MINIREVIEW
a-Conotoxins as tools for the elucidation of structure and function
of neuronal nicotinic acetylcholine receptor subtypes
Annette Nicke
1
, Susan Wonnacott
2
and Richard J. Lewis
3
1
Max Planck-Institute for Brain Research, Frankfurt, Germany;
2
Department of Biology & Biochemistry, University of Bath, UK;
3
Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia
Cone snails comprise 500 species of venomous molluscs,
which have evolved the ability to generate multiple toxins
with varied and often exquisite selectivity. One class,
the a-conotoxins, is proving to be a powerful tool for
the differentiation of nicotinic acetylcholine receptors
(nAChRs). These comprise a large family of complex
subtypes, whose significance in physiological functions and
pathological conditions is increasingly becoming apparent.
After a short introduction into the structure and diversity of
nAChRs, this overview summarizes the identification and
characterization of a-conotoxins with selectivity for neur-
onal nAChR subtypes and provides examples of their use in
defining the compositions and function of neuronal nAChR
subtypes in native vertebrate tissues.
Keywords:a-conotoxins; neuronal nicotinic acetylcholine
receptor subtypes; pharmacology; venom peptides; Xenopus
oocytes.
Neuronal nicotinic acetylcholine receptors
The nicotinic acetylcholine receptor family
The nicotinic acetylcholine receptor (nAChR) at the neuro-
muscular junction was first described as the Ôreceptive
substanceÕin Langley’s
1historic experiments which lead to
the formulation of the receptor concept [1]. nAChRs have
been amongst the earliest receptors to be investigated
by pharmacological, biochemical, electrophysiological and
molecular biological approaches, and to date represent one
of the most intensively investigated membrane proteins.
While the identification and pharmacological distinction of
nAChR subtypes at the neuromuscular endplate (causing
muscle contraction) and those in sympathetic and para-
sympathetic ganglia (mediating neurotransmission) was
made relatively early, the existence of nAChRs in the brain
was controversial until cloning of the first neuronal nAChR
isoforms in the mid 1980s [2,3]. nAChRs are ligand-gated
ion channels that belong to the Cys-loop receptor super-
family which includes GABA
A
,glycineand5HT
3
neuro-
transmitter receptors.
The electric organs of the electric ray Torpedo and
eel Electrophorus provided a rich source of nAChRs that
facilitated their early structural characterization. The
nAChR from Torpedo californica is the best investigated
ligand-gated ion channel so far and considered as a
prototype. By electron microscopy techniques [4], high
resolution images down to 4 A
˚have been obtained from
semicrystalline arrays of this receptor in Torpedo mem-
branes. These studies revealed the pentameric quaternary
structure of this protein (Fig. 1) and have provided valuable
information about the channel architecture and dimensions.
A deeper insight into the molecular structure, in particular
the acetylcholine (ACh) binding pocket, has become
available after crystallization of an ACh binding protein,
which has high homology to the extracellular domain of
the nAChR (Fig. 1) [5,6
2]. The Torpedo nAChR and the
nAChR in embryonic vertebrate muscle share the same
heteropentameric structure composed of four homologous
subunits which are arranged in the order a1ca1db1 around
the central ion-conducting channel [7,8] (Fig. 2A). In
addition, 11 nAChR subunits (a2–a7, a9, a10, b2–b4) have
been cloned from neuronal and sensory mammalian tissues.
A mammalian homologue of the avian a8 subunit has not
been found [2,3,9].
Subunit assembly of neuronal nAChRs
The a7, a8anda9 subunits represent a subclass of neuronal
nAChRs that is able to form functional homomeric
channels upon heterologous expression [2,3]. Coexpression
of a7anda8, as well as of a9 and the highly homologous
a10 subunit [10] has been shown to generate heteromeric
channels with properties distinct from those of the respective
homopentamers. The association of a7withbsubunits in
native nAChRs has been controversial [11]. The a2, a3, a4
and a6 subunits require coexpression of at least one b(b2or
b4) subunit to form functional channels [2,3,9]. However,
pairwise combinations of the a6withtheb2orb4 subunit
resulted in protein aggregation or very inefficient expression
of functional channels [12], indicating that at least two other
subunits are required for effective channel formation. In
Correspondence to A. Nicke, Max Planck-Institute for Brain Research,
Deutschordenstr. 46, D-60528 Frankfurt, Germany.
