α-Conotoxins

The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 www.elsevier.com/locate/ijbcb Review a-Conotoxins Hugo R. Arias *, Michael P. Blanton Departments of Pharmacology and Anesthesiology, School of Medicine, Texas Tech Uni6ersity Health Sciences Center, Lubbock, TX 79430, USA Received 16 May 2000; accepted 2 August 2000 Abstract a-Conotoxins (a-CgTxs) are a family of Cys-enriched peptides found in several marine snails from the genus Conus. These small peptides behave pharmacologically as competitive antagonists of the nicotinic acetylcholine receptor (AChR). The data indicate that (1) a-CgTxs are able to discriminate between muscle- and neuronal-type AChRs and even among distinct AChR subtypes; (2) the binding sites for a-CgTxs are located, like other cholinergic ligands, at the interface of a and non-a subunits (g, d, and o for the muscle-type AChR, and b for several neuronal-type AChRs); (3) some a-CgTxs differentiate the high- from the low-affinity binding site found on either a/non-a subunit interface; and that (4) specific residues in the cholinergic binding site are energetically coupled with their corresponding pairs in the toxin stabilizing the a-CgTx-AChR complex. The a-CgTxs have proven to be excellent probes for studying the structure and function of the AChR family. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: a-Conotoxins; Competitive antagonists; Nicotinic acetylcholine receptors Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018 2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018 3. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020 Abbre6iations: AChR, nicotinic acetylcholine receptor; 5-HT, 5-hydroxytryptamine; 5-HT3R, 5-hydroxytryptamine type 3 receptor; NMDA, N-methyl-D-aspartate; a-BTx, a-bungarotoxin; a-CgTx, a-conotoxin; aA-CgTx, aA-conotoxin; m-CgTx, m-conotoxin; v-CgTx, v-conotoxin; k-CgTx, k-conotoxin; d-CgTx, d-conotoxin; mO-CgTx, mO-conotoxin; s-CgTx, s-conotoxin. * Corresponding author. Tel.: + 1-806-7432425, ext.: 244; fax: + 1-806-7432744. E-mail address: phrhra@ttuhsc.edu (H.R. Arias). 1357-2725/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 5 1 - 0 1018 H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 4. Biological function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1023 5. Possible medical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025 1. Introduction From an evolutionary point of view, snails from the genus Conus are among the most successful marine invertebrates (reviewed in [1]). The genus Conus is formed by over 500 venomous species. These marine mollusks prey on other marine species by means of a very large number of small peptides (10– 50 amino acids in length) with specific pharmacological activities. Due to the remarkably fast interspecific divergence of peptide sequences, each Conus species has a repertoire of 50– 200 different peptides (reviewed in [2]). These peptides, called conotoxins, affect the functioning of different voltage-gated ion channels and neurotransmitter-gated receptors (reviewed in [3,4]). Based on the molecular form, the approximately 50 000 conotoxins can be grouped into a minimum of seven superfamilies; the A[e.g. a-conotoxins (a-CgTxs), aA-, and kACgTxs], M- (e.g. m- and c-CgTxs), O- (e.g. v-, k-, d-, and mO-CgTxs), P- (e.g. Spasmodic peptide; [5]), R-, S- (e.g. s-CgTxs), and T-superfamilies (e.g. tx5a, p5a, au5a, and au5b; [6]) (reviewed in [4,7]). This review will focus on the structure and pharmacological activity of only the conotoxin family (e.g. a-CgTx and aA-CgTxs) which specifically bind to several nicotinic acetylcholine receptors (AChRs) (reviewed in [4,8]). The AChR is the prototype of a superfamily of ion channel-coupled receptors which are gated by specific neurotransmitters. This superfamily also includes the type A g-aminobutyric acid, glycine, and type 3 5-hydroxytryptamine (5-HT) receptors (reviewed in [8,9]). There are two main AChR types, the muscle- and neuronal-type. The muscletype AChR is a pentamer comprised of two a1 subunits, one b1, one d and, one g or o subunit, depending on whether the receptor is in an embry- onic or adult stage, respectively. The a1 subunits contain two adjacent cysteines at position 192 and 193 (sequence number