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

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

(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 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].

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

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/d interface of the
mammalian muscle nAChR and is the only ligand

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
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.
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2307
Table 1.
21,21
21,21
Summary of neuronally active a-conotoxins and their
21,21
21,21
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

IC
50
(n
M
) in native tissues and
suggested native AChRs targeted
ImI
GCCSDPRCAWR C a7 220 [34], 100 [23], 191 [35], 1040 [106] fNJr 250–500 [46] rBm EC
50
(B) 1560 [35]
ha7 132 [85] rHr 86 [48] a7 ha7/5HT3 EC
50
(B) 407 [35]
a9 1800 [34] SCLC  10 [101] a7 ha7/5HT3 K
d
(B) 2380 [84], 4000 [107]
a3b4 no effect at 3–5l
M
[23,34] bCCc  300 [23] a7, 2500 [52] a3b4(a5)
ImII
ACCSDRRCRWR C a7 441 [35] not competitive with a-BTX [35]
PnIA
GCCSLPPCAANNPDYC a7 252 [55] rIGr 14 [56] a7* + additional component ha7/5HT3 K
d
(B) 61 200 [58]
ca7 349 [59]
ca7L247T 194 [59]
a3b2 9.6 [55]
[A10L]PnIA
GCCSLPPCALNNPDYC a7 13 [55] rIGr 1.4 [56] a7* ha7/5HT3 K

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]YGCCSNPVCHLEHSNLC a3b2 K
d
d
1.9 [64] rSs K
d
0.63 [66], 0.83 [66], NA a3/a6b2b3*

a3b2 74.2 [39]
a6/a3b4 30.5 [39]
ha6/a3b4 12.6 [39]
a6b4 33.5 [39]
a3b4 518 [39]
AnIB
GGCCSHPACAANNQDYC a7 76 [83]
a3b2 0.3 [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.
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

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-

inhibited an a-BTX insensitive secretory response, attrib-
uted to an a3b4* nAChR, with an IC
50
of 2.5 l
M
[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
M
) in these cells as compared to oocyte-expressed
receptors (1.6 n
M
) [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

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
50
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
50
values of 0.7 and 2 l
M
,
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

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
10
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
50
0.5–8 n
M
) 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.
a-Conotoxin
IC

[A10L]PnIA 99 12.6 7.9 [55] CALNNP –CH
2
–CH–(CH
3
)
2
PnIB 1970 61 32 [55] CALSNP
EpI >4000 30 >100 [61] CNMNNP –CH
2
–CH
2
–S–CH
3
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].
This
125
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].

M
) 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
M
AuIB) at oocyte-expressed a7 receptors. AuIB
(1–5 l
M
) 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
50
value of 1.2 n
M
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-

12
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
IC
50
value of 1–3 l
M
(Table 1). In competition binding
experiments on chromaffin cell membranes Vc1.1 showed
 1000-fold higher affinity (K
i
of 2.3 n
M
) for one of two
nAChR populations labelled by the relatively nonselective
nAChR ligand [
3
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-

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.
AnIB
The most recent addition to the fast growing list of
neuronally active conotoxins is AnIB from Conus anemone
14
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
selectivity
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-

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
accumbens)
15
(Fig. 3A).
As mentioned above, the determination of the subunit
composition of the nAChRs involved has been hindered by

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
IC
50
values (24.3 and 17.3 n
M
in synaptosomes and slices,
respectively) as in oocyte-expressed a3b2 receptors (8 n
M
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].
BasedonmeasurementsofCa
2+
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

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 [
3
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
M
)
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
125
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/
125
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
[
125

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
M
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
16
and
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

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
50
value of 1.2 n
M
[78] which is about 500-fold to
1000-fold lower than the IC
50
values of 750 n
M
[41] and
966 n
M
[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
50

nAChR [60]. In contrast, EpI showed little activity on
oocyte-expressed a3b2ora3b4 combinations but blocked
oocyte-expressed a7 receptors with an IC
50
value of 30 n
M
[61]. Even 100 n
M
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].
Conclusion
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

