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Tài liệu Báo cáo khoa học: a-Conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes doc

a-Conotoxins as tools for the elucidation of structure and function
of neuronal nicotinic acetylcholine receptor subtypes
Annette Nicke
, Susan Wonnacott
and Richard J. Lewis
Max Planck-Institute for Brain Research, Frankfurt, Germany;
Department of Biology & Biochemistry, University of Bath, UK;
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
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
substanceÕ in Langley’s
historic 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
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
]. 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 a7withb subunits 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) Ó 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 a and b subunits, the
stoichiometry (a)
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-
The ACh binding site has been located at the interface
between an a subunit (+ face) and an adjacent subunit
(– face), that may be a d, c or e subunit (muscle nAChR),
b subunit (heteromeric neuronal nAChR) or, in the case
of the homomeric channels, another a subunit (– face)
[6,7]. The a1, a2, a3, a4, a6, a7, a9anda10 subunits, as
well as the nona subunits, c, d, e (which replaces c in
adult muscle), b2andb4, can contribute to the ACh
binding site. In contrast, a5, b1andb3 subunits appear to
play a more ÔstructuralÕ role 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
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 a and 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) Ó 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
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])
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/d interface of the
mammalian muscle nAChR and is the only ligand
selective for the Torpedo a/d interface [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/c or 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)
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/b interface can
be expected to show agonist potencies that are different
from those of the simple combinations of only one type of a
and b subunits 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
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.
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2307
Table 1.
Summary of neuronally active a-conotoxins and their
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
indicate chimeric receptors between nicotinic a subunits and nicotinic a7
and the 5-hydroxytryptamine receptor, respectively. a7/5HT
constructs were expressed in human embrionic kidney (HEK) cells. IC
values > 10 l
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
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
Functional Data
Binding Data (n
) on recombinant nAChRs
) in native tissues and
suggested native AChRs targeted
GCCSDPRCAWR C a7 220 [34], 100 [23], 191 [35], 1040 [106] fNJr 250–500 [46] rBm EC
(B) 1560 [35]
ha7 132 [85] rHr 86 [48] a7 ha7/5HT3 EC
(B) 407 [35]
a9 1800 [34] SCLC  10 [101] a7 ha7/5HT3 K
(B) 2380 [84], 4000 [107]
a3b4 no effect at 3–5l
[23,34] bCCc  300 [23] a7, 2500 [52] a3b4(a5)
ACCSDRRCRWR C a7 441 [35] not competitive with a-BTX [35]
GCCSLPPCAANNPDYC a7 252 [55] rIGr 14 [56] a7* + additional component ha7/5HT3 K
(B) 61 200 [58]
ca7 349 [59]
ca7L247T 194 [59]
a3b2 9.6 [55]
GCCSLPPCALNNPDYC a7 13 [55] rIGr 1.4 [56] a7* ha7/5HT3 K
(B) 630 [58]
ca7 168 [59] bCCc 2000/1500 [57] a3b4
ca7L247T acts as an agonist [59]
a3b2 99 [55]
GCCSLPPCAASNPDYC a7 1710 [55] rIGr 375 [56] a7* + additional component ha7/5HT3 K
(B) 148 000 [58]
a3b2 241 [55]
GCCSLPPCALSNPDYC a7 61 [55] rIGr 33 [56] ha7/5HT3 K
(B) 29 600 [58]
a3b2 1970 [55] bCCc 700/1000 [57] a3b4
GCCSDPRCNMNNPCYC a7 30 [61] rIGr 1.6 [60] a3b2/a3b4
ca7/5HT3 (HEK293) 103 [61] bCCc 84/210 [60] a3b4
GCCSNPVCHLEHSNLC a3b2 0.5 [37], 3.5 [102], 8.0 [62], 1.7 [39], rIGr 10 [100] a3b2 rSi 1.3 [21] a6b2*
0.35 [64] rSYd 24 [62], 17 [62]a3b2* mS,SCm 1.4 [71] a6b2*
a6/a3b2b3 0.4 [39] mSd 2 [72] pSs 19 C [93], 12 P [93] a6b2, b3, or b4
ha6/a4b4 (HEK293) 24 [104] cCGr 33 [77] a3b2b4a5 cRi 66 [40] a6b4*
a7  100 [37] rHc < 150 [76] a3b2b4* mBm 2.7 [64] a3b2*
a4b2 430 [39] rCCr 35 [105] a3b2*
bCCc 710 [103] a3b4(a5)*
2308 A. Nicke et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Table 1. (Continued).
a-Conotoxin Sequence
Functional Data
Binding Data (n
) on recombinant nAChRs
) in native tissues and
suggested native AChRs targeted
1.9 [64] rSs K
0.63 [66], 0.83 [66], NA a3/a6b2b3*
pSs K
0.93 [67], C 0.92 [67] P a6b2(b3)
mSCm K
4.9 [64]
GCCSYPPCFATNPD-C a3b4 750 [41], 966 [61], K
500 [41] rIGr 1.2 [78]
a7 10 000 [41] cCGr 350 [77] a3b4a5*
RHc  2200 [76] a3b2b4*
rCCr 105 [105] a3b4*
AuIB (ribbon)
GCCSYPPCFATNPD-C a3b4 27.500 [61] rIGr 0.1 [78]
ha3b4 755 [38]
ha4b2 309 [38]
a3b2 3.1 [80]
a4b2 152 [80]
GCCSDPRCNYDHPEIC bCCc 1000–3000 [81] a3b4* bCCc 2.3 and 3700 (2 sites) [81] a3b4*
RDPCCSNPVCTVHNPQIC a6/a3b2 0.69 [39]
a6/a3b2b3 0.95 [39]
ha6/a3b2b3 1.72 [39]
a3b2 74.2 [39]
a6/a3b4 30.5 [39]
ha6/a3b4 12.6 [39]
a6b4 33.5 [39]
a3b4 518 [39]
a3b2 0.3 [83]
Sequence disulfide connectivity: underlined-underlined and bold-bold.