Fax: + 49 69 96769 441, Tel.: + 49 69 96769 262,
E-mail: nicke@mpih-frankfurt.mpg.de
Abbreviations: ACh, acetylcholine; nAChR, nicotinic acetylcholine
receptor; a-BTX, a-bungarotoxin; all a-conotoxins are abbreviated,
e.g. MII instead of a-conotoxin MII.
(Received 22 January 2004, revised 17 March 2004,
accepted 6 April 2004)
Eur. J. Biochem. 271, 2305–2319 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04145.x
support of this, higher expression levels could be obtained
by addition of the a5 and/or b3 subunit [12]. The a5andb3
subunits are very similar in sequence and both appear
unable to form functional channels in any pairwise combi-
nation [13–15].
From analysis of single channel conductances obtained
upon coinjection of wild-type and mutant subunits, and
from quantification of radiolabelled aand bsubunits, the
stoichiometry (a)
2
(b)
3
has been proposed for oocyte-
expressed neuronal nAChRs [16,17]. However, there is only
limited knowledge of the stoichiometry of native neuronal
nAChRs. Combinations of three and even four different
subunits (including a5, b3) have been described in both
heterologous expression systems and native tissues (e.g. [18–
21]) further complicating the determination of stoichio-
metries.
The ACh binding site has been located at the interface
between an asubunit (+ face) and an adjacent subunit
(– face), that may be a d,cor esubunit (muscle nAChR),
bsubunit (heteromeric neuronal nAChR) or, in the case
of the homomeric channels, another asubunit (– face)
[6,7]. The a1, a2, a3, a4, a6, a7, a9anda10 subunits, as
well as the nonasubunits, c,d,e(which replaces cin
adult muscle), b2andb4, can contribute to the ACh
binding site. In contrast, a5, b1andb3 subunits appear to
play a more ÔstructuralÕrole but may additionally modu-
late channel function and/or influence membrane trans-
port and targeting of nAChRs [9].
The subunit composition of different nAChRs deter-
mines the pharmacological and physiological properties of
the channel. In situ hybridization and immunohisto-
chemistry data show overlapping distributions for a variety
of subunits, and electrophysiological and other functional
studies in native tissues have revealed a great diversity of
nAChR subtypes with distinct pharmacological, electrical
and physiological properties even within single cells [2,3].
To decipher the physiological roles played by the different
nAChRs, a range of subtype specific inhibitors are
needed.
Neuronal nAChRs as targets for the development of
subtype specific drugs
Neuronal nAChRs are present throughout the central and
peripheral nervous system, at both pre- and postsynaptic
localizations. The most prevalent subunits in brain are a4,
b2anda7whereasa3andb4 predominate in peripheral
ganglia. Because more complex combinations may exist,
an asterisk is used to denote the potential presence of
additional subunits, as in a4b2* and a3b4* nAChRs [22].
The a7 subunit is widespread in the central nervous system
and a variety of peripheral tissues. The a7* receptors are
characterized by very fast inactivation kinetics and long
lasting desensitization, which makes their functional iden-
tification difficult [23].
Different neuronal nAChR subtypes have been shown
to be involved in learning, antinociception, nicotine
addiction and neurological disorders such as Parkinson’s
and Alzheimer’s disease. For the nonselective nAChR
agonist nicotine, analgesic, anxiolytic and cytoprotective
properties are seen, as well as beneficial effects in
Alzheimer’s disease, Parkinson’s disease, Tourette’s syn-
drome and certain forms of epilepsy and schizophrenia
[24,25]. However, the therapeutic use of nicotine is
hindered by its adverse effects on the cardiovascular and
Fig. 2. Subunit compositions of the muscle-type nAChR and assumed
subunit compositions of neuronal nAChRs targeted by a-conotoxins. (A)
The composition of neuronal nAChRs can be similarly complex to
that of the muscle-type nAChR. Note that the muscle-type specific
a-conotoxins MI and GI have opposite selectivities at nAChRs from
Torpedo and mammalian muscle. a-Conotoxins with selectivity for
heterologously expressed pairwise combinations of neuronal aand b
subunits, such as AuIB and MII (B), provide valuable tools to decipher
the complex assemblies of native neuronal nAChRs (C) and investigate
their physiological function. Although some a-conotoxins show
activity on a4b2nAChRs(e.g.GID),ana4b2selectivea-conotoxin
has not yet been described.