from Torpedo AChR), which are involved in the recognition and binding of cholinergic agonists and competitive antagonists. Based on the presence or the absence of these two cysteines, the neuronal-type AChR subunit classes are designated a (when they contain both cysteines) or b (when they do not). To date, eight a subunits (a2– a9) and three b subunits (b2– b4) have been identified. The a7, a8, and a9 polypeptides are the only subunits capable of forming homo-oligomeric ion channels. Interestingly, the two a subunits display non-equivalence for the binding of several cholinergic agonists and competitive antagonists in both muscle- and neuronal-type AChRs. In this regard, a-CgTxs have become one of the most powerful tools to support this non-equivalence (reviewed in [8,9]). 2. Structure The initial purification and chemical characterization of an a-CgTx was performed by Olivera and co-workers in 1981 [10]. Since then, a great deal of structural information has been obtained. Most of them exhibit four Cys residues in the following conserved arrangements; CCX3CX5C (the a3/5 subfamily), CCX4CX3C (the a4/3 subfamily), and CCX4CX7C (the a4/7 subfamily). Each alternate Cys pair forms a disulfide loop (i.e. loops I and II, respectively). In each case, the X represents the number of amino acids between the Cys residues. The only representative of the a3/5/3 subfamily, which has three Cys bonds, is a-CgTx SII. The spacing between disulfide bonds is an important determinant of backbone structure. Additionally, a conserved Pro residue is found H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 between the second and the third Cys in almost all a-CgTxs that have been characterized to date. The exception is the aA-CgTx subfamily (e.g. aA-PIVA, aA-EIVA, and aA-EIVB), which has a core sequence that is very different than the other 1019 subfamilies [11– 13]. Table 1 shows the primary and secondary structural features of some a- and aA-CgTxs as examples of each subfamily. The three-dimensional structures of several aCgTxs have been determined using either X-ray Fig. 1. Surface models of a-CgTxs PnIA (A) and MII (B) and backbone representations of a-CgTxs PnIA (C), MII (D), and GI (E) (taken from [16], with permission). Hydrophobic, polar, positive-charged, and negative-charged residues are displayed in purple, yellow, red, and blue, respectively. Backbone conformations of a-CgTxs PnIA (C), MII (D), and GI (E) are shown in ribbons. Surface distributions in dots with the same color codes. Arrows indicate carboxyl (blue) and amino (yellow) terminal residues. 1020 H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 crystallography or NMR spectroscopy [11,14 – 26]. These studies provide support for the idea that these small polypeptides achieve their conformational stability by means of disulfide bonding. Although a-CgTxs are apparently very rigid structures, two or more interconvertible conformers may exist in solution (reviewed in [4]). The overall shapes of the different a-CgTx subfamilies are as follow: the a4/7 subfamily is more rectangular, the a3/5 subfamily is more triangular, whereas the aA subfamily has been described as an iron. The structural comparison of several a-CgTxs specific for neuronal-type AChRs, such as a-CgTx ImI, PnIA, PnIB, MII, and EpI (Table 3), exhibit remarkable similarity in local backbone conformations and relative solvent-accessible surface areas [21]. A model of the tertiary structure of a-CgTxs PnIA, MII, and GI is shown in Fig. 1 (taken from [16]). The backbones of the a-CgTxs PnIA and MII structures (panels C and D, respectively) are very similar. Nevertheless, the surfaces of both the polypeptides are unique. The protrud- ing Tyr residue on the surface of the a-CgTx PnIA structure (panel C, in purple) contrasts with the flat hydrophobic surface of a-CgTx MII (panel D, residues in purple). Furthermore, charged residues are exposed on the a-CgTx MII surface (panel D, negative residues in blue and positive residues in red), whereas they are not exposed on the a-CgTx PnIA surface (panel C). In addition, both the backbone and the surface of a-CgTx GI (panel E), which is specific for muscletype AChRs (see Table 2), diverge significantly from the other PnIA and MII a-CgTxs. 