Nicotinic ACh Receptors

20 Pages·2007·3.08 MB·English

Nicotinic
ACh Receptors
Susan Wonnacott and Jacques Barik
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
Susan Wonnacott is Professor of Neuroscience in the Department of Biology and Biochemistry at the University of
Bath. Her research focuses on understanding the roles of nicotinic acetylcholine receptors in the mammalian brain
and the molecular and cellular events initiated by acute and chronic nicotinic receptor stimulation. Jacques Barik was
a PhD student in the Bath group and is continuing in addiction research at the Collège de France in Paris.
Introduction and there followed detailed studies of the properties
of nAChRs mediating synaptic transmission at
The nicotinic acetylcholine receptor (nAChR) is the
these sites. nAChRs at the muscle endplate and
prototype of the cys-loop family of ligand-gated ion
in sympathetic ganglia could be distinguished
channels (LGIC) that also includes GABA , GABA ,
A C
by their respective preferences for C10 and C6
glycine, 5-HT receptors, and invertebrate glutamate-,
3
polymethylene bistrimethylammonium compounds,
histamine-, and 5-HT-gated chloride channels.1,2
notably decamethonium and hexamethonium.7 This
nAChRs in skeletal muscle have been characterised
provided the first evidence that muscle and neuronal
in detail whereas mammalian neuronal nAChRs DD
nAChRs are structurally different.
in the central nervous system have more recently RR
become the focus of intense research efforts. This In the 1970s, elucidation of the structure and function II
was fuelled by the realisation that nAChRs in the brain of the muscle nAChR, using biochemical approaches, VV
and spinal cord are potential therapeutic targets for was facilitated by the abundance of nicotinic synapses II
a range of neurological and psychiatric conditions. akin to the muscle endplate in electric organs of the NN
The generation of transgenic mice with deleted or electric ray, Torpedo, and eel, Electrophorus. High GG
mutated nAChR subunits3 and the development affinity snake α-toxins, including α-bungarotoxin
of subtype-selective ligands to complement the (α-Bgt), enabled the nAChR protein to be purified RR
generous armamentarium of natural products that and subsequently resolved into 4 different subunits, EE
target nAChRs,4 support this research. Progress is designated α,β,γ and δ.8 An additional subunit, ε, was SS
being made in understanding the physiological roles subsequently identified in adult skeletal muscle. In EE
of nAChRs in the brain and the underlying molecular the early 1980s, these subunits were cloned and the AA
and cellular mechanisms, and the contribution of era of the molecular analysis of nAChRs commenced. RR
nAChRs to pathological conditions. The muscle endplate nAChR has the subunit CC
Muscle nAChR combination and stoichiometry (α1)2β1εδ, whereas HH
nAChRs in vertebrate skeletal muscle have been tinh efo eetxatrl aojru dnectnioenrvaal tendA CmhuRsc l(eα,1 a)2nβd1 γ(mδ upsrceldeo-dmeirnivaeteds) F F
studied for over a century; this preparation was electric organs. The high density of nAChRs in Torpedo UU
pivotal in Langley’s formulation of the concept electric organ has facilitated high resolution structural RR
of a ‘receptive substance’.5 In these studies he studies using electron microscopy.9 Together with TT
showed that ‘nicotine causes tonic contraction of biochemical and biophysical approaches to studying HH
certain muscles of fowl, frog and toad, and that this
structure-function relationships, this has resulted in a EE
contraction is prevented .... by curare’. This was
detailed molecular description of the nAChR.1 RR
the first notion that the action of a neurotransmitter
or pharmacological agonist is transduced into an Molecular Architecture of the nAChR
intracellular response by interaction with a molecular (Figure 1)
entity (‘receptor’) in the membrane of the responsive Each of the five subunits comprising the nAChR span
cell. Dale distinguished the actions of muscarine the lipid bilayer to create a water-filled pore. Each
and nicotine, leading to the recognition of two subunit consists of 4 transmembrane segments,
pharmacologically distinct (and structurally and the second transmembrane segment (M2) lines the
functionally unrelated) families of receptors for the ion channel. The extracellular N-terminal domain
neurotransmitter acetylcholine (ACh), that take their of every subunit contains a ‘cys-loop’ that is the
names from these natural products.6 Neuromuscular signature sequence of this LGIC family: two cysteine
and ganglionic preparations lend themselves to residues, separated by 13 amino acids (Cys 128,
physiological and pharmacological investigations, 142, Torpedo α subunit numbering), form a disulphide
Tocris Bioscience Scientific Review Series
Tocris Bioscience Scientific Review Series
Figure 1 | General structure of nAChRs1
Non-competitive
Antagonist
K+
Positive Allosteric Agonist / Competitive
Complementary
Modulator Antagonist
binding site: γ/(δ)
Primary E D
Channel Blocker
binding site: α BW Y111Y117 W
149 55/(57)
Y
151
F
Nic
D
Y 180/(182)
190
CC
192
C Y
a) N b) 193 198 Y93 W86
A
ACh binding
protein
Cys-loopC
C
Ca2+,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 drugs in the extracellular domain or within the channel lumen are indicated. b) Agonist binding site loop
model. The agonist binding site is enlarged to show the contributing polypeptide loops forming the primary and
complementary components, with key amino acids indicated on the loops. c) The topography of a single subunit.