Fig. 1. Schematic representation of the membrane topology and qua-
ternary structure of the nAChR. Each nAChR subunit contains four
transmembrane domains, with five subunits assembling to form an ion
channel. The second transmembrane domain of each subunit contri-
butes to the formation of the hydrophilic pore. ACh binding protein
has structural and functional homology to the extracellular ligand
binding domain of the nAChR, and likewise assembles into pentamers.
2306 A. Nicke et al.(Eur. J. Biochem. 271)FEBS 2004
gastrointestinal systems as well as its addictive potential.
The combinatorial diversity of nAChRs with distinct
pharmacological and physiological properties opens up
an opportunity to develop selective nAChR agonists and
modulators for the specific treatment of neurological
disorders. A prerequisite for the development of selective
drugs is the identification and pharmacological character-
ization of the various receptor subtypes, and the deter-
mination of their precise subunit composition and
physiological function(s). Compared to the muscle
nAChR, relatively little is known about the function and
composition of the neuronal nAChRs. This objective has
been greatly hampered by a lack of selective ligands. The
snake neurotoxin a-bungarotoxin (a-BTX) is one of the
first and most powerful tools for the purification, subtype
differentiation and histologic labelling of nAChRs con-
taining the muscle a1 or the neuronal a7–a9 subunits.
However, the a3* selective neuronal bungarotoxin
(n-BTX) is not generally available, and the antagonists
mecamylamine and dihydro-b-erythroidine are relatively
undiscriminating between different heteromeric neuronal
nAChRs. Thus, further and more specific inhibitors are
needed to probe neuronal nAChRs in native tissues.
a-Conotoxins as selective ligands for nAChR
subtypes
Among the most selective ligands targeting distinct nAChRs
are peptides isolated from the venom of cone snails [26].
Each of the 500 or so species contains in its venom a mixture
of 50–200 peptides, giving a total of 50 000 potential
pharmacologically active peptides. However, only a small
portion (< 0.1%) of these peptides has been pharmacolo-
gically characterized so far. The great variability of the
conotoxins and their highly specific action on different ion
channel subtypes derives from the structure of the peptides
which have evolved conserved and hypervariable regions
[27–30]. The conserved regions comprise the signal sequence
which is characteristic for the respective toxin superfamily
and generally defines the pattern of disulfide connectivities.
The loops between the cysteine residues represent the
hypervariable regions that define the pharmacological
diversity of conopeptides. This hypervariability has gener-
ated a wide diversity of a-conotoxins with activity at
neuronal nAChR subtypes.
Conotoxins targeting nAChRs
To date, three different conotoxin families targeting
nAChRs have been identified [26]. Each family is defined
by a common binding site on the nAChR as well as by
their structure (for nomenclature of a-conotoxins see [31])
3.
The w-conotoxin PIIIE has a structure similar to the
voltage-gated Na
+
channel-blocking l-conotoxins and
acts as a noncompetitive antagonist (perhaps a pore
blocker) of the muscle-type nAChR. The other two
families, aA- and a-conotoxins, function as competitive
antagonists at the ACh binding site, but differ in their
disulfide framework. The three aA-conotoxins identified
so far also target the muscle-type nAChR. The largest
family are the a-conotoxins which can be further divided
into a3/5, a4/3, a4/6 and a4/7 structural subfamilies
depending on the number of amino acids between the
second and the third cysteine residues (loop I) and the
third and the fourth cysteine residues (loop II), respectively
[32] (Table 1). It appears that these differences in structure
are paralleled by their selectivity for different nAChR
subtypes, with all known a3/5-conotoxins being selective
for the muscle-type nAChR, while the only published
a4/6-conotoxin and most a4/7-conotoxins are selective for
neuronal nAChRs. One exception is a4/7-conotoxin EI,
which preferentially targets the a/dinterface of the
mammalian muscle nAChR and is the only ligand
selective for the Torpedo a/dinterface [33] (Fig. 2A).
However, information on the activity of EI at neuronal
subtypes is lacking. The a4/3-conotoxins, represented by
ImI and ImII, are a7 selective [34,35]. Interestingly, these
peptides differ by only three amino acids and have been
shown to block the homomeric a7 nAChR with similar
potency but appear to have nonoverlapping binding sites
as only ImI competes with a-BTX binding [35]. Thus,
ImII may act in a noncompetitive manner. The example of
ImII shows that it is important to distinguish competitive
from noncompetitive modes of action for newly discovered
a-conotoxin-like peptides.
