue green algae,Anabaena flos aqua .57 Anatoxin A is a nAChR with high affinity (Ki = 1-11 nM) whereas the a7 nAChR is 1000-fold less sensitive (althougha 8* potent, semi-rigid, stereoselective agonist:58 nAChRs are more sensitive thana 7 nAChRs to activity resides in the (+)-enantiomer which nicotine and other agonists).49 Nicotine activates activatesab42 nAChRs at sub-micromolar concentraions (EC = 48-93 nM).43,59 Anatoxin A ab42nAChRs with an EC of 1m M, compared with 50 50 binds to this nAChR subtype with higher affinity 500mm M foraaa7 nAChRs and 200 M for 7/ 8 than nicotine. It has been exploited to characterise nAChRs.27 aaa 9 and 9/ 10 nAChRs are unique in that nicotinic currents in brain neurones and nAChR- nicotine is an antagonist at these subtypes (IC50 = 31 mediated release from striatal mM and 4m M respectively).13,26 Nicotine is widely preparations.60,61 used in behavioural studies: it crosses the brain barrier readily and its and Epibatidine, originally obtained from skin extracts of the Amazonian frog,Epidobates Tricolor is the 4 ab Table 1. Binding affinities (Ki ) and functional potencies for nicotinic agonists at native or recombinantab 4 2 and aab7 nAChRs and recombinant 3 4 nAChRs. References are quoted in brackets, beneath the figures.

nAChR subtype/Binding affinity Functional potency compound Ki50 (nM) EC (µM) ab42* a 7 ab 34 ab 42* a 7 ab 34 Acetylcholine 6.8-57 4000-10830 560-881 0.48-3 79-316 53-210 (47, 78, 170, 171) (45, 78, 172, 197) (65, 171) (20, 43, 59, 64, 90, (59, 64, 78, 89, 90, (64, 85, 90, 93, 173, 174, 175) 175, 176, 177) 178, 179) Carbachol 207-582 18000-580000 3839 2.5-29 296 n.d. (44, 170, 180, 181)(45, 172, 197) (65) (43, 44) (173) MCC 3.8 10580 n.d. 3.2 n.d. n.d. (47) (94) (43) (-)-Nicotine 1-11 400-8900 300-475 0.3-15 18-91 5-410 (44, 45, 47, 51, 52, (45, 51, 52, 78, 94, (65, 171) (43, 44, 52, 59, 64, (52, 59, 64, 89, 90, (64, 85, 90) 78, 170, 171, 180, 166,172, 176, 182, 67, 68, 90, 173, 174, 175, 176, 177, 183, 181, 182) 197) 175,180, 183) 184) Anabasine 76-260 58 2100 n.d. 16.8 n.d. (52, 171) (52) (171) (52) (-)-Cytisine 0.14-2.7 1400-3883 56-195 0.019-71.4 n.d. 72-134 (47, 78, 170, 171, (45, 78, 172, 197) (65, 171) (43, 44, 90, 174, 176, (85, 90, 178) 173, 180, 181) 180) (+)-Anatoxin A 3.5 63.3-380 52.9 0.048-0.093 0.58 n.d. (185) (185, 197) (65) (43, 59) (59) (-)-Anatoxin A 390 21000 n.d. n.d. n.d. n.d. (186) (186) (±)-Anatoxin A 1.3-4.2 707-1840 n.d. n.d. n.d. n.d. (47, 67) (67, 94) (+)-Epibatidine 0.019-0.041 9.8-590 n.d. 0.005-0.01 1.2-2 0.02-0.07 (63, 64) (64) ( 43, 64) (64) (64) (-)-Epibatidine 0.01-0.06 3.1-350 n.d. 0.004-0.02 1.1-2.2 0.009-0.02 (63, 64) (64) (43, 64) (64) (64) (±)-Epibatidine 0.01-0.05 20.6-233 0.38 n.d. 1.3-3.5 n.d. (47, 67, 170, 182) (67, 94, 182, 197) (65) (177, 184) (±)-UB-165 0.27-0.44 2790 n.d. partial agonist 6.9 0.27 (67) (67) (67) (67) (67) RJR 2429 1 n.d. n.d. >100 n.d. n.d. (68) (68) ABT-418 3-44 >100000 n.d. 0.5-14 264 n.d. (47, 69, 121) (69, 174) (184) RJR 2403 26 36000 n.d. 0.73-16 240 n.d. (51) (51) (51, 187) (187) SIB-1508Y 4.6 n.d. n.d. 2.6 n.d. 41 (72) (72) (72) ABT-594 0.04 1560-13800 n.d. n.d. n.d. n.d. (75) (75) A-85380 0.05 148 73.6 0.7 8.9 n.d. (188) (188) (65) (188) (188) GTS-21 (DMXB) 85 212-652 n.d. No activation 26-109 n.d. (78) (78, 197) (174) (78, 171, 184) Anabaseine 32-75 58-759 n.d. 4.2 6.7 n.d. (52, 78) (52, 78, 197) (52) (52) (±)-AR-R17779 16000 190 n.d. n.d. n.d. n.d. (79) (79) (-)-AR-R17779 16000 92 n.d. n.d. 21 n.d. (79) (79) (79) 112000 2380000 n.d. n.d. 1600 n.d. (81) (94) (82) DMPP 9.4-400 160-2300 1300 0.07-18 19-75 10-92 (44, 170, 171, 186) (172, 186) (171) (43, 44, 90) (89, 90, 176, 177) (85, 90, 178) Lobeline 4-50 11000-13100 480 n.d. No activation n.d. (171, 180) (153, 197) (171) (89)