3. Synthesis The Conus venom is biosynthesized and stored in the venom duct of the venom apparatus (reviewed in [27]). When the snail is hunting prey, the venom, by contraction of the muscular venom bulb, is delivered through a harpoon-like radula tooth located in the proboscis. Table 1 Primary and secondary structure of each conotoxin subfamily a-Conotoxin Conus species Subfamily Primarya and secondaryb structure MI Conus magus a3/5 GRCCHPACGKNYSCc PnIA Conus pennaceus ? ? ? ? Conus imperialis ? a4/7 GCCSLPPCAANNPDYdCc a4/3 GCCSDPRCAWRCc ? Conus striatus a3/5/3 ? Conus purpurascens aA [56] ? ? ? GCCCNPACGPNYGCCGTSCS ? aA-PIVA [55] ? ? SII [30] ? ? ImI References [57] ? ? ? GCCGSYONAACHOCSCKDROSYCGc ? ? ? [13] ? a The sequence alignment of the different a-CgTxs is shown in the standard one-letter amino acid code. The letter O represents trans-4-hydroxyproline. b a-Conotoxin subfamilies a3/5, a4/7, and a4/3 are formed by two disulfide bonds, while the aA-CgTx subfamily and a-CgTx SII (a3/5/3 subfamily) have three disulfide bonds. c Amidated COOH-terminus. Instead, a-CgTx SII presents a free COOH-terminus. d Sulfated residue. H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 1021 Table 2 a-Conotoxin specificity for different muscle-type nicotinic acetylcholine receptors a-Conotoxin AChR Source a IC50 References (subunit interface) MI BC3H-1 Mouse Torpedo Human (a2bod) BC3H-1 GI Mouse Torpedo CnIA SI Human (a2bod) Frog Fish (Eigenmannia) Torpedo BC3H-1 Human (a2bod) Torpedo SIA BC3H-1 Torpedo EI BC3H-1 Torpedo BC3H-1 Torpedo Torpedo Mouse SII aA-EIVA aA-EIVB aA-PIVA a BC3H-1 Torpedo Torpedo High-affinity binding site NM Low-affinity binding site mM 1.50 9 0.24 (ad) 0.42 9 0.15 (ad) 0.40 9 0.01b (a2bgd) 0.40 90.17b (abd) 1.4 9 0.1b (kinetic) 0.55 9 0.06 (a2bgd) 1.34 (abd) 5.3 (a2bd) 1.9 (ad) 12b (ad) 2.89 1.3 (ad) 2.69 1.0 (ag) 1209 10 (ag) 4.509 1.60 (ag) 1.69 1.4 (a2bgd) 32 9 2 (abg) 9 9 1 (ad) 4.9 9 1.9 (ad) 1.3 9 0.3 (ad) 20b (ad) 360940 (ag) 4.59 1.3 (ag) 41.39 8.2c (ag) 2 91 (ad) 22.0 9 1.1 (ag) 23.09 4.1 (ag) 50-100b 190 6809 160 (ad) 13009 430 (ad) 809 14 (ao) 7.70 9 0.14 (ad) 5909 40 (ag) 420 9.40 9 1.20 (ad) 0.41 9 0.09 (ad) 189 5 (abg) 18.39 0.93 (a2bgd) 11.7 (abg) 139 1.3 (a2bgd) 15 (ag) 7.8 9 1.3 (ag) 2.39 0.7 (ad) 2191 (ad) 0.489 0.10 (ad) 1.09 0.3 (a2bgd) 1.29 0.1 (abd) 0.099 0.01 (ao) 589 12 (ag) 609 1 (ag) 249 2 (ad) 0.099 0.02 (ad) 0.149 0.01 (ao) 2-4b 2209 45 (ag) 2909 110 (ag) 8.319 1.2 (ad) 0.17 9 0.04 (ag or ad) 16 9 1 (ag or ad) 1 2009 8.6 (ag) \260 (ad) 0.28 9 0.03 (ag) 0.199 0.02 (ag) 18 9 6.6 8 [31] [31] [30] [57] [12] [12] 17b 11b (a2bgd) 11b (abg) 32b (kinetic) 15b (abd) 37b (kinetic) 150910 18b [12] [12] [13] 1 These values were obtained by inhibition of [125I]a-BTx binding except those determined by the following: or fluorescence spectroscopy. c [30] [31] [12] [12] [12] [42] [42] [39] [32] [58] [33] [30] [45] [31] [40] [40] [48] [30] [43] [58] [45] [43] [59] [48] [56] [56] [19] [30] [43] [48] [44] [45] [57] [30] [45] b electrophysiology 1022 H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 Table 3 a-Conotoxin specificity for different neuronal-type nicotinic acetylcholine receptors a-Conotoxin AChR Subtype CnIA MII a7 a3b2 EpI a4b2 a3b4 a3b4 PnIA AuIB PnIB a3b2/a3b4 a7/? a7 a7 (human)/5-HT3R (rat) a3b2 a3b4 a3b4 a7 a7/? a7 a7 (human)/5-HT3R (rat) a3b2 a3b4 ImI a7 (rat) a7 a7 a7 (human) a9 a3b4/a3b4a5 a3b4 Aplysia a IC50 nM References 14 800 b 3.5 8.0 9 1.1 24.3 9 2.9c (synaptosomes) 17.3 9 0.1c (slices) 400 3000d (noradrenaline) 84 9 19d (adrenaline) 2109 30d (noradrenaline) 1.6 14 252 176 (Kd) 61 2009 1 100b 9.56 21 000–28 000d (noradrenaline) 500 9 140 (Kd) 750 20 000d (noradrenaline) \7000 33 61.3 84.9 (Kd) 29 6009 600b 1970 700b 700d (noradrenaline) 1000d (adrenaline) 220 100 300d 86.2 9 1.2 2450 9 100b 1800 2500 91200e \3000 47 (desensitizing Cl− response) \20 000 (sustained Cl− response) 150 922 (cationic response) [19] [46] [36] [36] [36] [46] [62] [54] [54] [54] [50] [51] [51] [53] [51] [52] [37] [37] [52] [37] [50] [51] [51] [53] [51] [30] [52] [52] [58] [60] [61] [48] [58] [34] [60] [35] a These values were obtained by electrophysiological techniques except those determined by the following: b inhibition of [125I]a-BTx binding, inhibition of agonist–induced, c