Unless otherwise indicated, data are from rat subunits expressed in Xenopus oocytes (h, human; c, chick subunits).
otherwise indicated, K
values for inhibition of epibatidine binding are shown; B, inhibition of a-BTX binding.
Indicates cases where K
values were obtained from oocyte-expressed receptors.
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2309
Identification and characterization
of neuronally active a-conotoxins
Assay-based and cDNA-based strategies
The first a-conotoxins were identified using bioassays such
as intraperitoneal (neuromuscular nAChRs) or intracranial
(neuronal nAChRs) injections into mice [32]. Identification
of a-conotoxins with selectivity for distinct neuronal
nAChR subtypes required more specific test systems such
as characterized native tissues or recombinant nAChRs.
Due to its high efficiency in protein expression, the
apparent absence of endogenous nAChR subunits, the
comparable ease of producing subunit combinations and
its suitability for electrophysiological measurements, the
Xenopus oocyte expression system is ideally suited to study
nAChRs. However, the functional properties of nAChRs
expressed in oocytes and mammalian cell lines have been
reported to differ [43]. A distinct membrane lipid compo-
sition and differences in maturation and folding events, or
of post-translational processing in oocytes may account for
the differences observed. But also nAChRs expressed in
mammalian cells have been reported to differ from
the assumed native receptors [20]. This might reflect the
presence of more complex subunit combinations than the
simple pairwise combinations generally studied in hetero-
logous expression systems. Still to be identified endogenous
subunits or splice variants may also participate in the
formation of native or expressed receptors, and interactions
with other membrane proteins, adapter proteins or cyto-
skeletal elements might modulate the nAChR properties as
seen for other receptors. Such proteins might be absent or
not sufficiently expressed in certain expression systems. In
cells of non-neuronal origin, specific neuronal proteins
required for nAChR folding might either be absent or not
synthesized in amounts sufficient for effective processing of
the highly overexpressed nAChR polypeptides. Indeed,
assembly and/or membrane expression of certain nAChR
subtypes, notably a7 homomeric nAChR, is notoriously
difficult in non-neuronal mammalian cells [44].
Because the signal sequence, the intron immediately
preceding the toxin sequence and the 3¢ untranslated region
of the a-conotoxins are highly conserved, new conotoxin
sequences can be identified by PCR amplification of cDNA
from venom duct or genomic DNA from other cone snail
tissues. The analysis of the DNA of different Conus species
has already revealed a large number of a-conotoxin
sequences [45] and the identification of further specific
nAChR ligands is likely. The advantage of a molecular
biology approach compared to conventional venom frac-
tionation is that only small amounts of tissue are required.
In addition, conotoxins with low expression levels that
would escape detection in functional assays can be identi-
fied. Because the most prevalent activity found in functional
assays is at a7 and/or a3b2 nAChRs (A. Nicke, unpublished
observation), these receptors probably resemble a prefer-
ential target for prey capture. However, the genetic
information for ÔunderdevelopedÕ a-conotoxins targeting
other nAChR subtypes might still be present in the snails
and could supply novel ligands for mammalian nAChRs
(for evolution, diversity and biosynthesis of a-conotoxins
see [30,31]).
ImI and ImII
The first a-conotoxin showing activity at neuronal nAChRs
was the a4/3-conotoxin ImI from Conus imperalis. It was
originally discovered in a mammalian bioassay where it
caused seizures in mice and rats upon intracranial injection,
but in contrast to muscle selective a-conotoxins and the
snake toxin a-BTX, had no paralytic effect upon intraperi-
toneal injections [46]. However, ImI was active on neuro-
muscular preparations from frog [46] and had affinity for
the muscle nAChR from chick [47], suggesting that species
differences can influence selectivity. Pereira et al.[48]
suggested that ImI acts as an open channel blocker at
receptors and muscle nAChRs from the rat. Interest-
ingly, even in extremely divergent organisms such as
molluscs (Aplysia) [49] and insects (Locusta migratoria)
[50] ImI showed selectivity for fast inactivating neuronal
nAChRs. Characterization on Xenopus laevis oocyte-
expressed rat nAChR subtypes revealed that ImI is selective
for the mammalian a7anda9 subtypes [34] (Table 1 shows
values). In several subsequent studies, ImI was used to
identify native a7* receptors for example in rat hippocampal
slices [48] and rat striatal slices [51]. These studies revealed
potencies for ImI that are comparable to those found at
oocyte-expressed rat a7 receptors, suggesting that the
binding site of the native a7* channel resembles that of
the heterologously expressed a7 channel. Thus ImI repre-
sents a useful tool for the characterization of native a7*
receptors. ImI was also used to define a functional a7
nAChR component in bovine chromaffin cells (IC
300 n
[23]), but in another study on these cells, ImI
inhibited an a-BTX insensitive secretory response, attrib-
uted to an a3b4* nAChR, with an IC
of 2.5 l
[52]. In the
latter study, an a7 response was not detected, probably due