(Bold text denotes compounds available from Tocris) aBinding affinities reported for competition binding assays of [3 H]-agonist binding (except [3 H]-epibatidine) to brain membranes (ab 4 2* nAChRs) and [3 H]-agonist binding to heterologously expressedab42 nAChRs, [125 I]-Bgt or [ 3 H]- MLA binding to brain membranes or heterologously expresseda 7 nAChR, [3 H]-epibatidine binding to heterologously expressedab3 4 nAChRs. bFunctional potencies reported at brain thalamic synaptosomes (ab42 * nAChRs) or heterologously expressed nAChRs.

Values are the range from the references cited below each entry.

5 most potent natural agonist at nAChRs yet known.62 shown potent properties that, together with Its potent non- analgesic activity awakened its weaker interaction with peripheral nAChRs, make interest in neuronal nAChRs as targets for analgesic it a promising therapeutic lead for the treatment of drugs.63 Like anatoxin A, epibatidine also has a pain.77 bicyclic moiety that confers some rigidity to its A further, structurally disparate group of compounds structure but, in this case, it is a smaller azabicycloheptane ring, coupled to a chloropyridyl exhibit some degree of preference for thea 7 nAChR. moiety. Epibatidine has exceptionally high binding GTS-21 (also known as DMXB) is a synthetic, functionally selective agonist fora 7 nAChRs (Ki = 212 affinity atab42 nAChRs (Ki = 19 pM) and about 10- fold lower affinity atab32 nAChRs (K = 230 pM) and nM; EC50 = 26.21-109m M). Despite binding toab 4 2* i nAChRs with a reasonable affinity (K = 85 nM), GTS- a3ba 4 nAChRs (K = 380 pM).64,65 Binding affinity at 7 i i 21 displays negligible agonist activity at this nAChRs is about 10,000-fold lower than at ab42 subtype.78 GTS-21 is a 3-substituted derivative of nAChRs (Ki ~200 nM). In contrast to anatoxin A, anabaseine, an alkaloid that occurs naturally in epibatidine is not particularly stereoselective and its nemertines (a marine worm). Anabaseine differs from 63,64 enantiomers show equivalent biological activities. the tobacco alkaloid anabasine only in bond order, but The functional potency of epibatidine is also this difference seems to confer a functional selectivity exceptionally high, with sub-micromolar EC values 50 fora 7 nAChRs that is also preserved in GTS-21. forab42 , ab 32 , ab 34 and a 8 neuronal nAChR Anabaseine has a moderate binding affinity atab42 * subtypes.64 Onlya 7 and muscle nAChRs exhibited nAChRs (Ki = 32 nM) and a relatively high affinity at EC50 values in the low micromolar range. 52 a7 nAChRs (Ki = 58 nM). UB 165 is a novel hybrid molecule, comprising the AR-R17779, a novel, conformationally restricted azabicyclononene bicycle of anatoxin A and the analogue of ACh, is also reported to be a selective chloropyridyl moiety of epibatidine.66 UB 165 exhibits stereoselectivity comparable to anatoxin A. It binds ligand foraab 7 over 4 2 nAChRs, with respective Ki values of 190 nM and 16000 nM for the racemate.79 with high affinity toab42 nAChRs but is a partial The (-) enantiomer was shown to be a full and potent agonist at this nAChR type, in contrast to its full efficacy in activating other neuronal nAChR subtypes agonist at rata 7 homomers expressed in Xenopus 79 expressed inXenopus oocytes.67 This property was oocytes. It has anxiolytic properties and improves 80 exploited to infer the participation of presynaptic learning and memory performance in rats. ab42* nAChRs in the modulation of dopamine release Choline, the product of ACh hydrolysis, is a selective 81,82 in rat striatal preparations. agonist ofa 7 nAChRs (EC50 = 1.6 mM). Other RJR 2429 resembles UB 165 both structurally and nAChR subtypes are not activated by choline, and it functionally. It is a quinuclidine derivative of nicotine, has been employed to selectively stimulatea 7 and displays high binding affinity atab42 nAChRs but nAChRs.82 Whereas concentrations above 200m M is unable to activate this nAChR subtype in an assay activatea 7 nAChRs, low concentrations (35m M) of rubidium efflux from rat thalamic synaptosomes.68 desensitise thea 7 nAChR: as the local However, in contrast to UB 165, RJR 2429 does not concentrations of choline in the brain are not precisely show selectivity for neuronal nAChRs over muscle known, the physiological implications of this potential nAChRs: it is a potent activator of human muscle endogenous ligand are a matter of debate.82 nAChRs (EC50 = 59nM) but is markedly less potent in The synthetic compounds dimethylphenylpiperazine activatinga 3* nAChRs in the rat PC12 cell line (EC50 (DMPP) and methylphenylpiperazine (MPP) are both = 1.1m M).68 agonists at nAChRs. DMPP has a long history as a In addition to UB 165 and RJR 2429, several other ganglionic agonist but shows little selectivity between synthetic nicotinic agonists have been described that neuronal nAChR subtypes, with respect to binding show improved nAChR subtype selectivity, compared affinity and potency of activation. However, DMPP is with nicotine. These include ABT-418 and SIB-1508Y. a partial agonist at chickena 7 nAChRs expressed in ABT-418, an isoxazole isostere of nicotine, has high Xenopus oocytes but is fully efficacious in eliciting affinity forab42 nAChRs while displaying reduced Type IA currents (attributed toa 7 nAChRs) in rat functional potencies at other nAChR subtypes which hippocampal neurones.60,83,84 DMPP was also shown could mediate peripheral side effects.69 This molecule to be a partial agonist at rata 3b 4 nAChRs stably has cognition enhancing and anxiolytic properties, expressed in HEK-293 cells.85 It is membrane with reduced side effects compared to nicotine.70,71 impermeant, due to the quaternary nitrogen atom, SIB-1508Y, a 5-substituted ethynyl analogue of and has been used to selectively block cell surface nicotine, retains potency atab42 nAChRs (binding nAChRs.86 with low nanomolar affinity, comparable to nicotine) Lobeline, an alkaloid produced by the Indian tobacco but has slightly reduced functional potency atab 3 4 inflata deserves a mention because of its andab 4 4 nAChRs stably expressed in HEK-293 cell anomalous behaviour. Lobeline binds with high affinity lines.72 It exhibits similar potency but increased toab 4 2* nAChRs. It was originally considered to be a efficacy, compared with nicotine, in stimulating , but it is now proposed to be an dopamine release from rat striatal slices, which led to antagonist.87 Although it was found to stimulate its development as a potential therapy for Parkinson’s dopamine release from rat striatal slices at disease.73 concentrations above 1m M, this effect is not nAChR- The 3-pyridyl comprise a distinct group of mediated as it cannot be blocked by the nAChR molecules that are structurally more distant from antagonist, . Indeed lobeline, at nicotine, incorporating the linkage found in ACh. concentrations below 1m M, inhibits nicotine- These molecules exhibit very high affinity forab42 * stimulated dopamine release.88 In addition, it has also nAChRs (comparable to epibatidine) and also display been shown to inhibit nicotine-stimulated rubidium high selectivity, in terms of binding affinity, for ab42 efflux from rat thalamic synaptosomes, an effect nAChRs overaa 3* and 7 nAChRs. Two recently ascribed toab42 * nAChRs, and fails to activate described examples of this class of compound are A- humana 7 nAChRs expressed in Xenopus 85380 and ABT-594, the latter being a chloropyridyl oocytes.88,89 derivative of A-85380.74,75 The 5-iodo derivative of A- 85380 is proposed to be anab42 selective ligand versusaa 7 and 3* nAChRs.76 ABT-594 has also