dopamine, d catecholamine (e.g. adrenaline or noradrenaline), e 5-HT release. Cone snails generate novel polypeptide sequences by amino acid hypermutation (reviewed in [2,7]). The synthesis of conotoxins can be compared with a combinatorial library search strategy. From studies with the O-superfamily, conotoxins have been considered to be initially translated as larger prepropeptide precursors 70– 120 amino acids in length with a single copy of the toxin present at the C-terminal end. Other neuropeptide precursors encode either multiple H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 copies of a specific peptide or several distinct toxins. The high number of polypeptide structures observed in different cone snails that inhibit a specific target are thought to have evolved by hypermutation of the amino acids located closer to the C-terminal. Exceptions are the Cys residues located in the toxin proper. The rest of the precursor sequence remains highly conserved. The signal sequence is the polypeptide region with the highest level of sequence conservation. Between the signal sequence and the mature toxin there exists an intervening pro-region 40 amino acids in length which exhibits a low mutation rate. 4. Biological function The pioneering studies done in Dr Baldomero Olivera’s laboratory provide the initial methodology for determining the biological activity of aCgTxs. Initial pharmacological data demonstrated that, in general, the family of a-CgTxs behave as competitive antagonists of the AChR (reviewed in [7,8]). The a-CgTxs compete for the binding sites of acetylcholine and cholinergic agonists. However, a distinct conotoxin from Conus purpurascens, called c-CgTx PIIIE, inhibits the functional activity of the AChR in a non-competitive manner [28]. The earliest studies showed that a-CgTxs inhibited muscle-type AChRs. Table 2 shows the a-CTx specificities for muscle-type AChRs from different species. Nonequivalent binding of some a-CgTxs at the two agonist/competitive antagonist binding domains in Torpedo AChR [29– 31] is also summarized in Table 1. Although early studies focused on a-CgTxs action in muscle-type AChRs, more recent efforts have identified pharmacological effects of these compounds in neuronal-type AChRs. Table 3 shows the a-CgTx specificities for different neuronal-type AChRs. In order to determine which subunits of the Torpedo AChR are involved in the a-CgTx binding site, purified a-CgTx MI was crosslinked to the AChR with bivalent succinimide reagents of different lengths [29]. With a 12-atom crosslinker, all the four subunits were labeled, whereas a 4-atom crosslinker labeled the b and g subunits. In addition, two azidosalicylate a-CgTx GIA derivatives 1023 were used for photoaffinity labeling of the AChR [29]. These studies showed that depending on the a-CgTx derivative used, the specifically labeled AChR subunits were b and g, or d and g. However, labeling of detergent-solubilized AChR was exclusive for residues 121 and 183 of the g subunit. The p-benzoylphenylalanine derivative of a-CgTx GI also labeled the a subunits [38]. In order to identify the determinants of a-CgTx MI selectivity, Dr Steven Sine’s laboratory used subunit chimeras and site-directed mutagenesis [33,39]. From these studies, it was found that the high affinity of subtype MI for the ad subunit interface of mammalian AChRs is determined by amino acids S36, Y113, and I178 from the d subunit, while the low affinity for the ag interface is determined by residues K34, S111, Y117, L119, and F172 of the g subunit. Since dY113 and gS111 are exchanged for Arg and Tyr in the Torpedo AChR, these two natural differences may account for the observed site-specificity between the two species. This idea is corroborated by the fact that the mutation dR113Y in the a2bd2 or the mutation gY111R in a2bg2 Torpedo AChR results either in an enhancement of or a decrease in a-CgTx MI affinity, respectively [40]. The pairs gK34/dS36 and gF172/dI178, as primary determinants for a-CgTx MI selectivity in mouse AChR, coincide with that for the selectivity of carbamylcholine [41]. In contrast, neither the gS111Y nor dY113S mutation affected carbamylcholine affinity, suggesting that agonists and at least the a-CgTx MI subtype do not