to the experimental conditions which would have allowed
desensitization of the receptor due to slow solution
exchange. These conflicting results indicate that ImI is less
selective in the bovine preparation, and species differences
between rat and bovine nAChRs may account for these
inconsistencies. Hence the exquisite specificity of conotoxins
may limit extrapolations between species. Alternatively, a
heteromeric a7-containing receptor with distinct pharma-
cological properties might be present in bovine chromaffin
cells as a-BTX also showed an unusual low activity
(300 n
) in these cells as compared to oocyte-expressed
receptors (1.6 n
) [23]. Recently, a second peptide with a4/
3-conotoxin structure, ImII, was discovered by PCR
amplification of a-conotoxin genes from C. imperalis
genomic DNA and cDNA [35]. Despite having 75% amino
acid identity and showing similar activity in bioassays and
on oocyte-expressed a7 receptors, ImI and ImII appear to
target different binding sites of the homomeric a7nAChR
or perhaps different microdomains within the same binding
site [35]. The proline residue in position 6, which is
conserved in all other a-conotoxins, appears to be the
major determinant of the abilities of ImI and ImII to
interact with a-BTX binding
PnIA and PnIB
PnIA and PnIB from Conus pennaceus
were the first a4/7-
conotoxins identified. They differ by only two amino acids
2310 A. Nicke et al.(Eur. J. Biochem. 271) Ó FEBS 2004
and were discovered in a bioassay probing the paralysing
activity of venom fractions on molluscs [54]. Further
characterization on Aplysia neurons confirmed that they
targeted neuronal a-BTX-insensitive nAChRs, albeit with
comparably low (micromolar) affinity. As the bioassays on
fish and insects as well as intracranial injections into rats
showed no detectable effects, PnIA and PnIB were origin-
ally reported to be mollusc-specific. A subsequent study
on oocyte-expressed nAChR subtypes, however, revealed
nanomolar activities on the a7anda3b2 nAChRs (Table 1,
Table 2), with PnIA showing a preference for the a3b2
subtype and PnIB a preference for the a7 subtype [55].
Interestingly, replacement of the alanine residue in position
10 of PnIA with a leucine residue, [A10L]PnIA, the
corresponding amino acid in PnIB, not only switched
subtype selectivity, but produced the most potent
a-conotoxin on oocyte-expressed a7nAChRs(compare
PnIA, PnIB and their analogues [A10L]PnIA and
[N11S]PnIA were also investigated in a patch clamp study
on dissociated rat intracardiac ganglion neurons [56] and for
their ability to inhibit catecholamine release from bovine
chromaffin cells [57]. In intracardiac neurons, the A10L
mutation in PnIA again caused an increase in potency as
well as a shift in selectivity: while PnIA inhibited an a-BTX-
sensitive as well as an a-BTX-insensitive component of an
ACh-induced current, [A10L]PnIA selectively inhibited the
a-BTX-sensitive component assumed to originate from an
a7* nAChR. However, in this preparation IC
values for
a-BTX and [A10L]PnIA were at least one order of
magnitude lower than those found in oocyte-expressed a7
receptors (Table 1), suggesting that the a7* receptors in
intracardiac ganglion neurons are not homomers, or that
the heterologously expressed a7 receptor differs structurally
from the native form. Neither PnIA nor [N11S]PnIA
showed significant activity on bovine chromaffin cells [57]
whereas PnIB and [A10L]PnIA inhibited catecholamine
release from these cells with IC
values of 0.7 and 2 l
respectively. These comparatively high values indicate that
nAChRs other than a7* and a3b2*, most probably an
a3b4* subtype, were targeted in this preparation.
Mutagenesis studies on PnIA and PnIB have provided
useful information on the binding mode of a-conotoxins
[53,58] and the activation states of the nAChR. At the
a7[L247] nAChR (a single point mutant that does not show
desensitization), [A10L]PnIA but not PnIA, surprisingly
acts as an agonist [59]. Thus, PnIA and [A10L]PnIA seem to
be selective for different states of the receptor and it was
hypothesized that PnIA stabilizes the nonconducting resting
state, whereas [A10L]PnIA stabilizes a desensitized state
which, in the case of the a7[L247] mutant, is conducting.
The a4/7-conotoxin EpI from Conus episcopatus
was first
identified in an analytical approach using HPLC in
combination with mass spectrometry [60]. After sequencing
and synthesis, the activities of EpI and its nonsulfated
analogue [Y15]EpI
on nAChRs were tested in three native
nAChR models, one muscular and two neuronal prepara-
tions. In concentrations up to 10 n
neither peptide
inhibited muscle twitches in a rat diaphragm preparation.
However, both peptides inhibited nicotine-induced cate-
cholamine release in bovine adrenal chromaffin cells, which
contain predominantly a3b4 nAChRs. The peptides also
inhibited ACh-evoked membrane currents in isolated neu-
rons from rat intracardiac ganglia, which are believed to
arise primarily from a3b2anda3b4 nAChRs. Activity on
a7 nAChRs was excluded for two reasons: (a) EpI and
[Y15]EpI failed to block an a-BTX-sensitive current in
intracardiac ganglia neurons and (b) EpI was able to inhibit
both adrenaline and noradrenaline release in bovine
chromaffin cells, whereas only adrenaline releasing cells
are proposed to contain a7 nAChRs. Surprisingly, at
oocyte-expressed rat nAChRs, EpI was found to be a7
selective and did not show significant activity at a3b2and
a3b4 subunit combinations [61].