Deutsche Forschungsgemeinschaft (NI 592/2-1). We thank Heinrich
Betz for his support and for critical reading of the manuscript.
References
1. Langley, J.N. (1907) On the contraction of muscle, chiefly in
relation to the presence of receptive substances. Part 1. J. Physiol.
36, 347–384.
2. Sargent, P.B. (1993) The diversity of neuronal nicotinic acetyl-
choline receptors. Annu.Rev.Neurosci.16, 403–443.
3. McGehee, D.S. & Role, L.W. (1995) Physiological diversity of
nicotinic acetylcholine receptors expressed by vertebrate neurons.
Annu. Rev. Physiol. 57, 521–546.
4. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. (2003) Structure and
gating mechanism of the acetylcholine receptor pore. Nature 423,
949–955.
17
5. Brejc, K., van Dijk, W.J., Klaassen, R.V., Schuurmans, M., van
Der Oost, J., Smit, A.B. & Sixma, T.K. (2001) Crystal structure of
an ACh-binding protein reveals the ligand-binding domain of
nicotinic receptors. Nature 411, 269–276.
6. Dutertre,S.&Lewis,R.J.(2004)Computationalapproachesto
understand a-conotoxin interactions at neuronal nicotinic recep-
tors. Eur. J. Biochem. 271, 2327–2334.
18
7. Hucho, F., Tsetlin, V.I. & Machold, J. (1996) The emerging three-
dimensional structure of a receptor. The nicotinic acetylcholine
receptor. Eur. J. Biochem. 239, 539–557.
8. Karlin, A. (2002) Emerging structure of the nicotinic acetylcholine
receptors. Nature Rev. Neurosci. 3, 102–114.
9. Le Novere, N., Corringer, P.J. & Changeux, J.P. (2002) The
diversity of subunit composition in nAChRs: evolutionary origins,

structure. J. Biol. Chem. 266, 11192–111198.
17. Cooper, E., Couturier, S. & Ballivet, M. (1991) Pentameric
structure and subunit stoichiometry of a neuronal nicotinic
acetylcholine receptor. Nature 350, 235–238.
18. Conroy, W.G. & Berg, D.K. (1995) Neurons can maintain mul-
tiple classes of nicotinic acetylcholine receptors distinguished by
different subunit compositions. J. Biol. Chem. 270, 4424–4431.
19. Colquhoun, L.M. & Patrick, J.W. (1997) a3, b2, and b4form
heterotrimeric neuronal nicotinic acetylcholine receptors in Xen-
opus oocytes. J. Neurochem. 69, 2355–2362.
20.Sivilotti,L.G.,McNeil,D.K.,Lewis,T.M.,Nassar,M.A.,
Schoepfer, R. & Colquhoun, D. (1997) Recombinant nicotinic
receptors, expressed in Xenopus oocytes, do not resemble native rat
sympathetic ganglion receptors in single-channel behaviour.
J. Physiol. 500, 123–138.
21. Zoli, M., Moretti, M., Zanardi, A., McIntosh, J.M., Clementi, F.
& Gotti, C. (2002) Identification of the nicotinic receptor subtypes
expressed on dopaminergic terminals in the rat striatum. J. Neu-
rosci. 22, 8785–8789.
22. Lukas, R.J., Changeux, J.P., Le Novere, N., Albuquerque, E.X.,
Balfour, D.J., Berg, D.K., Bertrand, D., Chiappinelli, V.A.,
Clarke, P.B., Collins, A.C., Dani, J.A., Grady, S.R., Kellar, K.J.,
Lindstrom, J.M., Marks, M.J., Quik, M., Taylor, P.W. & Won-
nacott, S. (1999) International Union of Pharmacology. XX.
Current status of the nomenclature for nicotinic acetylcholine
receptors and their subunits. Pharmacol. Rev. 51, 397–401.
23. Lopez, M.G., Montiel, C., Herrero, C.J., Garcia-Palomero, E.,
Mayorgas, I., Hernandez-Guijo, J.M., Villarroya, M., Olivares,
R., Gandia, L., McIntosh, J.M., Olivera, B.M. & Garcia, A.G.
(1998) Unmasking the functions of the chromaffin cell a7 nicotinic