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 agonists 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
transition from the closed, resting conformation to
agonist binding site (see Figure 1).11 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
constitutes a barrier to ion permeation. Agonist
respect to ligand affinity.1 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 aaggoonniisstt
exist.14 In the active (open) conformation, the nAChR RREESSTTIINNGG AACCTTIIVVEE
CChhaannnneellcclloosseedd CChhaannnneellooppeenn AAggoonniissttbbiinnddss
binds agonists with low affinity (Figure 2; e.g. K for wwiitthhlloowwaaffffiinniittyy
d
ACh ~50 μM). The desensitised states display higher
affinity for agonist binding (K for ACh ~1-5 μM), thus
d
the desensitised nAChRs can retain bound agonist
despite its non-conducting state. DDEESSEENNSSIITTIISSEEDD
FFaassttoonnsseett
Sites on the Muscle nAChR for Ligand
AAggoonniissttbbiinnddss
Interactions (Figure 1) wwiitthhhhiigghhaaffffiinniittyy
In addition to agonists binding to the agonist binding
sites in the extracellular domain, competitive DDEESSEENNSSIITTIISSEEDD
antagonists also bind at or close to these sites, SSlloowwoonnsseett
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 amine from blue-geen algae that is a potent, enantio-selective ACh-like agonist lacking EC = 50 nM
50
significant activity at muscarinic receptors or AChE.88,237
(-)-Nicotine The natural tobacco alkaloid is ~6 times more potent than its unnatural enantiomer at muscle ED = 20 µM
20
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 channel blocker of muscle
and neuronal nAChR subtypes.241
α-Bungarotoxin Polypeptide snake toxin from Bungarus multicinctus; most potent of the ‘long’ α-neurotoxins. Binds K = 0.01-10 nM
d
(α-Bgt) pseudo-irreversibly, reflecting very slow dissociation kinetics. Interacts potently with α subunit
sequence around Tyr -X-Cys -Cys .1,242
190 192 193
α-Conotoxin MI One of several α-conotoxins from Conus sp. that specifically block muscle nAChRs. MI exhibits a K ~ 0.1-1 nM
d
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 ED = 0.12 µmol/kg;
95
nicotine and other agonists. It is more accurately classified as a partial agonist.7,244 0.03 mg/kg
Pancuronium Used clinically as a non-depolarizing muscle relaxant.245,246 IC ~ 5 nM
50
d-Tubocurarine As a photoaffinity label, it discriminates αδ and αγ agonist binding sites.1,246,247 K = 30 nM,
d1
K = 8 µM
d2
IC ~ 50-100 nM
50
Channel Blockers
Chlorpromazine This neuroleptic drug also blocks the nAChR channel, interacting with hydrophobic residues in the IC >300 nM
50
Torpedo M2 channel lining.248,249,250
Histrionicotoxin First isolated from frogs of the dendrobatid genera.251,252,253 K ~ 0.1-1 µM
i
(Bold Text Denotes Compounds Available From Tocris)
aEC , effective concentration producing 50% of maximum activation; ED , effective dose producing 20% of maximum response (activation); ED , effective dose
50 20 95
producing 95% of maximum response (blockade); K, Afinitiy for binding to muscle or Torpedo preparations or purified nAChR; IC , concentration producing 50%
d 50
inhibition; dependent on experimental conditions; K, Inhibition constant
i
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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 ) 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 tyrosine from loop C (Tyr190, Torpedo α1
realisation that the tobacco smoking 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.