MII and AuIB
The a4/7-conotoxin MII from Conus magus
and the a4/6-
conotoxin AuIB from Conus aulicus were discovered in an
approach aimed to directly identify selective ligands for the
a3b2anda3b4 nAChR subunit interfaces. Both toxins were
isolated by assay-directed fractionation of venoms using
oocyte-expressed rat nAChRs [37,41].
a-Conotoxin MII was shown to have low nano-
molar affinity (EC
0.5–8 n
) and high selectivity for
Table 2. Comparison of a common motif in loop II of a4/7-conotoxins and their activity on oocyte-expressed a7anda3b2 nAChRs. The length/
hydrophobicity of the amino acid that corresponds to position 10 (bold) in PnIA correlates with the a3b2overa7 selectivity. Italic letters in the
sequence show residues where variations in the AXNNP sequence occur. O, hydroxyproline. Note that GIC is included tentatively as its activity on
the a7 nAChR is not published. The corresponding residues of the consensus sequence are 8–13 in PnIA.
Ratio IC
a3b2/a7 Ref.
Side chain
in position 10
GIC 1.1 – – [38]
AnIB 0.3 76 0.004 [83]
PnIA 9.6 252 0.04 [55] CAANNP
[N11S]PnIA 241 1710 0.14 [55] CAASNP
[R12A]GID 10 48 0.2 [80] CAVNNO –CH–(CH
GID 3.1 4.5 0.7 [80] CRVNNO
[A10L]PnIA 99 12.6 7.9 [55] CALNNP –CH
PnIB 1970 61 32 [55] CALSNP
EpI >4000 30 >100 [61] CNMNNP –CH
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2311
oocyte-expressed a3b2 nAChRs [37,62]. In mammalian
striatal [62] and avian ciliary ganglion [63] preparations, it
showed potent and selective inhibition of nAChR subpop-
ulations. Among the a-conotoxins, MII has found the
widest application in the characterization of a range of
native nAChRs (Table 1). Because of its relatively slow
dissociation kinetics, MII is suitable as a radioligand. An
N-terminal tyrosine was added to the sequence to provide
an iodination site that did not decrease toxin potency [64].
I-labelled analogue of MII was used to visualize a
population of nAChRs that differed in pharmacology and
distribution from previously characterized nAChRs in the
brain [64] and has proven to be a powerful radioligand in
numerous binding and autoradiography studies [64–70].
Binding studies on the a6-rich chick retina [40] and
electrophysiological investigation of oocyte-expressed
human a6b2anda6b4 interface containing nAChRs and
chimeras [12], showed that MII also recognizes the a6
subunit, which is highly homologous to the a3 subunit,
particularly around its agonist binding site. Surprisingly,
subsequent studies on knockout mice revealed that most
I-labelled MII binding sites were conserved in a3
knockout mice [65], whereas high-affinity
I-labelled MII
binding sites completely disappeared in a6 knockout mice
[71]. The observation that a3b2 binding sites apparently are
not detected by MII argues against a role for a3inthe
formation of native MII binding sites, but may reflect the
scarcity of these sites in the investigated brain tissues and/or
the formation of low affinity binding sites that are not
detected by autoradiography. As expected, formation of
MII-sensitive receptors was strongly dependent on expres-
sion of the b2 subunit [72,73] but more surprisingly, also on
expression of b3 subunits [74] (see also Characterization of
nAChR subtypes in the striatum).
AuIB is the most potent of three highly homologous
a-conotoxins (AuIA, AuIB and AuIC) identified in C. auli-
culus and is the only a4/6-conotoxin described to date [41].
It blocks oocyte-expressed rat a3b4nAChRswitha
relatively low affinity (IC
value of 750 n
) and is at least
100 times less potent at other a/b combinations. However,
AuIB also showed significant activity (30–40% block
at 3 l
AuIB) at oocyte-expressed a7 receptors. AuIB
(1–5 l
) reduced nicotine stimulated noradrenaline release
from rat hippocampal synaptosomes but did not affect
dopamine release from striatal synaptosomes [41]. It was
subsequently used to characterize a3b4* nAChRs in rat
medial habenula neurons, the locus coerulus and chick
ciliary ganglion neurons, where similar potencies as in the
oocyte system were observed [75–77]. An exceptionally high
potency was found in isolated rat intracardiac ganglion
neurons, where an IC
value of 1.2 n
was obtained for
AuIB (discussed further in Correlation between native and
heterologously expressed nAChRs). Surprisingly, a disulfide
bond isomer was even 10-fold more potent than AuIB [78].
a-AuIB and a-MII were used in combination to identify
receptor populations sensitive to both toxins, presumably
a3b2b4* and a6/a3b2b4* nAChRs in canine intracardiac
ganglia, rat medial habenula neurons and in locus coerulus
neurons [75,76,79] (Fig. 2B). Interestingly, a (H12A)ana-
logue of MII, which was not active on the pairwise a3b2or
a3b4 combinations blocked nAChRs in rat medial habenula
neurons and oocyte-expressed a3b2b4nAChRs[75].An
explanation for this could be that the presence of two
different b subunits constrains one of the interfaces in such a
way that it can accommodate the mutated peptide.