6 Competitive antagonists (Table 2) andab 3 2 nAChRs but it is 10-50-fold less potent at ab3 4 and a 7 nAChRs expressed in Xenopus Competitive antagonists interact reversibly with the 90,93 nAChR at, or close to, the agonist binding site, oocytes. In hippocampal neurones, 100 nM DHb E stabilising the receptor in a conformation with the blocked Type II currents, attributed toab42 * nAChRs, channel closed and preventing access for agonists. whereas Type I currents mediated bya 7 nAChRs Inhibition by competitive antagonists is surmountable were insensitive to DHb E concentrations below 10 with increasing agonist concentration. Like agonists, mM.60 most competitive antagonists originate from a wide The most well-established subtype-selective nicotinic variety of natural sources. Unfortunately, there is a very limited range of subtype-selective agents and not antagonist isa -Bgt, an 8kDa peptide isolated from the all are commercially available. Binding affinities for of the Taiwanese banded krait, Bungarus competitive antagonists are given in Table 2 and these Multicinctus. It was instrumental in the isolation and approximate more closely to functional potency, unlike purification of muscle andTorpedo nAChRs but is also a highly potent and selective antagonist ata 7 agonists. IC50 concentrations for functional blockade are not listed as these values will depend on the nAChRs, to which it binds with an affinity (Ki ) of agonist concentration used. Typical concentrations for approximately 1 nM.94 However, its association nAChR blockade are indicated in the text. binding kinetics are very slow and typically a pre- incubation of one hour with the would be A classic antagonist is d-tubocurarine (d-TC), which is necessary to achieve a complete blockade, using a produced by the South American shrub low nanomolar concentration ofa -Bgt (10 nM). This is Chondodendron tomentosum. It is well known as the commonly circumvented by increasing the arrow tip poison used by South American Indians and concentration and decreasing the preincubation time. has had a long history as a .2 d-TC This strategy is possible because, even at micromolar does not discriminate appreciably between nAChR concentrations,a -Bgt does not appear to interact with subtypes and it is able to fully antagonise functional aa/b heteromeric nAChRs. -Bgt also exhibits very responses mediated by a wide variety of neuronal slow dissociation kinetics, such that functional 90 nAChRs at a concentration of approximately 10m M. blockade is not reversed by washout within the It should be noted that the mechanism of inhibition by timescale of a typical experiment (e.g. 1 hour). The d-TC can be complex, also involving non-competitive avianaa 8* nAChRs bind, and are blocked by, -Bgt (K interactions.83,90 In addition, d-TC is also a 5-HT i 3 ~ 2 nM), anda 9* nAChRs of cochlear hair cells are : it inhibits 5-HT3 receptor- mediated currents in hippocampal interneurones (full reversibly inhibited by nanomolar concentrations of a- 13,27 blockade at 1m M) and in HEK-293 cells stably Bgt. transfected with the 5-HT3 receptor (IC50 = 2.8 The venom ofBungarus Multicinctus also contains a mM).91,92 number of minor components with pharmacological activity. One such fraction exhibits specificity for A purely competitive antagonist for neuronal nAChRs inhibiting at least somea 3* subtypes of neuronal is dihydro-beta-erythroidine (DHb E), an alkaloid 95 originating fromErythrina seeds. Sub-micromolar nAChRs, in addition toa 7 nAChRs. This toxin has been given a variety of names but is now referred to concentrations of DHbab E block human and rat 42 as neuronal bungarotoxin (n-Bgt).a 3b 2 nAChRs are