ααααα99999 ααααα99999
ααααα99999 ααααα99999 ααααα1111100000 ααααα1111100000
α9
ααααα99999 ααααα99999 ααααα99999 ααααα99999
α10 NNNeeeuuurrrooonnnaaalll,,,
hhhooommmooommmeeerrriiiccc
α7 ααααα77777 ααααα88888 ααααα77777
pppCCCaaa222+++///NNNaaa+++
ααααα77777 ααααα77777 ααααα88888 ααααα88888 ααααα88888 ααααα88888
α8 ~~~111000
ααααα77777 ααααα77777 ααααα88888 ααααα88888 ααααα77777 ααααα77777
α1
β1
MMMuuussscccllleee,,, ε/γ
δ α1 α1
pppCCCaaa222+++///NNNaaa+++
γ
000...222 δ β1
εεε
α2 βββββ22222
ααααα44444 ααααα44444 xxxxX = β2,α4, α5,…
α4
α3 βββββ22222 xxxxX
NNNeeeuuurrrooonnnaaalll,,,
α6
hhheeettteeerrrooommmeeerrriiiccc
βββββ22222 βββββ22222
α5 pppCCCaaa222+++///NNNaaa+++ ααααα66666 ααααα44444 ααααα66666 ααααα66666
~~~111---111...555
β3 βββββ22222 βββββ33333 βββββ22222 βββββ33333
β2
βββββ22222 βββββ22222 βββββ44444
β4 ααααα33333 ααααα33333 ααααα33333 ααααα33333 ααααα33333 ααααα33333
βββββ22222 YYYYY βββββ44444 YYYYY βββββ44444 YYYYY
YYYYY = 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 neuromuscular junction 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 cholinergic
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 methyllycaconitine (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 heteromers 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 choline, in the case of α7 nAChRs).42
more reversible blockade by α-Bgt. The related α10
The α7 nAChR is particularly prominent in the
subunit is only incorporated into a functional nAChR
hippocampus, where it is found on GABAergic
when co-expressed with the α9 subunit.26,27
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 schizophrenia, 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) (β2) has been
2 3
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) (β2) nAChRs are also viable,
3 2
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.
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Tocris Bioscience Scientific Review Series
Table 2 | Potencies of selected compounds that interact preferentially with α7 nAChR
Potency
Drug Comment
Binding Function
Agonist K a EC b
i 50
AR-R17779 A synthetic, structurally rigid spirooxazolidone with 100-fold great potency for binding to α7 0.2 µM 10-20 µM
nAChRs than α4β2 nAChRs. No activation of α4β2, α3β4, α3β2, 5-HT receptors.254,255
3
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 IC of 15 and 370 µM. IC for desensitisation of α7 nAChRs ~40
50 50
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
(aka DMXB) Also interacts with α4β2 nAChRs (K = 84 nM versus [3H]-cytisine): very low partial agonist
i
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-HT receptors (K = 0.9 µM). When 26 nM 128 nM
3 i
administered at 1 mg/kg i.v. it restored amphetamine-induced sensory gating deficit and
augmented hippocampal theta oscillation in anaesthetised rats.271,272,273
SSR180711 A synthetic α7-selective partial agonist (E = 36-50% of maximum response to ACh). ~20 nM 1-4 µM
max
Elicits central effects after i.p. or oral administration.274,275
Competitive antagonists Ka IC c
i 50
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 (IC 1.8 µM) and muscle nAChRs (50 µM), with no action at heteromeric
50
nAChRs. Open channel blocker of 5-HT receptor.139,277,278 May be species selective, as
3
reported to block bovine α3β4* nAChRs (IC 2.5 µM).279 Also blocks certain invertebrate
50
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
Strychnine This glycine receptor antagonist 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
neuronal nAChRs (IC = 7-30 µM).27,66,283,284,285,286
50
Allosteric modulators EC b
50
5-Hydroxyindole A metabolite of 5-HT that potentiates α7 nAChR responses to ACh: increases potency and efficacy of 2.5 mM
ACh without affecting desensitisation. Effective at 100 µM in brain preparations.287,288,289
Ivermectin 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 GABA and glycine
A
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)
aK, Inhibition constant, from competition binding assays for [125I]-α-Bgt or [3H]-MLA binding to brain membranes or heterologously expressed human or rat α7 nAChR;
i
bEC , effective concentration producing 50% of maximum activation; from electrophysiological recording of whole cell currents or intracellular Ca2+ responses, from
50
native receptors in hippocampal neurones or cell lines or heterologously expressed human or rat α7 nAChR;
cIC , concentration of competitive antagonist producing 50% inhibition of functional responses; dependent on experimental conditions, especially agonist
50
concentration and whether antagonist is co- or pre-applied (and, in the case of α-Bgt, preincubation time, due to slow association kinetics);
n.d. = not determined
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Nicotinic Receptors
Transgenic knockout of either of these subunits 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
Table 2. Selected pan-acting or less discriminating
expressed in the thalamus. As a consequence of
agonists and antagonists are summarised in Tables
their putative role in thalamo-cortical circuitry, gain of
3 and 4 respectively, and some are briefly discussed
function mutations in the M2 domain of either the α4
below. More comprehensive accounts of the families
or β2 subunit give rise to some forms of autosomal
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
Agonists (Table )
distribution in the CNS, for example in medial
habenula and locus coeruleus they are often, but not Structurally diverse naturally occurring nicotinic
always, co-expressed.19 agonists include: (-)-nicotine, (-)-cytisine,
(+)-anatoxin A, (+)-epibatidine, anabasine and
α6 and β3 subunits are largely restricted to
anabaseine. 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 dopaminergic
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 (K values) are typically 2-3 orders
i
effects of the efferent olivocochlear system on of magnitude lower, in terms of concentration,
auditory processing.26,61 than EC values for nAChR activation (Table 3).
