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

MINIREVIEW a-Conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes Annette Nicke 1 , Susan Wonnacott 2 and Richard J. Lewis 3 1 Max Planck-Institute for Brain Research, Frankfurt, Germany; 2 Department of Biology & Biochemistry, University of Bath, UK; 3 Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia Cone snails comprise  500 species of venomous molluscs, which have evolved the ability to generate multiple toxins with varied and often exquisite selectivity. One class, the a-conotoxins, is proving to be a powerful tool for the differentiation of nicotinic acetylcholine receptors (nAChRs). These comprise a large family of complex subtypes, whose significance in physiological functions and pathological conditions is increasingly becoming apparent. After a short introduction into the structure and diversity of nAChRs, this overview summarizes the identification and characterization of a-conotoxins with selectivity for neur- onal nAChR subtypes and provides examples of their use in defining the compositions and function of neuronal nAChR subtypes in native vertebrate tissues. Keywords: a-conotoxins; neuronal nicotinic acetylcholine receptor subtypes; pharmacology; venom peptides; Xenopus oocytes. Neuronal nicotinic acetylcholine receptors The nicotinic acetylcholine receptor family The nicotinic acetylcholine receptor (nAChR) at the neuro- muscular junction was first described as the Ôreceptive substanceÕ in Langley’s 1 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 A ,glycineand5HT 3 neuro- transmitter receptors. The electric organs of the electric ray Torpedo and eel Electrophorus provided a rich source of nAChRs that facilitated their early structural characterization. The nAChR from Torpedo californica is the best investigated ligand-gated ion channel so far and considered as a prototype. By electron microscopy techniques [4], high resolution images down to 4 A ˚ have been obtained from semicrystalline arrays of this receptor in Torpedo mem- branes. These studies revealed the pentameric quaternary structure of this protein (Fig. 1) and have provided valuable information about the channel architecture and dimensions. A deeper insight into the molecular structure, in particular the acetylcholine (ACh) binding pocket, has become available after crystallization of an ACh binding protein, which has high homology to the extracellular domain of the nAChR (Fig. 1) [5,6 2 ]. The Torpedo nAChR and the nAChR in embryonic vertebrate muscle share the same heteropentameric structure composed of four homologous subunits which are arranged in the order a1ca1db1 around the central ion-conducting channel [7,8] (Fig. 2A). In addition, 11 nAChR subunits (a2–a7, a9, a10, b2–b4) have been cloned from neuronal and sensory mammalian tissues. A mammalian homologue of the avian a8 subunit has not been found [2,3,9]. Subunit assembly of neuronal nAChRs The a7, a8anda9 subunits represent a subclass of neuronal nAChRs that is able to form functional homomeric channels upon heterologous expression [2,3]. Coexpression of a7anda8, as well as of a9 and the highly homologous a10 subunit [10] has been shown to generate heteromeric channels with properties distinct from those of the respective homopentamers. The association of 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) 2 (b) 3 has been proposed for oocyte- expressed neuronal nAChRs [16,17]. However, there is only limited knowledge of the stoichiometry of native neuronal nAChRs. Combinations of three and even four different subunits (including a5, b3) have been described in both heterologous expression systems and native tissues (e.g. [18– 21]) further complicating the determination of stoichio- metries. The ACh binding site has been located at the interface between an 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 needed. Neuronal nAChRs as targets for the development of subtype specific drugs Neuronal nAChRs are present throughout the central and peripheral nervous system, at both pre- and postsynaptic localizations. The most prevalent subunits in brain are a4, b2anda7whereasa3andb4 predominate in peripheral ganglia. Because more complex combinations may exist, an asterisk is used to denote the potential presence of additional subunits, as in a4b2* and a3b4* nAChRs [22]. The a7 subunit is widespread in the central nervous system and a variety of peripheral tissues. The a7* receptors are characterized by very fast inactivation kinetics and long lasting desensitization, which makes their functional iden- tification difficult [23]. Different neuronal nAChR subtypes have been shown to be involved in learning, antinociception, nicotine addiction and neurological disorders such as Parkinson’s and Alzheimer’s disease. For the nonselective nAChR agonist nicotine, analgesic, anxiolytic and cytoprotective properties are seen, as well as beneficial effects in Alzheimer’s disease, Parkinson’s disease, Tourette’s syn- drome and certain forms of epilepsy and schizophrenia [24,25]. However, the therapeutic use of nicotine is hindered by its adverse effects on the cardiovascular and Fig. 2. Subunit compositions of the muscle-type nAChR and assumed subunit compositions of neuronal nAChRs targeted by a-conotoxins. (A) The composition of neuronal nAChRs