Two neuronally active a4/7-conotoxins, GIC and GID,
were identified in Conus geographus
by amplification from
genomic DNA and in an oocyte-based assay, respectively
[38,80]. This makes a total of six a-conotoxins, four muscle
active and two neuronally active forms, that have been
isolated from this single species so far. GIC was character-
ized on oocyte-expressed human nAChR subunit combina-
tions and seems to have a similar selectivity and activity as
MII on the rat a3b2 combination [38]. However, its activity
on a6-containing receptors and on a7 receptors has not yet
been reported. GID differs from other neuronally active
a-conotoxins in having a four amino acid N-terminal tail
[80]. It inhibits a7anda3b2 nicotinic nAChRs with similar
low nanomolar potencies and also potently blocks the a4b2
subtype (Table 1). This wide spectrum of activities makes it
less useful as a tool for pharmacological characterization of
native receptors. Nevertheless, GID represents a useful
template from which to define determinants of subtype
selectivity [53].
PCR amplification of Conus victoriae
venom duct cDNA
led to the discovery of the peptide sequence of Vc1.1 [81].
The synthetic peptide was not active on neuromuscular
nAChRs. Its competitive antagonistic activity on neuronal
nAChRs was tested on bovine chromaffin cells where it
inhibited nicotine-induced catecholamine release with an
value of 1–3 l
(Table 1). In competition binding
experiments on chromaffin cell membranes Vc1.1 showed
 1000-fold higher affinity (K
of 2.3 n
) for one of two
nAChR populations labelled by the relatively nonselective
nAChR ligand [
H]epibatidine. It was suggested that
Vc1.1acts on a3b4* receptors containing a5 and/or a7
subunits (Table 1). Interestingly, Vc1.1 was able to inhibit
in vivo a vascular response to pain and was effective in
alleviating chronic pain and accelerating functional recovery
in an animal model of neuropathy. These data are in
agreement with an important role of nAChRs in pain
perception, although typically nicotinic agonists, rather
than antagonists, have antinociceptive effects [82]. Never-
theless, a-conotoxins may represent valuable tools to
investigate the mechanisms of nicotinergic pain transmis-
sion and could serve as templates for the development of
selective pain blockers.
PIA from Conus purpurascens
was again identified in a
cloning approach making use of the high conservation of
the 3¢ untranslated region and the intron preceding the
sequence of the a-prepropeptide [39]. The peptide was
characterized on oocyte-expressed nAChRs and found to be
the first a-conotoxin that discriminates between the closely
related a3anda6 subunits. Because the a6 subunit did not
form functional nAChRs, either in combination with b2or
2312 A. Nicke et al.(Eur. J. Biochem. 271) Ó FEBS 2004
with b2plusb3 subunits, and was not reliably expressed in
combination with b4 subunits, an a6/a3 chimera consisting
of the extracellular ligand-binding domain of the a6 subunit
and the transmembrane and intracellular domains of the a3
subunit was used in this study. PIA selectively blocks rat
and human nAChRs that contain a6b2 interfaces (with
potencies of about 1 n
) and with 10–30-fold lower potency
a6b4 interfaces. The a3 containing combinations, rat a3b2
and a3b4, were blocked with about 100- and two-fold
lower potency, respectively. In addition to the differences
in potency, a3b2anda6b2 binding sites could also be
distinguished by the different dissociation rates of PIA:
while recovery from block for receptors with an a6b2
interface took about 10 min, the block of a3b2nAChRs
was reversed within one minute. Interestingly, the dissoci-
ation rate from both a3- and a6-containing receptors
was greatly slowed when the b2 subunit was replaced by the
b4 subunit.
The most recent addition to the fast growing list of
neuronally active conotoxins is AnIB from Conus anemone
which was identified through a combined approach of LC/
MS analysis and assay-directed fractionation [83]. It has
subnanomolar potency at the a3b2 nAChR and is 200-fold
less active on the a7 nAChR (Table 1). AnIB is sulfated at
tyrosine 16 and has, like most a-conotoxins, an amidated
C-terminus. To investigate the influence of these postrans-
lational modifications on potency and subtype selectivity, its
nonamidated and nonsulfated analogues were synthesized
and characterized on oocyte-expressed nAChRs. Removal
of the modifications increased the selectivity for a3b2
nAChRs. The two N-terminal glycine residues were dem-
onstrated to be important for the binding affinity.
Correlating the sequence and subtype
The Xenopus oocyte expression system has been widely used
to characterize neuronally active a-conotoxins. Together
with the three dimensional structures that are available
for most a-conotoxins [53], this provides the necessary
structural basis to study structure-activity relationships.
a-Conotoxins with nanomolar potency for only one inter-
face or a wider range of activities have been identified.
Although the less selective peptides might be less useful
as pharmacological tools, they provide information for
structure-activity studies. Comparison of their primary
structures with those of more ÔspecialisedÕ a-conotoxins
can reveal first clues for critical determinants of subtype
selectivity, and ultimately may lead to the engineering of
a-conotoxins with tailored selectivity.
Information on the binding mode of neuronally active
a-conotoxins and the factors that determine subtype
selectivity is currently emerging [6,53]. Through double-
cycle mutagenesis and binding studies, different binding
modes were found for ImI, ImII and PnIB [35,58,84,85],
suggesting that various neuronally active a-conotoxins with
different attachment points might have evolved to target
different microdomains that overlap around the conserved
ACh binding site of nAChRs. Thus, it might be useful to
subgroup the neuronally active a-conotoxins based on their
subunit specificity and sequence similarity in order to
compare structures that are likely to have similar binding
modes. One such subgroup might be represented by
a-conotoxins with a common NNP/O/Q motif and activity
at a7 and/or a3b2 nAChRs (Table 2). Substitution experi-
ments [53,55,56] and sequence comparison of these pep-
tides implicate increasing length of the aliphatatic sidechain
at position 10 (or 13 for GID) as an important determinant
of selectivity for a7vs.a3b2 nAChR (Table 2).