50
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 atropine, ~1 µM) and an
possibility that they might be involved in anti- acetylcholinesterase 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 (carbachol) is formed
Due to the critical roles of muscle and ganglionic by the modification of ACh to a carbamate,
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 muscarinic agonist.
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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 K (nM) Functional potency EC (µM)
i 50
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
(-)-Cotinine 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
Lobeline 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)
*IC (drug inhibits rather than activating nAChR responses)
50
**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 dopamine 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 (T = 5-6 hours, compared with
½
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 pyrrolidine 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 (IC values are 31 µM and
50
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
Selective α4β2 Ligands
α7 nAChRs appear to be less stereoselective.
Nicotine crosses the blood brain barrier readily
and its pharmacokinetics and metabolism MeHN
TC 2559 N
are well documented.72 For a comprehensive
Cat. No. 2737
discussion of nicotine doses used in vivo in .2C4H4O4
OEt
various vertebrate and invertebrate organisms,
see ref. 343. Behavioural responses typically RJR 2403 NHMe
Cat. No. 1053
show a bell-shaped dose-response profile with .C4H4O4
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)(β2) receptor stoichiometry and acts as a
delivery of 2-4 mg/kg/day in rats reproduces the 2 3
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 (T <10
½ α2β4 – 14.0
min) than rats (T ~55 min) and consequently
½ α3β4 >1000 >30
require higher doses.76,77,78 However, high doses
α1β1γδ >1000 –
of nicotine (>1 mg/kg s.c. in rats) produce
adverse effects, most likely due to ganglionic α3β2 15 >100
or neuromuscular actions, although higher α7 36 (K) >100
i
concentrations elicit convulsions. These doses EC values (in µM). Data taken from Bencherif et al (1996) and Chen et al
50
refer to the free base concentration of nicotine; (2003).
3-times higher concentrations of the tartrate salt Bencherif et al (1996) RJR-2403: a nicotinic agonist with CNS selectivity I. In
vitro characterization. J.Pharmacol.Exp.Ther. 279 1413. Lippiello et al (1996)
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-
Its ability to displace nicotinic radioligands from 2559, increases dopamine neuronal activity in the ventral tegmental area of rat
midbrain slices. Neuropharmacol. 45 334. Zwart et al (2006) 5-I A-85380 and
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.
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Tocris Bioscience Scientific Review Series
• TC 2559 is the 5-ethoxy derivative of trans- affinity binding (K ~1 nM), but its differential
i
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 scopolamine-induced cognitive 3-position of the pyridine 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
• Varenicline (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 smoking cessation.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
Agonist in Xenopus oocytes (IC = 6 nM) than it is at
50
eliciting responses (EC = 3 µM), interpreted
50
(±)-Anatoxin A Me
O as a reflection of the higher affinity for agonists
NH
Cat. No. 0789 .C4H4O4 shown by the desensitised state of nAChRs (α4β2
nAChRs in particular) (see Figure 2). Varenicline
(±)-Anatoxin A is a nicotinic receptor agonist (K values is effective in vivo at doses of 0.01-3.0 mg/kg
i
are 3.5 and 380 nM for α4β2 and α7 nicotinic receptors (given s.c. or p.o.)100 and has a half life of 4 and
respectively). The agonist stimulates [3H]-dopamine release
17 hours in rats and humans respectively, with
from rat striatal synaptosomes (EC = 136 nM) with a higher
50 little metabolism.101
potency than (-)-nicotine. (±)-Anatoxin A displays powerful
behavioural effects in the rat; it decreases locomotor activity
in nicotine-tolerant and non-tolerant rats and decreases • 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 pharmacology of anatoxin analogs. II. Side binding to multiple heteromeric nAChRs with
chain structure-activity relationships at neuronal nicotinic ligand binding sites. sub-nanomolar affinities.103 Like anatoxin A,
J.Pharmacol.Exp.Ther. 259 387. Stolerman et al (1992) Behavioural effects of
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 some rigidity to its structure, but in this case it is
receptors. J.Neurochem. 60 2308. Sharples et al (2000) UB-165 implicates
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 EC values for heteromeric
50
comparable to nicotine, with respect to its high
10 |