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

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 nACh

M I N I R E V I E W

a-Conotoxins as tools for the elucidation of structure and function

of neuronal nicotinic acetylcholine receptor subtypes

1

Max Planck-Institute for Brain Research, Frankfurt, Germany;2Department of Biology & Biochemistry, University of Bath, UK;

3

Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia

which have evolved the ability to generate multiple toxins

with varied and often exquisite selectivity One class,

the a-conotoxins, is proving to be a powerful tool for

the differentiation of nicotinic acetylcholine receptors

(nAChRs) These comprise a large family of complex

subtypes, whose significance in physiological functions and

pathological conditions is increasingly becoming apparent

After a short introduction into the structure and diversity of

nAChRs, this overview summarizes the identification and characterization of a-conotoxins with selectivity for neur-onal nAChR subtypes and provides examples of their use in defining the compositions and function of neuronal nAChR subtypes in native vertebrate tissues

Keywords: a-conotoxins; neuronal nicotinic acetylcholine receptor subtypes; pharmacology; venom peptides; Xenopus oocytes

Neuronal nicotinic acetylcholine receptors

The nicotinic acetylcholine receptor family

The nicotinic acetylcholine receptor (nAChR) at the

neuro-muscular junction was first described as the Ôreceptive

substanceÕ in Langley’s

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

neuro-transmitter receptors

The electric organs of the electric ray Torpedo and

eel Electrophorus provided a rich source of nAChRs that

facilitated their early structural characterization The

nAChR from Torpedo californica is the best investigated

ligand-gated ion channel so far and considered as a prototype By electron microscopy techniques [4], high resolution images down to 4 A˚ have been obtained from semicrystalline arrays of this receptor in Torpedo mem-branes These studies revealed the pentameric quaternary structure of this protein (Fig 1) and have provided valuable information about the channel architecture and dimensions

A deeper insight into the molecular structure, in particular the acetylcholine (ACh) binding pocket, has become available after crystallization of an ACh binding protein, which has high homology to the extracellular domain of the nAChR (Fig 1) [5,6

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 subunithas not been found [2,3,9]

Subunit assembly of neuronal nAChRs The a7, a8 and a9 subunits represent a subclass of neuronal nAChRs that is able to form functional homomeric channels upon heterologous expression [2,3] Coexpression

of a7 and a8, 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 a7 wit h b subunits in native nAChRs has been controversial [11] The a2, a3, a4 and a6 subunits require coexpression of atleastone b (b2 or b4) subunit to form functional channels [2,3,9] However, pairwise combinations of the a6 wit h t he b2 or b4 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 toA 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)

support of this, higher expression levels could be obtained

by addition of the a5 and/or b3 subunit[12] The a5 and b3

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

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 adjacentsubunit (– 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, a9 and a10 subunits, as well as the nona subunits, c, d, e (which replaces c in adultmuscle), b2 and b4, can contribute to the ACh binding site In contrast, a5, b1 and b3 subunits appear to play a more ÔstructuralÕ role butmay additionally modu-late channel function and/or influence membrane trans-port and targeting of nAChRs [9]

er-mines the pharmacological and physiological properties of the channel In situ hybridization and immunohisto-chemistry data show overlapping distributions for a variety

of subunits, and electrophysiological and other functional studies in native tissues have revealed a great diversity of nAChR subtypes with distinct pharmacological, electrical and physiological properties even within single cells [2,3]

To decipher the physiological roles played by the different nAChRs, a range of subtype specific inhibitors are needed

Neuronal nAChRs as targets for the development of subtype specific drugs

Neuronal nAChRs are present throughout the central and peripheral nervous system, at both pre- and postsynaptic localizations The most prevalent subunits in brain are a4, b2 and a7 whereas a3 and b4 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]

Differentneuronal 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 a4b2 nAChRs (e.g GID), an a4b2 select ive a-conotoxin

has notyetbeen described.

Fig 1 Schematic representation of the membrane topology and

qua-ternary structure of the nAChR Each nAChR subunitcontains 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.