Other groups with similar sequences and selectivities for
recombinant receptors could be represented by PIA and
MII (SNPV motif in the first loop and nanomolar activity
on a3/a6 containing nAChRs) and EpI and ImI (SDPR
motif in the first loop and nanomolar activity on a7
nAChRs). It remains to be determined if these a-conotoxins
share a common binding mode.
Use of selective a-conotoxins to characterize
neuronal nAChRs in native systems
Characterization of nAChR subtypes in the striatum
In the central nervous system, distinct subtypes of pre-
synaptic nAChRs appear to modulate the release of different
neurotransmitters, e.g. noradrenaline in the hippocampus
or dopamine in the striatum [86]. In the striatum, a dense
local innervation from cholinergic interneurones closely
interacts with dopaminergic projections, principally from
the substantia nigra (nigrostriatal pathway), and also from
the ventral tegmental area (mesolimbic pathway) (Fig. 3A).
Dopaminergic mechanisms in the dorsal and ventral
striatum are involved in motor coordination, learning,
psychotic and addictive behaviour and play a role in
Tourette’s syndrome, nicotine addiction and Parkinson’s
disease. Thus, nAChRs modulating the dopamine release
gain increasing interest as drug targets, and identification of
the nAChR subtypes involved is crucial for the development
of pharmacological agents. The dopaminergic neurons
express both somatodendritic (subtantia nigra, ventral
tegmental area) and presynaptic nAChRs (striatum, nucleus
(Fig. 3A).
As mentioned above, the determination of the subunit
composition of the nAChRs involved has been hindered by
the lack of selective ligands and imperfect correlations
between the characteristics of native and heterologously
expressed nAChRs. For presynaptic nAChRs, the deter-
mination of subunit composition has been particularly
challenging because of the impossibility of direct electro-
physiological recordings and their incomplete pharmacolo-
gical characterization. Furthermore, the distance of the
projection areas from the cell bodies and the indistinct
correlation between subunit mRNA levels and functional
surface nAChRs hampers the interpretation of studies at the
transcriptional level. a-Conotoxin MII has found its widest
application and served as an important tool in the elucida-
tion of nAChR subtypes and function in the dopaminergic
system. The following will focus on the investigation of
presynaptic nAChRs on dopaminergic nerve terminals in
the striatum.
In situ hybridization and single-cell PCR studies on
midbrain dopaminergic neurons revealed a3, a4, a5, a6, a7,
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2313
b2, b3 and to a minor extent b4 subunits [9,86,87], as
possible candidates. Initial pharmacological studies using
the agonists nicotine and cytisine and the a3 selective
antagonist n-BTX in striatal synaptosome preparations
suggested an a4b2* nAChR with a possible involvement of
the a3 subunit [86]. Subsequent studies [62,88] showed that
34–50% of agonist-evoked dopamine release in rat striatal
synaptosomes could be blocked by MII, indicating the
presence of at least two receptor subtypes, one of them
having at least one a3b2 interface (Fig. 3B). The contribu-
tion of a presynaptic a7 receptor was excluded by the
absence of ImI activity [88]. A smaller fraction of the
response (21–29%) was blocked by MII in slice prepara-
tions, indicating an additional indirect mechanism via an
MII-insensitive receptor [62] (Fig. 3C). However, similar
values (24.3 and 17.3 n
in synaptosomes and slices,
respectively) as in oocyte-expressed a3b2 receptors (8 n
determined in the same study) were obtained [62]. A further
study using a new agonist (UB-165) in combination with
MII concluded that the MII-insensitive nAChR was an
a4b2* subtype [89]. The finding that MII binds with high
affinity a6-containing nAChRs from chick retina and
blocks heterologously expressed human a6-containing
receptors installed the a6 subunit as another possible
subunit conferring MII-sensitivity [12,40].
changes in individual
rat striatal synaptosomes by laser scanning confocal micro-
scopy and immunocytochemical studies, Nayak et al.[90]
hypothesized that a4anda3(ora6) subunits are present on
separate nerve terminals in the striatum, and that a
mecamylamine- and MII-sensitive population of a3(or
a6) subunits in combination with b2 and possibly b3
subunits exists beside a mecamylamine-insensitive,
a4-containing subtype that includes b2 subunits. The
finding that
I-labelled MII binding is absent in basal
ganglia of a6 knockout mice [71] but basically unchanged in
a3 knockout mice [65] finally confirmed the involvement of
the a6 subunit rather than the a3 subunit in MII-sensitive
nAChRs. The presence of two b2 containing populations is
supported by the fact that agonist-stimulated dopamine
release from striatal synaptosomes is abolished in b2 null
mutants [72]. Immunoprecipitation and ligand binding
studies [21] confirmed that a4b2* (with possible inclusion
of a5 subunits) and a6b2* (with possible inclusion of a4
and b3 subunits) are the main nAChR populations present
on dopaminergic terminals in rat striatum.
In recent studies on a4, a6, a4a6andb2 knockout mice
[91,92], MII and
I-labelled MII were used in autoradio-
graphy and binding studies on immunoimmobilized recep-
tors as well as in functional studies in synaptosomal
preparations and recordings from dopaminergic neurons.