have identical selectivity determinants. Residues gK34, gS111, and gF172 contribute to loops D, F, and G, respectively, of the agonist/competitive antagonist binding site (reviewed in [8]). Since other residues from the a subunit are considered to be involved in the agonist/competitive antagonist binding sites (reviewed in [8,9]), Sugiyama et al. [42] examined the contribution of some of these residues to the binding of a-CgTx MI. Mutations aY190F and aY189F do not affect a-CgTx MI affinity, whereas removal of aromaticity by exchanging these residues for Thr has a marked influence on a-CgTx association. This suggests that aromaticity may be required to stabilize the cationic peptide. The effect elicited by substitutions of Y93 (loop A; see [8]) and D152 residue (loop B; see [8]) indicate that both peptide and nonpeptidic ligands bind to the same site in 1024 H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 the AChR, but unique though overlapping sets of amino acids contribute to the binding domain. In addition, substitution of Y12A on the MI toxin dramatically reduces its affinity for the highaffinity site (ad) with little effect on toxin potency at the low-affinity site (ag) [43]. This and additional data suggest that the orientation of residue Y12 is important in the formation of the a-CgTx MI-AChR complex. In order to determine the existence of a linkage relationship between mutations in a and d/g subunits, a mutant cycle analysis was employed [42]. From this study, a high coupling energy between S36 and I178 of the d subunit was demonstrated. In contrast, a relatively low linkage between residues aY93/V188 and pairs gK34/dS36, gS111/dY113, and gF172/dI178 is evident. Taking into account that the energetic contributions of amino acids in the a chain to a-CgTx MI association with the AChR seem to be independent from the ones at the d/g subunits, it is postulated that one of the surfaces of the neurotoxin molecule interacts with the a subunit, whereas the other surface interacts with the d or the g subunit. In this regard, recent conformational sudies using [H1]-NMR spectroscopy suggest that both the faces of the aCgTx GI are involved in the orientation of the molecule within the ad subunit interface [25]. The binding face of a-CgTx GI, a toxin closely related to the MI subtype in structure, interacts by means of residues C2, N4, P5, A6, and C7 (from loop I) with the a1 subunit, whereas the selectivity face comprising amino acids R9 and H10 (from loop II) is oriented towards the d subunit (the subunit forming the high-affinity a-CgTx GI locus in mammalian AChRs). Residues R9 and H10 were found to be responsible for the high differential selectivity and affinity between both the cholinergic ligand binding sites [44,45]. The lack of effect of the mutation P9 to the neutral residue Ala in the a-CgTx SI suggests that the cationic group of A9 in the a-CgTx GI plays a major role in ag selectivity in the Torpedo receptor. The critical difference between a-CgTx GI and SI has been ascribed to position 9 (reviewed in [24]). The importance of a cationic group for high selectivity is further substantiated by the fact that mutations on the a-CgTx MI at position K10, the ho- mologous residue of A9, resulted in a loss of selectivity [45]. Taking into account that neuronal receptors containing the subunit composition a4b2, a2b2, or a3b4 are more than 200-fold less sensitive to a-CgTx MII than a3b2 AChRs, the determinants of a-CgTx selectivity were identified using chimeric subunits and subunits with single residue substitutions [46]. Residues b2T59, a3K185, and a3I188 were identified as specific determinants for a-CgTx MII sensitivity. The amino acid a3K185 may electrostatically interact with E11 from aCgTx MII. Regarding a-CgTx ImI specificity, the pairs a7W55/a1R55, a7S59/a1Q59, and a7T77/a1K77 have been considered as components conferring high affinity binding to a7/5-HT3R compared with a1/ 5-HT3R homooligomeric chimeras [47] (reviewed in [8]). The third pair (a7T77/a1K77) may be considered as a new loop or an allosterically coupled loop. Experiments performed in parallel show that two regions in the a-CgTx ImI molecule are essential for binding to the a7/5-HT3R chimera [48]: a region comprising residues