a Table 2. Binding affinities (Ki ) for nicotinic antagonists in competition binding assays at native or recombinant ab4 2* and a 7 nAChRs and recombinant ab 3 4 nAChRs.

nAChR subtype/ Ki (nM) compound ab42* a 7 ab 34 DHb E 13.9-1900 25000-57900 218622 (46, 47, 156, 170, 186, 195) (186, 197) (65) d-TC 1000-25000 3400-7700 22929 (44, 47, 170, 181) (45, 172, 181) (65) (MLA) 3700-6100 0.69-10.3 3700b (47, 186) (94, 166, 172, 186, 197) a-Bgt > 1000 0.35-3.5 n.d. (186) (45, 94, 166, 172, 176, 186, 197) a- ImI n.d. 2400 n.d. (194) Hexamethonium 28000-> 1000000 800000-880000 > 1000000 (47, 181, 182, 186) (45, 186) (65) Decamethonium 460-120000 124000-200000 n.d. (47, 182, 186) (45, 186) Mecamylamine 822000-> 1000000 > 1000000 > 1000000 (47, 181, 182, 186) (186) (65) 686000 n.d. n.d. (182)

(Bold text denotes compounds available from Tocris) aBinding affinities reported for competition binding assays of [3 H]-agonist binding (except [3 H]-epibatidine) to brain membranes (ab 4 2* nAChRs) and [3 H]-agonist binding to heterologously expressedab42 nAChRs, [125 I]- a Bgt or [3 H]- MLA binding to brain membranes or heterlogously expresseda 7 nAChRs, [3 H]-epibatidine binding to heterologously expressedab 3 4 nAChRs. bSharples, unpublished observation.

Values are the range from the references cited below each entry.