These extensive studies further established that (non-
a6)a4b2* nAChRs represent the major subtype on the
neuronal soma whereas a combination of a6b2* and a4b2*
nAChRs modulates dopamine release at the nerve termi-
nals. Deletion of the b3 gene [74] strongly reduced MII-
sensitive dopamine release and almost completely abolished
I-labelled MII binding in the nerve terminals, indicating
Fig. 3. Presynaptic nAChR modulating dopamine release in the rat striatum. (A) Nicotine acts at somatodendritic nAChR in the substantia nigra
pars compacta and at presynaptic nAChR in the striatum. (B) a-Conotoxin MII was one of the first antagonists that differentiated pharmaco-
logically between receptor populations in the striatum. The [
H]dopamine release from rat striatal synaptosomes, evoked by the nicotinic agonist
anatoxin-a, is almost completely blocked in the presence of mecamylamine. Maximally effective concentrations of a-conotoxin MII (112 n
produced only about 50% inhibition, indicative of nAChR heterogeneity [62]. (C) Model showing current views for the localization and com-
position of nAChR subtypes, with at least two heteromeric nAChRs on dopaminergic terminals. This model is based on the results from a variety of
binding studies using MII and the radioligand
I-labelled MII on knockout mice [74,92] and immunoprecipitation studies using rat synaptosomes
[21], as well as pharmacological studies such as those shown in (B). In slices, an a7* nAChR on adjacent glutamate terminals was found to indirectly
influence dopamine release via the release of glutamate [51].
2314 A. Nicke et al.(Eur. J. Biochem. 271) Ó FEBS 2004
that the b3 subunit is an essential element of these a6b2*
nAChRs (Figs 2C and 3C).
Quantitative autoradiography and competition binding
studies on monkeys [67,93,94] and rodents [69,70] revealed
that MII/
I-labelled MII binding to high affinity sites in
the striatum was selectively reduced after admistration of
the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine, which causes selective damage of dopaminergic
neurons in the nigrostriatal system and represents a model
for Parkinsonism. In contrast, multiple receptor popula-
tions were decreased in the substantia nigra [68]. The loss of
I]MII binding sites closely corresponded to the reduction
in nicotine-evoked dopamine release [69] but had a poorer
correlation with the release of other neurotransmitters. This
is in agreement with a localization of a6 receptors on
dopaminergic nerve terminals and provides further evidence
that they play a key role in the pathogenesis of Parkinson’s
The newly discovered a6 selective PIA provides another
potentially useful tool for the specific localization and
further characterization of these important subtypes.
Characterization of nAChR subtypes in the avian ciliary
Another example where a-conotoxins have proved useful
in investigating the subunit composition of nAChRs in
native systems, are parasympathetic motoneurons of the
chick ciliary ganglion. These ganglia contain two kinds of
neurons, choroid and ciliary neurons, which innervate
vascular smooth muscles in the choroid layer and striated
muscle in the iris and ciliary body, respectively. Both types
of neurons are cholinergic and receive cholinergic input
from preganglionic terminals. The large size of these calyx-
like synapses makes the chick ciliary ganglion an attractive
model for studying synaptic mechanisms. At least five
different nAChR genes, a3, a5, a7, b2andb4, are
expressed in these neurons [95]. Studies using a monoclonal
antibody (mAb 35) that recognizes the a3anda5 subunits
and a-BTX distinguished two major classes of nAChRs:
a major population of a-BTX binding a7* nAChRs which
is mainly localized perisynaptically, and a less abundant
population of a3* nAChRs which contain a5andb4
subunits and in some cases b2 subunits, and is localized at
postsynaptic densities [18,96]. Functionally, a rapidly
decaying a-BTX-sensitive and a slowly decaying a-BTX-
resistant response could be separated that both contributed
to synaptic transmission [97]. This broad distinction of two
major nAChR types was pharmacologically confirmed by
the use of a-BTXandMIIwhichwereshowntoselectively
inhibit a rapidly decaying population (suggested a7*
nAChRs), and a slowly decaying population (suggested
a3b2* nAChRs that also contain a5andb4 subunits),
respectively [63]. Moreover, this study showed that a
combination of a-BTX and 50 n
MII abolished nearly all
evoked current and confirmed the contribution of a7*
receptors to synaptic transmission. A recent study based on
single channel recordings and the use of a-conotoxins MII
and AuIB further dissected and correlated the combinato-
rial and functional heterogeneity of the slowly decaying
population [77]. In this study, two long events of 25 pS
40 pS conductance could be resolved that were unaffected
by a-BTX. Both events were inhibited by AuIB but only
the 40 pS event was sensitive to MII. It was concluded that
the 25 pS event arises from the numerically dominant
a3b4a5 subtype whereas the 40 pS events arise from a
minor a3b2b4a5 subtype (Fig. 2C). Because calculations
based on the open probability and conductivity indicated a
far greater contribution (92%) of the 40 pS event to the
a3*-mediated membrane current than the 20 pS event
(8%), it was concluded that the b2 subunit strongly
enhances the function of a3* nAChR.