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

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

(n-BTX) is not generally available, and the antagonists

mecamylamine and dihydro-b-erythroidine are relatively

undiscriminating between different heteromeric neuronal

nAChRs Thus, further and more specific inhibitors are

needed to probe neuronal nAChRs in native tissues

a-Conotoxins as selective ligands for nAChR

subtypes

Among the most selective ligands targeting distinct nAChRs

are peptides isolated from the venom of cone snails [26]

Each of the 500 or so species contains in its venom a mixture

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

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 actin 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 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

T3

T3

Kd

Kd

Kd

Kd

Kd

Kd

Kd

Kd

Kd

Kd

Kd

a Sequ

b Un

c Unless

Ki

d Indica

Kd

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 subunitcombinations and

its suitability for electrophysiological measurements, the

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 mighteither be absentor 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

difficultin 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 ductor 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 Itwas originally discovered in a mammalian bioassay where it caused seizures in mice and rats upon intracranial injection,

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

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 a7 and a9 subtypes [34] (Table 1 shows

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

inhibited an a-BTX insensitive secretory response,

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

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

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 a7 nAChR

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

a4/7-conotoxins identified They differ by only two amino acids

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 a7 and a3b2 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

a-conotoxin on oocyte-expressed a7 nAChRs (compare

[53,55])

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-ina-BTX-sensitive component of an

ACh-induced current, [A10L]PnIA selectively inhibited the

a-BTX-sensitive component assumed to originate from an

a-BTX and [A10L]PnIA were atleastone 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

respectively These comparatively high values indicate that

nAChRs other than a7* and a3b2*, mostprobably 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 pointmutantthatdoes notshow

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 EpI

The a4/7-conotoxin EpI from Conus episcopatus

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

nAChR models, one muscular and two neuronal

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 ratintracardiac ganglia, which are believed to arise primarily from a3b2 and a3b4 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 notshow significantactivity ata3b2 and a3b4 subunitcombinations [61]

MII and AuIB The a4/7-conotoxin MII from Conus magus

a4/6-conotoxin AuIB from Conus aulicus were discovered in an approach aimed to directly identify selective ligands for the a3b2 and a3b4 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

Table 2 Comparison of a common motif in loop II of a4/7-conotoxins and their activity on oocyte-expressed a7 and a3b2 nAChRs The length/ hydrophobicity of the amino acid that corresponds to position 10 (bold) in PnIA correlates with the a3b2 over a7 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 notpublished The corresponding residues of the consensus sequence are 8–13 in PnIA.

a-Conotoxin

IC 50 (n M ) a3b2

IC 50 (n M ) a7

Ratio IC 50

Consensus sequence

Side chain

in position 10

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]

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

human a6b2 and a6b4 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,

subsequentstudies on knockoutmice revealed thatmost

binding sites completely disappeared in a6 knockoutmice

[71] The observation that a3b2 binding sites apparently are

not detected by MII argues against a role for a3 in t he

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] butmore 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

100 times less potentatother a/b combinations However,

AuIB also showed significant activity (30–40% block

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

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, ratmedial 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 a3b2 or

a3b4 combinations blocked nAChRs in rat medial habenula

neurons and oocyte-expressed a3b2b4 nAChRs [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 GIC and GID

Two neuronally active a4/7-conotoxins, GIC and GID, were identified in Conus geographus

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] Itinhibits a7 and a3b2 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]

Vc1.1 PCR amplification of Conus victoriae

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

experiments on chromaffin cell membranes Vc1.1 showed

nAChR populations labelled by the relatively nonselective

Vc1.1acts on a3b4* receptors containing a5 and/or a7 subunits (Table 1) Interestingly, Vc1.1 was able to inhibit

alleviating chronic pain and accelerating functional recovery

in an animal model of neuropathy These data are in

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 PIA from Conus purpurascens

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 a3 and a6 subunits Because the a6 subunitdid not form functional nAChRs, either in combination with b2 or

with b2 plus b3 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

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, a3b2 and a6b2 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 a3b2 nAChRs

was reversed within one minute Interestingly, the

dissoci-ation rate from both a3- and a6-containing receptors

was greatly slowed when the b2 subunitwas replaced by the

b4 subunit

AnIB

neuronally active conotoxins is AnIB from Conus anemone

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-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

differentmicrodomains thatoverlap around the conserved

ACh binding site of nAChRs Thus, itmightbe 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

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 a7 vs a3b2 nAChR (Table 2)

Other groups with similar sequences and selectivities for

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 accumbens)

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 funcelucida-tion in the dopaminergic system The following will focus on the investigation of presynaptic nAChRs on dopaminergic nerve terminals in the striatum

midbrain dopaminergic neurons revealed a3, a4, a5, a6, a7,

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] Subsequentstudies [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 atleastone 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

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

subunit conferring MII-sensitivity [12,40]

rat striatal synaptosomes by laser scanning confocal

micro-scopy and immunocytochemical studies, Nayak et al [90]

hypothesized that a4 and a3 (or a6) 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

a4-containing subtype that includes b2 subunits The

ganglia of a6 knockoutmice [71] butbasically unchanged in a3 knockoutmice [65] finally confirmed the involvementof the a6 subunit rather than the a3 subunitin 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 recentstudies on a4, a6, a4a6 and b2 knockoutmice

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

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 [ 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].