7 subject to pseudo-irreversible blockade by 100 nM n- behavioural studies.116,117 However, as the local Bgt whereasab 3 4 nAChRs are insensitive to this concentration of MLA achieved is not known in these concentration: analysis of chimeric subunits has studies, results must be interpreted with caution as shown that the kinetics and affinity of n-Bgt blockade this antagonist is selective, rather than specific, fora 7 resides in the N-terminal domain of theb subunit.96,97 nAChRs over other nAChR subtypes. This toxin is not generally available, and has been , generally regarded as a competitive superseded to some extent by the more stringent antagonist of glycine-gated LGIC, has been shown to specificities of theConus toxins. competitively inhibit currents mediated bya 7 nAChRs 118 The ofConus snails are a valuable source of on rat hippocampal neurones (IC50 = 1.2m M).a 9 subtype-selective antagonists for a variety of andaa 9/ 10 nAChRs are exquisitely sensitive to 13 pharmacological targets.a - are small strychnine (IC50 = 20 nM). In contrast, strychnine peptide toxins (14-17 amino acids) with a highly was concluded to block currents mediated byab 4 2* conserved disulphide bonding pattern, with specificity nAChRs in a voltage-dependent, non-competitive for nAChR subtypes.98 It is not yet established if they manner.118 all act in a truly competitive manner. Three well characteriseda -conotoxins specific for neuronal Non-competitive antagonists nAChRs areaa -conotoxins MII, ImI and AuIB. - Non-competitive antagonists (NCA) exert their effects Conotoxin MII was originally shown to be a potent by interacting with sites distinct from the agonist and selective antagonist ofa 3b 2 nAChRs expressed binding site, and therefore do not compete with inXenopus oocytes;ab32 nAChRs are fully blocked agonists for binding. Commonly a NCA inhibits by 100 nM toxin.99,100,101 At other heterologously nAChR activity by blocking the intrinsic channel pore, expressed nAChRs, the potency ofa -conotoxin MII is by binding to sites within or near the entrance to the 2-4 orders of magnitude lower. The partial inhibition of channel. As the action of a NCA is not surmountable nicotine- or anatoxin A-evoked dopamine release by agonist, it is easier to predict effective from rat striatal synaptosomes bya -conotoxin MII led concentrations required to achieve full blockade to the proposition that a nAChR containing at least under conditions of varying agonist concentrations. oneab32 interface contributed to this response.101,102 However, if the NCA is a , the nAChR This interpretation may require modification in light of must first be activated to allow the drug access to the the high sequence similarity between theaa 3 and 6 channel. Such NCAs typically show voltage- subunits, and the observation thata 6b 2 nAChRs dependent blockade of nAChRs. expressed inXenopus oocytes are susceptible to The synthetic compound mecamylamine is the inhibition bya -conotoxin MII.103 The absence of [ 125 I]- archetypal NCA for neuronal nAChRs. Mecamylamine a-conotoxin MII binding (see below) in transgenic blocks most neuronal nAChRs with IC50 values in the mice lacking theb 3 nAChR subunit suggests that this low micromolar range.a 7 nAChRs are somewhat less rare subunit (which shares a similar anatomical sensitive thanab / heteromers, requiring 10m M for full distribution withaa 6) may also participate in - blockade.90 In addition,N -methyl-D -aspartate (NMDA) conotoxin MII binding sites.104 receptors are also transiently inhibited by mecamylamine at high micromolar concentrations.119 a-Conotoxin AuIB partially inhibits nicotine-evoked Mecamylamine crosses the blood brain barrier freely [33 H]-noradrenaline and [ H]-ACh release from rat and is typically administered at a concentration of brain preparations and is a selective antagonist of around 1 mg/kg in rats to block CNS nAChRs in 105,106 ab3 4 nAChRs. It is less potent than a -conotoxin behavioural studies. MII and 10ma M -conotoxin AuIB is required to fully Figure 4. Chemical structures of some nAChR inhibitab 3 4 nAChRs. Another well characterised a - antagonists conotoxin isa -conotoxin ImI, which is a selective antagonist ofa 7 nAChRs, fully blocking responses at 1ma M.107,108 -Conotoxin ImI also blocks a 9 nAChRs Gly-Cys-Cys-Ser-Asn-Pro-Val-Cys-His-Leu- 107 with an affinity 8-fold less than ata 7 nAChRs. One Glu-His-Ser-Asn-Leu-Cys-NH2 caveat with regard toa -conotoxins is that their exquisite specificity may limit extrapolation between a-Conotoxin MII OMe (Cat. No. 1340) species: it is reported that althougha -conotoxin ImI OMe OMe inhibits rataa 7 nAChRs, it blocks a non- 7 nAChR in Me bovine chromaffin cells.109 N OH Methyllycaconitine (MLA) is a norditerpenoid alkaloid OH OMe produced byDelphinium sp . It is a competitive O antagonist, selective foraa 7 nAChRs and, unlike - Methyllycaconitine (Cat. No. 1029) N O O Bgt, discriminates betweena 7 and muscle O 53,110 nAChRs. MLA binds toa 7 nAChRs with a Ki of approximately 1 nM, and picomolar concentrations Me are reported to blocka 7 nAChR-mediated currents Cl recorded from hippocampal neurones or Xenopus Cl Me Chlorisondamine oocytes.111,112 Inhibition ofa 7 nAChRs by MLA is N+ + N Me3 (Cat. No. 1001) rapid and reversible, making it a useful complement Cl toaa -Bgt in the 7 pharmacological armoury. While Cl Me muscle anda 4b 2 nAChRs require micromolar O concentrations for inhibition,ab 3 2* nAChRs are about Me O 10-fold more sensitive to MLA.113 Recent evidence Me suggests that MLA at concentrations of 1-50 nM may Me H N+ N+ block a minor, non-a 7 nAChR in rat striatum.32,114 In Me addition, non-a 7 interactions of MLA have been H H indicated in avian preparations, where functional O Pancuronium H (Cat. No. 0693) responses sensitive to MLA, but nota -Bgt, have been Me O observed.34,115 MLA is also able to cross the blood- brain barrier and has been given intracerebrally in (Bold text denotes compounds available from Tocris) 8 Like mecamylamine, hexamethonium was also first nAChRs expressed in the SH-SY5Y cell line, with IC50 recognised as a ganglionic nAChR blocking agent but values in the high nanomolar to micromolar range.134 the hydrophilic nature of this polymethylene In addition, a similar range of steroids inhibited bistrimethylammonium compound prevents it from currents mediated by ratab 4 2 nAChRs stably 5 crossing the blood-brain barrier. Comparative studies expressed in the HEK-293 cell line, with IC50 values in of the effects of mecamylamine and hexamethonium the low micromolar range, and inhibited in vivo have been undertaken to establish if a ab4 2 nAChRs expressed inXenopus oocytes with an particular behaviour is centrally or peripherally 135,136 IC50 of 9m M. mediated, as peripherally administered hexamethonium is unable to block CNS nAChRs in Numerous diverse blockers of voltage-operated vivo. calcium channels have been reported to inhibit rat ab3 4 nAChRs expressed inXenopus oocytes, Chlorisondamine, a bisquaternary nicotinic whereasa 7 nAChRs were virtually unaffected.137 antagonist, was also originally used as a ganglionic These drugs include the N/P/Q-type . It is an irreversible open channel blocker for blocker,w -conotoxin MVIIC, the N-type blocker,w - neuronal nAChRs and is unique in exerting a conotoxin GVIA and the L-type blockers furnidipine, persistent blockade over a period of weeks when and . All inhibited nicotinic givenin vivo . For example, it effectively blocked currents with IC values in the low micromolar range. dopamine release from rat striatal synaptosomes in 50 vitro, even when administered in vivo (10 mg/kg/day) Thewab -conotoxins were found to block the 3 4 nAChR reversibly, whereas they exert a longer lasting several weeks before the sacrifice of the animal.120 It is hypothesised that this long lasting inhibiton may inhibition of calcium channels. This difference can be used to discriminate between these two targets.138 arise from an intracellular accumulation of the drug.121 Dihydropyridine calcium channel blockers , an alkaloid isolated from the African shrub (nimodipine, nifedipine, nitrendipine and furnidipine) Tabernanthe iboga, is reported to have anti-addictive have also been observed to abolish nAChR-mediated properties. It attenuates self-administration of 45Ca 2+ uptake in bovine chromaffin cells, an