can be similarly complex to that of the muscle-type nAChR. Note that the muscle-type specific a-conotoxins MI and GI have opposite selectivities at nAChRs from Torpedo and mammalian muscle. a-Conotoxins with selectivity for heterologously expressed pairwise combinations of neuronal 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 subtypes Among the most selective ligands targeting distinct nAChRs are peptides isolated from the venom of cone snails [26]. Each of the 500 or so species contains in its venom a mixture of 50–200 peptides, giving a total of  50 000 potential pharmacologically active peptides. However, only a small portion (< 0.1%) of these peptides has been pharmacolo- gically characterized so far. The great variability of the conotoxins and their highly specific action on different ion channel subtypes derives from the structure of the peptides which have evolved conserved and hypervariable regions [27–30]. The conserved regions comprise the signal sequence which is characteristic for the respective toxin superfamily and generally defines the pattern of disulfide connectivities. The loops between the cysteine residues represent the hypervariable regions that define the pharmacological diversity of conopeptides. This hypervariability has gener- ated a wide diversity of a-conotoxins with activity at neuronal nAChR subtypes. Conotoxins targeting nAChRs To date, three different conotoxin families targeting nAChRs have been identified [26]. Each family is defined by a common binding site on the nAChR as well as by their structure (for nomenclature of a-conotoxins see [31]) 3 . The w-conotoxin PIIIE has a structure similar to the voltage-gated Na + channel-blocking l-conotoxins and acts as a noncompetitive antagonist (perhaps a pore blocker) of the muscle-type nAChR. The other two families, aA- and a-conotoxins, function as competitive antagonists at the ACh binding site, but differ in their disulfide framework. The three aA-conotoxins identified so far also target the muscle-type nAChR. The largest family are the a-conotoxins which can be further divided into a3/5, a4/3, a4/6 and a4/7 structural subfamilies depending on the number of amino acids between the second and the third cysteine residues (loop I) and the third and the fourth cysteine residues (loop II), respectively [32] (Table 1). It appears that these differences in structure are paralleled by their selectivity for different nAChR subtypes, with all known a3/5-conotoxins being selective for the muscle-type nAChR, while the only published a4/6-conotoxin and most a4/7-conotoxins are selective for neuronal nAChRs. One exception is a4/7-conotoxin EI, which preferentially targets the a/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) 4 .Sofar,a-conotoxins selectively targeting mammalian a3b2(a-MII, a-GIC) a6b2 (a-MII, a-PIA), a3b4(a-AuIB) and a7(a-ImI) interfaces have been identified [12,34,37–41]. It appears that binding of only one toxin molecule is sufficient to block receptor function [33,42]. In contrast, two agonist molecules seem to be required to open the nAChR channel. As a consequence, native nAChRs with two different types of a/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 neurons; CG, ciliary ganglion neurons; S, striatum; SY, striatal synaptosomes; SC, superior colliculus; R, retina; NA, nucleus accumbens; C, caudate; P, putamen. Small letters at the end indicate the method: r, electrophysiological recordings; m, binding studies on membrane preparations; i, binding studies on immunoimmobilized receptors; s, quantitative autoradiography on tissue sections; d, quantification of agonist-evoked dopamine release; c, quantification of agonist-evoked catecholamine release. a6/a3anda7/5HT 3 indicate chimeric receptors between nicotinic a subunits and nicotinic a7 and the 5-hydroxytryptamine receptor, respectively. a7/5HT 3 constructs were expressed in human embrionic kidney (HEK) cells. IC 50 values > 10 l M and a-conotoxin mutants were generally not considered. Differences between expression systems and between heterologously expressed and native channels as well as species differences have been suggested to account for inconsistencies in IC 50 values. In addition, preparation inherent differences (e.g. dissociated neurons, synaptosomes or physiologically more intact systems such as slices) and methodological variations (e.g. different agonist concentrations, protocols for toxin application or determination of the toxin concentration) have to be considered. a-Conotoxin Sequence a Functional Data Binding Data (n M ) c IC 50 (n M ) on recombinant nAChRs b IC 50 (n M ) in native tissues and suggested native AChRs targeted ImI GCCSDPRCAWR 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 d (B) 630 [58] ca7 168 [59] bCCc 2000/1500 [57] a3b4 ca7L247T acts as an agonist [59] a3b2 99 [55] [N11S]PnIA GCCSLPPCAASNPDYC a7 1710 [55] rIGr 375 [56] a7* + additional component ha7/5HT3 K d (B) 148 000 [58] a3b2 241 [55] PnIB GCCSLPPCALSNPDYC a7 61 [55] rIGr 33 [56] ha7/5HT3 K d (B) 29 600 [58] a3b2 1970 [55] bCCc 700/1000 [57] a3b4 EpI GCCSDPRCNMNNPCYC a7 30 [61] rIGr 1.6 [60] a3b2/a3b4 ca7/5HT3 (HEK293) 103 [61] bCCc 84/210 [60] a3b4 MII GCCSNPVCHLEHSNLC a3b2 0.5 [37], 3.5 [102], 8.0 [62], 1.7 [39], rIGr 10 [100] a3b2 rSi 1.3 [21] a6b2* K d d 