choline receptor antagonists as pharmacological tools and
potential drug leads. Curr.Med.Chem.8, 327–344.
33. Martinez, J.S., Olivera, B.M., Gray, W.R., Craig, A.G., Groebe,
D.R., Abramson, S.N. & McIntosh, J.M. (1995) a-Conotoxin EI,
a new nicotinic acetylcholine receptor antagonist with novel
selectivity. Biochemistry 34, 14519–14526.
34. Johnson, D.S., Martinez, J., Elgoyhen, A.B., Heinemann, S.F.
& McIntosh, J.M. (1995) a-Conotoxin ImI exhibits subtype-
specific nicotinic acetylcholine receptor blockade: preferential
inhibition of homomeric a7anda9 receptors. Mol. Pharmacol. 48,
194–199.
35. Ellison, M., McIntosh, J.M. & Olivera, B.M. (2003) a-Conotoxins
ImI and ImII. Similar a7 nicotinic receptor antagonists act at
different sites. J. Biol. Chem. 278, 757–764.
36. Arias, H.R. & Blanton, M.P. (2000) a-Conotoxins. Int. J. Bio-
chem. Cell Biol. 32, 1017–1028.
37. Cartier, G.E., Yoshikami, D., Gray, W.R., Luo, S., Olivera, B.M.
& McIntosh, J.M. (1996) A new a-conotoxin which targets a3b2
nicotinic acetylcholine receptors. J. Biol. Chem. 271, 7522–7528.
38. McIntosh, J.M., Dowell, C., Watkins, M., Garrett, J.E., Yoshi-
kami, D. & Olivera, B.M. (2002) a-Conotoxin GIC from Conus
geographus, a novel peptide antagonist of nicotinic acetylcholine
receptors. J. Biol. Chem. 277, 33610–33615.
39. Dowell, C., Olivera, B.M., Garrett, J.E., Staheli, S.T., Watkins,
M., Kuryatov, A., Yoshikami, D., Lindstrom, J.M. &
McIntosh, J.M. (2003) a-Conotoxin PIA is selective for a6
subunit-containing nicotinic acetylcholine receptors. J. Neurosci.
23, 8445–8452.
40. Vailati, S., Moretti, M., Balestra, B., McIntosh, M., Clementi, F.
& Gotti, C. (2000) b3 subunit is present in different nicotinic

49. Kehoe, J. & McIntosh, J.M. (1998) Two distinct nicotinic
receptors, one pharmacologically similar to the vertebrate
a7-containing receptor, mediate Cl currents in Aplysia neurons.
J. Neurosci. 18, 8198–8213.
50. van den Beukel, I., van Kleef, R.G., Zwart, R. & Oortgiesen, M.
(1998) Physostigmine and acetylcholine differentially activate
nicotinic receptor subpopulations in Locusta migratoria neurons.
Brain Res. 789, 263–273.
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2317
51.Kaiser,S.&Wonnacott,S.(2000)a-Bungarotoxin-sensitive
nicotinic receptors indirectly modulate [
3
H]dopamine release in
rat striatal slices via glutamate release. Mol. Pharmacol. 58, 312–
318.
52. Broxton, N.M., Down, J.G., Gehrmann, J., Alewood, P.F.,
Satchell, D.G. & Livett (1999) a-Conotoxin ImI inhibits the
a-bungarotoxin-resistant nicotinic response in bovine adrenal
chromaffin cells. J. Neurochem. 72, 1656–1662.
53. Millard, E.L., Daly, N.L. & Craik, D.J. (2004) Structure-activity
relationships of a-conotoxins targeting neuronal nicotinic acetyl-
choline receptors. Eur. J. Biochem. 271, 2320–2326.
20
54. Fainzilber,M.,Hasson,A.,Oren,R.,Burlingame,A.L.,Gordon,
D., Spira, M.E. & Zlotkin, E. (1994) New mollusc-specific
a-conotoxins block Aplysia neuronal acetylcholine receptors.
Biochemistry 33, 9523–9529.
55. Luo, S., Nguyen, T.A., Cartier, G.E., Olivera, B.M., Yoshikami,
D. & McIntosh, J.M. (1999) Single-residue alteration in a-cono-
toxin PnIA switches its nAChR subtype selectivity. Biochemistry