Specificity of a-conotoxins for distinct nAChR interfaces
The a3/5 conotoxins GI, MI, SI, SIA and SII are amongst
the first nicotinic antagonists identified from cone snail
venoms [26,36]. They specifically target neuromuscular
receptors in a wide range of species but have no activity at
neuronal subtypes. The members of this subclass show
remarkable selectivity for the distinct interfaces (a/cor a/d)
within the muscle-type nAChR complex of different species
[26,36]. Like the muscle active a-conotoxins, several neuro-
nally active a-conotoxins show a similar specificity
for distinct interfaces within neuronal nAChR subunit
combinations (compare Fig. 2A–C)
4.Sofar,a-conotoxins
selectively targeting mammalian a3b2(a-MII, a-GIC) a6b2
(a-MII, a-PIA), a3b4(a-AuIB) and a7(a-ImI) interfaces
have been identified [12,34,37–41]. It appears that binding of
only one toxin molecule is sufficient to block receptor
function [33,42]. In contrast, two agonist molecules seem to
be required to open the nAChR channel. As a consequence,
native nAChRs with two different types of a/binterface can
be expected to show agonist potencies that are different
from those of the simple combinations of only one type of a
and bsubunits which are generally studied in heterologous
expression systems. The ability to differentiate pharmaco-
logically between nonequivalent binding sites within the
same receptor, together with the dominant inhibitory effect
obtained by binding of only one antagonist molecule,
represents a particular advantage of a-conotoxins. These
features make them useful tools for defining different
nAChR subtypes and their specific functions in native
tissues.
The a4/7-conotoxins are the most common nAChR
antagonists found in cone snail venoms. Identification of
further selective peptides, together with the investigation
and understanding of their structure-activity relationships,
may start to provide a rational way to develop additional
pharmacological tools for the elucidation of nAChR
structure and function.
FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2307
Table 1.
21,2121,21 Summary of neuronally active a-conotoxins and their
21,2121,21 activity on vertebrate nAChRs. Small letters at the beginning indicate the species: r, rat; m, mouse; h, human; c, chick; p, monkey; b, bovine;
f, frog. Capital letters indicate the tissue/cells: CC, chromaffin cells; NJ, neuromuscular junction; H, hippocampal neurons; SCLC, small cell lung carcinoma cells; B, brain; IG, intracardiac ganglion
neurons; CG, ciliary ganglion neurons; S, striatum; SY, striatal synaptosomes; SC, superior colliculus; R, retina; NA, nucleus accumbens; C, caudate; P, putamen. Small letters at the end indicate the
method: r, electrophysiological recordings; m, binding studies on membrane preparations; i, binding studies on immunoimmobilized receptors; s, quantitative autoradiography on tissue sections;
d, quantification of agonist-evoked dopamine release; c, quantification of agonist-evoked catecholamine release. a6/a3anda7/5HT
3
indicate chimeric receptors between nicotinic asubunits and nicotinic a7
and the 5-hydroxytryptamine receptor, respectively. a7/5HT
3
constructs were expressed in human embrionic kidney (HEK) cells. IC
50
values > 10 l
M
and a-conotoxin mutants were generally not
considered. Differences between expression systems and between heterologously expressed and native channels as well as species differences have been suggested to account for inconsistencies in IC
50
values.
In addition, preparation inherent differences (e.g. dissociated neurons, synaptosomes or physiologically more intact systems such as slices) and methodological variations (e.g. different agonist concentrations,
protocols for toxin application or determination of the toxin concentration) have to be considered.