activity and in rats and is an NCA for neuronal largely attributable toab 3 4* nAChRs.139 nAChRs.122 It is able to block functional responses The atropine, the GABA mediated by ganglionica 3* nAChRs in the PC12 and A receptor antagonist , and the 5HT human neuroblastoma SH-SY5Y cell lines.123,124 3 receptor antagonist ICS-205,930 inhibita 9* nAChRs Many NCAs for neuronal nAChRs have alternative 13 with IC50 values of 1m M or less. Other nAChR primary targets and cannot be considered to be subtypes are much less sensitive to these agents: specific nicotinic drugs. In compiling the list below, atropine blocksam 7 nAChRs with an IC of 120 M.45 compounds are included that would not be 50 deliberately chosen to antagonise nicotinic responses Particularly intriguing are recent reports from several but awareness of such an interaction may be groups thatba -amyloid peptide inhibits 7 nAChRs. It important when interpreting the effects of the drug. binds competitively with picomolar affinity toa 7 This list is not comprehensive; any positively charged 140 nAChRs. Recentlyb -amyloid1-42 was shown to compound small enough to approach the nAChR reversibly blocka 7 nAChRs on rat hippocampal channel may indeed block the lumen and inhibit ion neurones (IC50 = 7.5 nM) in a non-competitive conductance: this also applies to nicotinic agonists, manner, by an interaction with the N-terminal domain that become channel blockers in the high micromolar 141 of the receptor. Moreover,b -amyloid1-42 inhibited concentration range.125,126 whole cell and single channel nicotinic currents in rat MK 801 () is an anticonvulsant agent hippocampal interneurones at a concentration of 142 developed as a channel blocker of the NMDA 100 nM.b -Amyloid1-42 has also been shown to receptor. It is able to perform the same function on stimulate the mitogen-activated protein kinase neuronal nAChRs, where it is reported to be an open (MAPK) cascade in hippocampal slices viaa 7 143 channel blocker at theam 4ba 2 (IC50 = 15 M) and 7 nAChRs. This encourages speculation about the 127,128 role of nAChRs in the pathogenesis of Alzheimer’s nAChRs (IC50 = 15mm M). MK 801 (10-25 M) has also been shown to partially inhibit nicotine-evoked Disease. [3 H]-GABA release from rat hippocampal synaptosomes.129 Studies onTorpedo nAChRs Positive allosteric modulators indicate that MK 801 may also interact with a non- Positive allosteric modulators potentiate responses to luminal site, in addition to the channel.130 nicotinic agonists by acting at a site other than the Buproprion (ZybanTM ) was originally developed as an agonist binding site or channel area. Such effects are but is now marketed as an aid to well established for other receptor classes (e.g. . At low micromolar concentrations and GABAA receptors; glycine and it non-competitively inhibits ratab 3 2, ab 4 2 and a 7 NMDA receptors), but have only recently been nAChRs expressed inXenopus oocytes and nAChR- recognised for neuronal nAChRs. mediated rubidium efflux in the human SH-SY5Y cell Acetylcholinesterase inhibitors, including line.122,131 In the former study, inhibition by buproprion galanthamine and , have been shown was voltage-independent suggesting that it may not to potentiate the functional effects of sub-maximal act within the lumen of the channel. concentrations of nicotinic agonists on neuronal Other known NCAs for neuronal nAChRs include the nAChRs, without themselves causing significant neuroleptic, and a number of nAChR activation when applied alone.145 This dual psychoactive drugs.132 The dissociative anaesthetics action (acetylcholinesterase inhibition and nAChR (PCP) and are in this class.133 potentiation) has led to the development of these In addition there are several classical NCAs which agents for the treatment of Alzheimer’s Disease. have been used to study muscle nAChRs but whose Galanthamine (ReminylTM ) (1-10m M) potentiated activity at neuronal nAChRs has not been well ACh-evoked currents in HEK-293 cells stably characterised. This includes pancuronium, expressing the humanab 4 2 nAChR.144 At higher histrionicotoxin and quinacrine. concentrations (> 10m M) galanthamine inhibited responses, possibly by acting as a channel blocker; A range of steroids, including corticosterone, physostigmine behaves in a similar manner. The site progesterone, oestradiol, hydrocortisone and of action for their positive allosteric modulation is aldosterone, have been shown to inhibit neuronal thought to be at, or close to, the binding site for the 9 monoclonal antibody FK-1, near lys 125 in the Specific nicotinic labelling by [3 H]-ACh (in the extracellular N-terminal domain of the neuronal presence of atropine and an acetylcholinesterase nAChRa subunit.145 Other allosteric potentiators inhibitor) is comparable to that of [3 H]-nicotine in include the , the anthelminthic drug, anatomical distribution and pharmacological and the neurotransmitter 5-HT. Codeine characteristics.9,152,153 [ 3 H]-Cytisine also labels acts at the FK-1 antibody binding site to potentiate specific sites in the brain with an affinity (Kd = 0.96 currents activated by ACh in PC12 cells, while nM) and distribution comparable to [3 H]-nicotine;154 ivermectin was shown to enhance currents through immunoprecipitation analyses suggest that it also chick or humana 7 nAChRs expressed in Xenopus labelsa 4b 2* nAChRs.28 In addition, one or more oocytes.146,147 Sub-micromolar concentrations of 5-HT other, minor nAChR subtypes may be labelled by [3 H]- could mimic the potentiating effects of galanthamine cytisine.155 [3 H]-ABT-418 was also shown to bind to 145 in PC12 cells. Recently, the steroid,b -estradiol, rat brain with a low nanomolar affinity (Kd = 2.85 nM) was reported to potentiate human, but not rat,ab 4 2 and the distribution of [3 H]-ABT-418 binding sites in nAChRs expressed inXenopus oocytes distinct from the brain, determined by autoradiography, is the inhibitory effects of steroids.134,135,136,148 This is an comparable to that of [3 H]-cytisine.156 Similarly, [ 3 H]- example of positive allosteric modulation being MCC has been used to label sites in brain with high species specific. affinity (Kd = 1 nM) and a pharmacological profile resembling that of [33 H]-nicotine and [ H]-cytisine Radioligands (Table 3) binding sites.46,157 Nicotinic radioligands have provided invaluable tools Although each of these agonists can interact with a for the identification, pharmacological characterisation full range of neuronal nAChR subtypes (Table 1), as and localisation of neuronal nAChRs. Table 3 radioligands they predominantly label onlyab42 * summarises the binding affinities at relevant neuronal nAChRs. This is because, in a binding assay, the nAChR subtypes of the major nicotinic radioligands prolonged incubation with agonist converts the published. nAChR to a high affinity desensitised state (Figure 2). Of the various nAChR subtypes, the desensitised [3 H]-Nicotine binding to brain tissue was first ab42nAChR has the highest affinity (~ 1 nM) for described in detail by Romano & Goldstein.7 [3 H]- these particular agonists; lower affinity binding (> 10 Nicotine identifies a single population of sites in the nM) would not result in specific labelling under the CNS, in most reports, with a K of 1-10 nM and is d conditions used. considered to label primarilya 4b 2* nAChRs, as [3 H]- nicotine binding is almost totally absent from the In 1995, the binding of [3 H]-epibatidine was brains of transgenic mice lacking theab 4 or 2 characterised, and it was found to label 2 sites in the subunits.149 [ 3 H]-Nicotine binds to glass fibre filters, rat brain with sub-nanomolar affinities and very low and this is remedied by pre-soaking the filters in poly- non-specific binding.158 The highest affinity binding 47 3 L-lysine or polyethyleneimmine; non-specific binding site (Kd ~ 10 pM) accords withab42 nAChRs. [ H]- to tissue preparations can be diminished by including Epibatidine has proved to be a very useful radioligand a high concentration of Tris in the buffer.7,150,151 [ 3 H]- for monitoring a variety of defined nAChR subtypes in Nicotine does not store well and addition of heterologous expression systems. [3 H]-Epibatidine mercaptoacetic acid is advised.7 labelleda 3b 4 nAChRs expressed in HEK-293 cells and inXenopus oocytes with Kd values of 0.3 nM and