H]dopamine release from rat striatal
synaptosomes and slices. J. Neurochem. 70, 1069–1076.
63. Ullian, E.M., McIntosh, J.M. & Sargent, P.B. (1997) Rapid
synaptic transmission in the avian ciliary ganglion is mediated
by two distinct classes of nicotinic receptors. J. Neurosci. 17,
7210–7219.
64. Whiteaker, P., McIntosh, J.M., Luo, S., Collins, A.C. & Marks,
M.J. (2000)
125
I-a-conotoxin MII identifies a novel nicotinic
acetylcholine receptor population in mouse brain. Mol. Pharma-
col. 57, 913–925.
65. Whiteaker, P., Peterson, C.G., Xu, W., McIntosh, J.M., Paylor,
R., Beaudet, A.L., Collins, A.C. & Marks, M.J. (2002) Involve-
ment of the a3 subunit in central nicotinic binding populations.
J. Neurosci. 22, 2522–2529.
66. Mogg, A.J., Whiteaker, P., McIntosh, J.M., Marks, M., Collins,
A.C. & Wonnacott, S. (2002) Methyllycaconitine is a potent
antagonist of a-conotoxin-MII-sensitive presynaptic nicotinic
acetylcholine receptors in rat striatum. J. Pharmacol. Exp. Ther.
302, 197–204.
67. Quik, M., Polonskaya, Y., Kulak, J.M. & McIntosh, J.M. (2001)
Vulnerability of
125
I-a-conotoxin MII binding sites to nigrostriatal
damage in monkey. J. Neurosci. 21, 5494–5500.
68. Quik, M., Polonskaya, Y., McIntosh, J.M. & Kulak, J.M. (2002)
Differential nicotinic receptor expression in monkey basal ganglia:
effects of nigrostriatal damage. Neuroscience 112, 619–630.
69. Quik, M., Sum, J.D., Whiteaker, P., McCallum, S.E., Marks,

Stitzel, J.A., McIntosh, J.M., Boulter, J., Collins, A.C. & Heine-
mann, S.F. (2003) The b3 nicotinic receptor subunit: a component
of a-conotoxin MII-binding nicotinic acetylcholine receptors that
modulate dopamine release and related behaviors. J. Neurosci. 23,
11045–11053.
75. Quick, M.W., Ceballos, R.M., Kasten, M., McIntosh, J.M. &
Lester, R.A. (1999) a3b4 subunit-containing nicotinic receptors
dominate function in rat medial habenula neurons. Neurophar-
macology 38, 769–783.
76. Fu, Y., Matta, S.G., McIntosh, J.M. & Sharp, B.M. (1999)
Inhibition of nicotine-induced hippocampal norepinephrine
release in rats by a-conotoxins MII and AuIB microinjected into
the locus coeruleus. Neurosci. Lett. 266, 113–116.
77. Nai, Q., McIntosh, J.M. & Margiotta, J.F. (2003) Relating neu-
ronal nicotinic acetylcholine receptor subtypes defined by subunit
composition and channel function. Mol. Pharmacol. 63, 311–324.
78. Dutton, J.L., Bansal, P.S., Hogg, R.C., Adams, D.J., Alewood,
P.F. & Craik, D.J. (2002) A new level of conotoxin diversity, a
non-native disulfide bond connectivity in a-conotoxin AuIB
reduces structural definition but increases biological activity.
J. Biol. Chem. 277, 48849–48857.
79. Bibevski, S., Zhou, Y., McIntosh, J.M., Zigmond, R.E. & Dunlap,
M.E. (2000) Functional nicotinic acetylcholine receptors that
mediate ganglionic transmission in cardiac parasympathetic neu-
rons. J. Neurosci. 20, 5076–5082.
80. Nicke, A., Loughnan, M.L., Millard, E.L., Alewood, P.F.,
Adams, D.J., Daly, N.L., Craik, D.J. & Lewis, R.J. (2003) Isola-
tion, structure and activity of GID, a novel 4/7a-conotoxin with
an extended N-terminal sequence. J. Biol. Chem. 278, 3137–3144.
81. Sandall, D.W., Satkunanathan, N., Keays, D.A., Polidano, M.A.,