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 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* pSs K d 0.93 [67], C 0.92 [67] P a6b2(b3) mSCm K d 4.9 [64] AuIB GCCSYPPCFATNPD-C a3b4 750 [41], 966 [61], K d d 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] GIC GCCSHPACAGNNQHIC ha3b2 1.1 [38] ha3b4 755 [38] ha4b2 309 [38] GID IRDcCCSNPACRVNNOHVC a7 4.5 [80] a3b2 3.1 [80] a4b2 152 [80] Vc1.1 GCCSDPRCNYDHPEIC bCCc 1000–3000 [81] a3b4* bCCc 2.3 and 3700 (2 sites) [81] a3b4* PIA 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] 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 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 5HT 3 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 IC 50 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 50 of 300 n M [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 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 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 6 [35,53]. PnIA and PnIB PnIA and PnIB from Conus pennaceus 7 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 [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-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 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. EpI The a4/7-conotoxin EpI from Conus episcopatus 8 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 9 on nAChRs were tested in three native nAChR models, one muscular and two neuronal prepara- tions. In concentrations up to 10 n M 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 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 50 (n M ) a3b2 IC 50 (n M ) a7 Ratio IC 50 a3b2/a7 Ref. Consensus sequence Side chain in position 10 GIC 1.1 – – [38] CAGNNQ –H AnIB 0.3 76 0.004 [83] CAANNQ –CH 3 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 3 ) 2 GID 3.1 4.5 0.7 [80] CRVNNO [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]. 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 125 I-labelled MII binding sites were conserved in a3 knockout mice [65], whereas high-affinity 125 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 50 value of 750 n 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- 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. GIC and GID Two neuronally active a4/7-conotoxins, GIC and GID, were identified in Conus geographus 11 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]. Vc1.1 PCR amplification of Conus victoriae 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- 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 13 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 M ) 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. 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- 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 accumbens) 15 (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 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 finding that 125 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 125 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 125 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 [ 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 [...]... localization of a6 receptors on dopaminergic nerve terminals and provides further evidence that they play a key role in the pathogenesis of Parkinson’s disease 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 ganglion Another example where a-conotoxins. .. identify selective ligands for the characterization 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 combinations Nevertheless, the activity data obtained from oocyte-expressed receptors provide... undefined nAChR subunit of obtaining detailed information on their three dimencombination with very high affinity for AuIB and ribbon sional structures and the relative ease of synthesis, makes AuIB is present in intracardiac ganglia, or that the a3b4* them particularly useful templates for the design of receptors in intracardiac ganglia form a substantially optimized synthetic peptides for the subtype characterizadifferent... 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. .. International Union of Pharmacology XX Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits Pharmacol Rev 51, 397–401 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 receptor. .. study based on single channel recordings and the use of a-conotoxins MII and AuIB further dissected and correlated the combinatorial and functional heterogeneity of the slowly decaying 16 population [77] In this study, two long events of 25 pS 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... effects of MII and a-BTX were additive, the attenuation 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... 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... IC50 value of ribbon AuIB on oocyte expressed a3b4 nAChRs was 27.5 lM, about 30-fold higher than that for native AuIB and even 3 · 106-fold 2316 A Nicke et al (Eur J Biochem 271) Ó FEBS 2004 valuable basis for structure- activity studies The high higher than that determined on native receptors This result selectivity of the a-conotoxins, together with the possibility suggests that either an as yet undefined... 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 . MINIREVIEW a-Conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes Annette Nicke 1 ,. 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