a Table 3. Binding affinities (Kd ) of nicotinic radioligands.

nAChR subtype/ Kd (nM) radioligand ab42*a 7 ab 34 ab32* aab 354 aab 352 [3 H]-Nicotine 0.89-9 – –––– (44, 46, 181, 186, 189, 190) [3 H]-Acetylcholine 10 – –––– (191) [3 H]-Cytisine 0.15-0.96 – –––– (46, 154) [3 H]-ABT-418 2.85 – –––– (156) [3 H]-MCC 1.07 – –––– (46) [3 H]-Epibatidine 0.011-0.086 – 0.306-4.9 0.013-0.14 2.8 0.25 (47, 170, 171, 192) (159, 171) (64, 159, 171) (159) (159) 5-[125 I]-A-85380 0.010 – –––– (76) [125 I]-a -Conotoxin MII ––– 0.93-1.9 –– (161, 196) [125 I]--–a Bgt 0.4-1.66 –––– (45, 166, 172, 176, 186, 193) [3 H]-MLA – 1.86-2.2 –––– (94, 163) [125 I]-MLA – 1.8 –––– (167)

(Bold text denotes compounds available from Tocris) aBinding affinities derived from saturation binding experiments carried out on brain membranes/primary cultured neurones (forab 4 2* ora 7 nAChRs) or heterologously expressed nAChRs. Binding affinities of [125 I]-a- Conotoxin MII forab 3 2* were derived from quantitative autoradiography on brain slices.

Values are the range from the references cited below each entry.