D5-P6-R7 in the first loop and a second region in loop II formed by W10. The fact that D5 functions as an N-terminal cap and P6 as a helix-initiator suggests that the contribution of both the amino acids to binding may be due to their structural roles rather than due to direct interaction with the AChR [21]. The structural role of P6 was recently corroborated by mutagenesis studies [49]. Subsequent thermodynamic mutant cycle analyses demonstrated the existence of a dominant pairwise interaction between a-CgTx ImI R7 and a7Y195 (located in loop C; reviewed in [8]), and multiple weak interactions between a-CgTx ImI D5 and W149, Y151, and G153 of a7 (which are all located in loop B; reviewed in [8]), and between a-CgTx ImI W10 and a7T77 (which is probably located in a new loop) and a7N111 (located in loop F; reviewed in [8]) [49]. Although a-CgTxs PnIA and PnIB differ in only two amino acids (A10L and N11S, which are located on the helix face exposed to the solvent residues), the PnIA subtype preferentially inhibits the a3b2 AChR, whereas a-CgTx PnIB is selective for the a7 receptor [50] (Table 3). The fact that H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 the a-CgTx PnIA A10L mutant binds with higher affinity to the a7 receptor suggests that position 10 is important for the observed selectivity [50,51]. These studies also suggest that both A10 and N11 in a-CgTx PnIA independently interact with the a3b2 AChR whereas L10 in a-CgTx PnIB seems to be the only structural requirement for the binding to either a7 or a3b2 subtype [51], as well as for the inhibition of the nicotine-evoked catecholamine release [52]. Subsequent thermodynamic mutant cycle analyses demonstrated the existence of a dominant interaction between aCgTx PnIB L10 and a7W149 (located in loop B; reviewed in [8]), and weaker interactions between a-CgTx PnIB P6 and a7W149, and between both P6 and P7 of a-CgTx PnIB and a7Y93 (located in loop A; reviewed in [8]) [53]. The evidence from mutational experiments also suggests that the binding site for a-CgTx PnIA [50] or for a-CgTx PnIB [53] on the a7 receptor is different from the a-CgTx ImI site [49]. 5. Possible medical applications Nicotinic acetylcholine receptors appear to be important for a number of neurophysiological processes including cognition, learning, and memory. In addition, this receptor family has been implicated in the pathophysiology of several neuropsychiatric disorders including Alzheimer’s and Parkinson’s disease, schizophrenia, Tourette’s syndrome, nocturnal frontal lobe epilepsy, as well as nicotine addiction, myasthenia gravis, and various congenital myasthenic syndromes (reviewed in [63]). Thus, the identification of a ligand with high specificity for certain AChR subtype will be of great importance in the development of new drugs with potential medical uses. In the future, a-CTxs might be used as therapeutic agents in the treatment of some of the above mentioned diseases. In this regard, additional Conus toxins not discussed in this review are being examined for possible clinical use. For example, conantokin-R which inhibits the N-methyl-D-aspartate (NMDA)-type glutamate receptor might have use as an anticonvulsant agent [64]. The v-CTx MVIIA, which is highly specific for the voltage-gated calcium chan- 1025 nels containing the a1B subunit, is being used in clinical trials for the treatment of certain chronic pain syndromes (e.g. intractable pain resulting from cancer, traumatic nerve demage, or amputation) and it has also proved to be useful as a neuroprotector of cerebral ischemia provoked by stroke, cardiac arrest, or head trauma (reviewed in [65]). Finally, an immunoprecipitation assay with [125I]v-CTx is used to diagnose the Lambert – Eaton myastenic syndrome (reviewed in [65]), an autoimmune disease in which antibodies recognize endogenous calcium channels. Acknowledgements This work was supported in part by NINDS Grant R29 NS35786 from the National Institutes of Health (to M.P. Blanton). We thank Dr Tina Machu for her critical reading of the manuscript. References [1] D. Röckel, W. Korn, A.J. Kohn, Manual of Living Conidae; I: Indo-Pacific Region. Verlag Christa Hemmen, Wiesbaden, Germany. [2] B.M. 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