Correlation between native and
heterologously expressed nAChRs
The examples presented above clearly demonstrate the
usefulness of a-conotoxins in the determination of the
structure and function of native nAChRs, and indicate
that selectivities and potencies found on oocyte-expressed
nAChRs can be extrapolated to native systems. There are,
however, also native systems where the activity and
selectivity of a-conotoxins appear to deviate from the
data obtained with oocyte-expressed nAChRs. One such
example is intracardiac ganglia. These ganglia receive
cholinergic innervation that is predominantly mediated by
nAChRs, and coordinate efferent parasympathetic input
to the heart. A single cell PCR study on rat intracardiac
ganglia neurons [98] revealed the presence of eight
different subunits (a2–a5, a7andb2–b4) that were
heterogeneously expressed in nine examined cells. Overall,
the a3, a7, b2andb4 subunits appeared to dominate. The
presence of a3, a7, b2andb4 subunits was indicated by
immunohistochemistry in canine intracardiac ganglia [79].
In in vivo experiments on dogs in which the intracardiac
ganglia were locally perfused with antagonists, an attenu-
ation of sinus cycle length could be found with a-BTX,
AuIB [79]. Whereas
the effects of MII and a-BTX were additive, the attenu-
ation by AuIB was not observed in animals pretreated
with MII. Therefore, it was concluded that the ganglionic
transmission is mediated primarily by a3b2* nAChRs and,
to a smaller extent, by a7* nAChRs. Because of the
nonadditive effect of AuIB, inclusion of b4 subunits in
some a3b2* nAChRs rather than the presence of a distinct
a3b4 nAChR population was suggested. However, given
the experiences with bovine chromaffin cells discussed
above, one needs to be cautious in extrapolating between
species on the basis of an a-conotoxin specificity defined in
one species only.
In dissociated neurons of rat parasympathic intracardiac
ganglia, AuIB was shown to block a nAChR population
with an IC
value of 1.2 n
[78] which is about 500-fold to
1000-fold lower than the IC
values of 750 n
[41] and
966 n
[61] reported for rat a3b4 nAChR expressed in
oocytes. Surprisingly, ribbon AuIB, an isomer in which the
disulfide connectivity was 1–2 and 3–4 instead of 1–3 and
2–4, was even more active (IC
of 0.1 n
than native AuIB [78].
This was explained by a higher structural flexibility of the
molecule possibly allowing a more complementary fit in the
binding site. In contrast, the IC
value of ribbon AuIB on
oocyte expressed a3b4 nAChRs was 27.5 l
, about 30-fold
higher than that for native AuIB and even 3 · 10
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2315
higher than that determined on native receptors. This result
suggests that either an as yet undefined nAChR subunit
combination with very high affinity for AuIB and ribbon
AuIB is present in intracardiac ganglia, or that the a3b4*
receptors in intracardiac ganglia form a substantially
different interface than the oocyte expressed a3b4nAChRs.
Alternatively, these receptors might be modulated by a
factor or subunit that greatly enhances their AuIB
Another paradoxical result was obtained with EpI. This
a-conotoxin was originally characterized on rat intracardiac
ganglia neurons and bovine chromaffin cells and assumed to
be selective for a3b2anda3b4 interfaces and unable to
inhibit an a-BTX-sensitive current attributed to an a7*
nAChR [60]. In contrast, EpI showed little activity on
oocyte-expressed a3b2ora3b4 combinations but blocked
oocyte-expressed a7 receptors with an IC
value of 30 n
[61]. Even 100 n
EpI that caused a 75% block of the a7
nAChR in the oocyte system, was without effect on a-BTX-
sensitive receptor in intracardiac ganglia [60]. It must be
mentioned, however, that the a-BTX-sensitive current in
intracardiac ganglia decays much more slowly than the
ÔclassicalÕ a7* current recorded from, for example, chicken
ciliary ganglia, PC12 cells and hippocampus. Moreover, the
a-BTX block is rapidly reversible in intracardiac ganglia
while it is long lasting in the other models [99]. This suggests
that not yet identified nAChR subunits or splice variants
participate in the formation of EpI-resistant a7 receptors, or
that the functional properties of the nAChR are modified in
a cell-specific way. The fact that an anti-a7 mAb selectively
inhibited the a-BTX-sensitive current in intracardiac ganglia
argues against the possibility that a subunit other than a7
accounts for the a-BTX sensitive current in intracardiac
ganglia [99].
a-Conotoxins provide a degree of specificity that is superior
to most other nAChR ligands and have proven to be
valuable tools to characterize nAChR subtypes in native
tissues and to investigate their physiological role. In
particular, the use of radiolabelled a-conotoxin MII has
enabled the localization of distinct nAChR subtypes in the
brain and helped to decipher their composition, which was
found to be much more complex than the pairwise
combinations generally studied in heterologous expression
It has to be considered however, that the high specificity
of conotoxins might limit the extrapolation between
species. Also, the pharmacology of native nAChRs might
be modulated by factors other than subunit stoichiometry
and assembly. Understanding the cause of differences
observed between native and recombinant receptors is
crucial for future studies that use heterologous expression
systems to identify selective ligands for the characteriza-
tion of native receptors. A thorough comparison and
characterization of various a-conotoxins in different
systems is therefore essential to prove the utility of the
oocyte system and the validity of correlations based on the
characterization of oocyte-expressed subunit combina-
tions. Nevertheless, the activity data obtained from
oocyte-expressed receptors provide an extensive and
valuable basis for structure-activity studies. The high
selectivity of the a-conotoxins, together with the possibility
of obtaining detailed information on their three dimen-
sional structures and the relative ease of synthesis, makes
them particularly useful templates for the design of
optimized synthetic peptides for the subtype characteriza-
tion of nAChRs.
A. N. was supported by an Emmy Noether research fellowship of the
Deutsche Forschungsgemeinschaft (NI 592/2-1). We thank Heinrich
Betz for his support and for critical reading of the manuscript.
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