A.C.,Washburn,M.,Wright,E.,Spencer,J.A.,Gallagher,T.,
Whiteaker, P. & Wonnacott, S. (2000) UB-165: a novel nicotinic
agonist with subtype selectivity implicates the a4b2* subtype in the
modulation of dopamine release from rat striatal synaptosomes.
J. Neurosci. 20, 2783–2791.
90. Nayak, S.V., Dougherty, J.J., McIntosh, J.M. & Nichols, R.A.
(2001) Ca
2+
changes induced by different presynaptic nicotinic
receptors in separate populations of individual striatal nerve
terminals. J. Neurochem. 76, 1860–1870.
91. Marubio, L.M., Gardier, A.M., Durier, S., David, D., Klink,
R.,Arroyo-Jimenez,M.M.,McIntosh,J.M.,Rossi,F.,
Champtiaux, N., Zoli, M. & Changeux, J.P. (2003) Effects of
nicotine in the dopaminergic system of mice lacking the a4 subunit
of neuronal nicotinic acetylcholine receptors. Eur. J. Neurosci. 17,
1329–1337.
92. Champtiaux, N., Gotti, C., Cordero-Erausquin, M., David, D.J.,
Przybylski, C., Lena, C., Clementi, F., Moretti, M., Rossi, F.M.,
Le Novere, N., McIntosh, J.M., Gardier, A.M. & Changeux, J.P.
(2003) Subunit composition of functional nicotinic receptors
in dopaminergic neurons investigated with knock-out mice.
J. Neurosci. 23, 7820–7829.
93. Kulak, J.M., McIntosh, J.M. & Quik, M. (2002a) Loss of nicotinic
receptors in monkey striatum after 1-methyl-4-phenyl-1,2,3,6-tetra-
hydropyridine treatment is due to a decline in a-conotoxin MII
sites. Mol. Pharmacol. 61, 230–238.
94. Kulak, J.M., Musachio, J.L., McIntosh, J.M. & Quik, M. (2002c)
Declines in different b2* nicotinic receptor populations in monkey
striatum after nigrostriatal damage. J. Pharmacol. Exp. Ther. 303,

336–342.
103. Tachikawa, E., Mizuma, K., Kudo, K., Kashimoto, T., Yamato,
S. & Ohta, S. (2001) Characterization of the functional subunit
combination of nicotinic acetylcholine receptors in bovine adrenal
chromaffin cells. Neurosci. Lett. 312, 161–164.
104. Evans,N.M.,Bose,S.,Benedetti,G.,Zwart,R.,Pearson,K.H.,
McPhie, G.I., Craig, P.J., Benton, J.P., Volsen, S.G., Sher, E. &
Broad, L.M. (2003) Expression and functional characterisation of
a human chimeric nicotinic receptor with a6b4 properties. Eur. J.
Pharmacol. 466, 31–39.
105. Di Angelantonio, S., Matteoni, C., Fabbretti, E. & Nistri, A.
(2003) Molecular biology and electrophysiology of neuronal
nicotinic receptors of rat chromaffin cells. Eur. J. Neurosci. 17,
2313–2322.
106. Utkin, Y.N., Zhmak, M.N., Methfessel, C. & Tsetlin, V.I. (1999)
Aromatic substitutions in a-conotoxin ImI. Synthesis of iodinated
photoactivatable derivative. Toxicon. 37, 1683–1695.
107. Servent,D.,Thanh,H.L.,Antil,S.,Bertrand,D.,Corringer,P.J.,
Changeux, J.P. & Menez, A. (1998) Functional determinants by
which snake and cone snail toxins block the a7 neuronal nicotinic
acetylcholine receptors. J. Physiol. Paris. 92, 107–111.
Ó FEBS 2004 Pharmacology of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2319