10 4.9 nM respectively, while affinity forab 3 2 nAChRs studies.9,94,153,162,163 Longer incubation times are expressed inXenopus oocytes was 0.12 nM.65,159 necessary to achieve equilibrium, but the very slow ab4 2 nAChRs in transfected cell lines are labelled dissociation kinetics of this ligand minimise loss of 47 binding during washing steps. In brain it labels with higher affinity thana 3* nAChRs (Kd = 12 pM). 153 In a comparative analysis of nicotinic radioligand a single population of sites with Kd ~ 1 nM. Both autoradiography in brains of transgenic mice lacking high (2000 Ci/mmol) and low(150 Ci/mmol) specific theb 2 subunit, Zoli and colleagues identified one high activity versions of [125 I]-a -Bgt are available affinity [3 H]-epibatidine binding site that persisted in commercially, depending on the sensitivity required, these animals, and tentatively assigned the subunit or it can be readily iodinated with intermediate compositionab 3 4*.155 One caution in using such a specific activity (~ 700 Ci/mmol) by the chloramine T high affinity radioligand is the possibility of ligand method.164,165,166 [ 3 H]-a -Bgt is also available but has depletion during an assay, such that the free generally not been used to characterise neuronal radioligand concentration is diminished and nAChRs. quantitative analysis becomes unreliable; this can be [3 H]-MLA has been developed as an alternative to avoided by decreasing tissue concentration and/or [125 I]-aa -Bgt for labelling 7 nAChRs. 94,163 This 47,158 increasing assay volumes. radioligand labels a single population of sites in 125 [ I]-Epibatidine (in which the chlorine atom of rodent brain with low nanomolar affinity (Kd ~ 1-2 nM) epibatidine is replaced by [125 I] with little change in and the distribution of binding sites closely parallels pharmacological properties) has recently become that of [125 I]-aa -Bgt. An -Bgt-insensitive fraction of available and provides a higher specific activity ligand [3 H]-MLA binding was observed but may be explained for labelling minor populations of binding sites.55,160 by steric factors preventing access of the largera -Bgt These sites were differentiated by their sensitivities to (but not MLA) to five binding sites on a homomerica 7 unlabelled cytisine and A-85380.55 Indeed 5-[ 125 I]-A- nAChR.94 An iodinated version of MLA, generated 85380, labels brainab 4 2 nAChRs with a low using the chloramine T method, has also been 76 167 picomolar affinity (Kd = 10 pM): it labels a single described. population of sites and thus offers improved binding There is also interest in developing isotopically selectivity forab 4 2 over a 3* nAChRs compared to labelled nicotinic ligands for non-invasive in vivo radiolabelled epibatidine (unlabelled 5-iodo-A-85380 imaging of brain nAChRs.168,169 displaced [3 H]-epibatidine binding toa 3* nAChRs in rat adrenal gland membranes with an affinity 1000- Future directions with nicotinic ligands fold less than epibatidine).76 Despite the growing nicotinic pharmacopoeia, there is A minor population of nAChR binding sites comprising still a lack of subtype-selective agonists and aab3/ 6 2* subunits has been identified directly, in antagonists, especially directed towards the less rodent and monkey brain, using an iodinated version abundant nAChR subtypes. Presently only thea - ofa -conotoxin MII.161,196 Conventional “grind and conotoxins provide this degree of specificity. The bind” assays with this conotoxin are confounded by design of more subtype-selective ligands will facilitate high non-specific binding. However, the labelled elucidation of the physiological roles and therapeutic peptide works effectively in quantitative potential of individual neuronal nAChR subtypes. This autoradiography: this technique was used to family of LGICs is increasingly perceived to have an demonstrate monophasic [125 I]-a -conotoxin MII important influence on central, as well as peripheral, labelling with an affinity (Kd ) of 0.9 nM and 1.9 nM for nervous system activities: the pharmacological monkey and rodent brain respectively.161,196 dissection of these roles and the development of [125 I]-a -Bgt has been widely used as a definitive nAChR subtype-selective are goals that will occupy researchers in this field for a good ligand for a distinct nAChR, now equated with thea 7 while longer yet! subtype, in both ligand binding and autoradiography

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Nicotinic Receptor Ligands available from Tocris Agonists 0352 4-Acetyl-1,1-dimethylpiperazinium ...... Nicotinic agonist 0351 1-Acetyl-4-methylpiperazine...... Nicotinic agonist 0789 (±)-Anatoxin A ...... Nicotinic agonist 1390 (-)-Cytisine ...... Potent, selective neuronal nicotinic agonist 0684 (±)-Epibatidine ...... Verypotent nicotinic agonist 1077 Lobeline ...... Nicotinic agonist 1053 RJR 2403 ...... CNSselective nicotinic agonist

Antagonists 0424 Benzoquinonium...... Nicotinic antagonist 1001 Chlorisondamine ...... Nicotinic antagonist; slow offset 1340*aabb -Conotoxin MII ...... Potent, 3 2 and 3 subunit selective antagonist 1029 Methyllycaconitine ...... Neuronal nicotinic receptor antagonist R1029 [3 H]-Methyllycaconitine b ...... Radiolabelled form of (1029) 0693 Pancuronium ...... Nicotinic (neuromuscular) antagonist

Modulators of Function 0388 Ambenonium ...... inhibitor 0422 Bromodechloroambenonium ...... 0686 Galanthamine...... Cholinesterase inhibitor 1260 Ivermectin...... Allosteric modulator ofa 7 nicotinic receptor 0537 MR 16728...... Stimulates ACh release 0657a -NETA ...... Potent, selective, fluorescent choline acetyltransferase inhibitor 0592b -NETA...... Selective, fluorescent choline acetyltransferase inhibitor 0750 PG-9 maleate ...... Presynaptic cholinergic modulator 0622 Physostigmine ...... Cholinesterase inhibitor 0751 SM-21...... Presynaptic cholinergic modulator 0965 ...... Cholinesterase inhibitor 0739 Tropanyl 2-phenylthiobutanoate...... Presynaptic cholinergic modulator 0653 (±)-Vesamicol ...... Inhibits ACh storage

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Neuronal Nicotinic Receptors, Tocris Reviews No. 19, October 2001

©2001 Tocris Cookson Published and distributed by Tocris Cookson, Bristol, UK

Editors: Samantha Manley, Ph.D., Natalie Barker, B.Sc. Design and Production: Jane Champness

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