a-Conotoxin Sequence
a
Functional Data
Binding Data (n
M
)
c
IC
50
(n
M
) on recombinant nAChRs
b
IC
50
(n
M
) in native tissues and
suggested native AChRs targeted
ImI GCCSDPRCAWR----Ca7220 [34], 100 [23], 191 [35], 1040 [106] fNJr 250–500 [46] rBm EC
50
(B) 1560 [35]
ha7132 [85] rHr 86 [48] a7ha7/5HT3 EC
50
(B) 407 [35]
a91800 [34] SCLC 10 [101] a7ha7/5HT3 K
d
(B) 2380 [84], 4000 [107]
a3b4no effect at 3–5l
M
[23,34] bCCc 300 [23] a7, 2500 [52] a3b4(a5)
ImII ACCSDRRCRWR----Ca7441 [35] not competitive with a-BTX [35]
PnIA GCCSLPPCAANNPDYCa7252 [55] rIGr 14 [56] a7* + additional component ha7/5HT3 K
d
(B) 61 200 [58]
ca7349 [59]
ca7L247T 194 [59]
a3b29.6 [55]
[A10L]PnIA GCCSLPPCALNNPDYCa713 [55] rIGr 1.4 [56] a7* ha7/5HT3 K
d
(B) 630 [58]
ca7168 [59] bCCc 2000/1500 [57] a3b4
ca7L247T acts as an agonist [59]
a3b299 [55]
[N11S]PnIA GCCSLPPCAASNPDYCa71710 [55] rIGr 375 [56] a7* + additional component ha7/5HT3 K
d
(B) 148 000 [58]
a3b2241 [55]
PnIB GCCSLPPCALSNPDYCa761 [55] rIGr 33 [56] ha7/5HT3 K
d
(B) 29 600 [58]
a3b21970 [55] bCCc 700/1000 [57] a3b4
EpI GCCSDPRCNMNNPCYCa730 [61] rIGr 1.6 [60] a3b2/a3b4
ca7/5HT3 (HEK293) 103 [61] bCCc 84/210 [60] a3b4
MII GCCSNPVCHLEHSNLCa3b20.5 [37], 3.5 [102], 8.0 [62], 1.7 [39], rIGr 10 [100] a3b2rSi 1.3 [21] a6b2*
K
dd
0.35 [64] rSYd 24 [62], 17 [62]a3b2* mS,SCm 1.4 [71] a6b2*
a6/a3b2b30.4 [39] mSd 2 [72] pSs 19 C [93], 12 P [93] a6b2, b3, or b4
ha6/a4b4(HEK293) 24 [104] cCGr 33 [77] a3b2b4a5cRi 66 [40] a6b4*
a7100 [37] rHc < 150 [76] a3b2b4* mBm 2.7 [64] a3b2*
a4b2430 [39] rCCr 35 [105] a3b2*
bCCc 710 [103] a3b4(a5)*
2308 A. Nicke et al.(Eur. J. Biochem. 271)FEBS 2004
Table 1. (Continued).
a-Conotoxin Sequence
a
Functional Data
Binding Data (n
M
)
c
IC
50
(n
M
) on recombinant nAChRs
b
IC
50
(n
M
) in native tissues and
suggested native AChRs targeted
[
125
I]MII [
125
I]YGCCSNPVCHLEHSNLCa3b2K
dd
1.9 [64] rSs K
d
0.63 [66], 0.83 [66], NA a3/a6b2b3*
pSs K
d
0.93 [67], C 0.92 [67] P a6b2(b3)
mSCm K
d
4.9 [64]
AuIB GCCSYPPCFATNPD-Ca3b4750 [41], 966 [61], K
dd
500 [41] rIGr 1.2 [78]
a710 000 [41] cCGr 350 [77] a3b4a5*
RHc 2200 [76] a3b2b4*
rCCr 105 [105] a3b4*
AuIB (ribbon) GCCSYPPCFATNPD-Ca3b427.500 [61] rIGr 0.1 [78]
GIC GCCSHPACAGNNQHICha3b21.1 [38]
ha3b4755 [38]
ha4b2309 [38]
GID IRDcCCSNPACRVNNOHVCa74.5 [80]
a3b23.1 [80]
a4b2152 [80]
Vc1.1 GCCSDPRCNYDHPEICbCCc 1000–3000 [81] a3b4* bCCc 2.3 and 3700 (2 sites) [81] a3b4*
PIA RDPCCSNPVCTVHNPQICa6/a3b20.69 [39]
a6/a3b2b30.95 [39]
ha6/a3b2b31.72 [39]
a3b274.2 [39]
a6/a3b430.5 [39]
ha6/a3b412.6 [39]
a6b433.5 [39]
a3b4518 [39]
AnIB GGCCSHPACAANNQDYCa776 [83]
a3b20.3 [83]
a
Sequence disulfide connectivity: underlined-underlined and bold-bold.
b
Unless otherwise indicated, data are from rat subunits expressed in Xenopus oocytes (h, human; c, chick subunits).
c
Unless
otherwise indicated, K
i
values for inhibition of epibatidine binding are shown; B, inhibition of a-BTX binding.
d
Indicates cases where K
d
values were obtained from oocyte-expressed receptors.
FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2309