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The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028


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


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


1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018

2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018

3. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020

Abbre6iations: AChR, nicotinic acetylcholine receptor; 5-HT, 5-hydroxytryptamine; 5-HT3R, 5-hydroxytryptamine type 3 recep-
tor; 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: (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 onic or adult stage, respectively. The a1 subunits

contain two adjacent cysteines at position 192 and
From an evolutionary point of view, snails 193 (sequence number from Torpedo AChR),
from the genus Conus are among the most suc- which are involved in the recognition and binding
cessful marine invertebrates (reviewed in [1]). The of cholinergic agonists and competitive antago-
genus Conus is formed by over 500 venomous nists. Based on the presence or the absence of
species. These marine mollusks prey on other these two cysteines, the neuronal-type AChR sub-
marine species by means of a very large number unit classes are designated a (when they contain
of small peptides (10 – 50 amino acids in length) both cysteines) or b (when they do not). To date,
with specific pharmacological activities. Due to eight a subunits (a2– a9) and three b subunits
the remarkably fast interspecific divergence of (b2– b4) have been identified. The a7, a8, and a9
peptide sequences, each Conus species has a reper- polypeptides are the only subunits capable of
toire of 50–200 different peptides (reviewed in forming homo-oligomeric ion channels. Interest-
[2]). These peptides, called conotoxins, affect the ingly, the two a subunits display non-equivalence
functioning of different voltage-gated ion chan- for the binding of several cholinergic agonists and
nels and neurotransmitter-gated receptors (re- competitive antagonists in both muscle- and neu-
viewed in [3,4]). Based on the molecular form, the ronal-type AChRs. In this regard, a-CgTxs have
approximately 50 000 conotoxins can be grouped become one of the most powerful tools to support
into a minimum of seven superfamilies; the A- this non-equivalence (reviewed in [8,9]).
[e.g. a-conotoxins (a-CgTxs), aA-, and kA-
CgTxs], M- (e.g. m- and c-CgTxs), O- (e.g. v-, k-,
d-, and mO-CgTxs), P- (e.g. Spasmodic peptide; 2. Structure
[5]), R-, S- (e.g. s-CgTxs), and T-superfamilies
(e.g. tx5a, p5a, au5a, and au5b; [6]) (reviewed in The initial purification and chemical characteri-
[4,7]). This review will focus on the structure and zation of an a-CgTx was performed by Olivera
pharmacological activity of only the conotoxin and co-workers in 1981 [10]. Since then, a great
family (e.g. a-CgTx and aA-CgTxs) which specifi- deal of structural information has been obtained.
cally bind to several nicotinic acetylcholine recep- Most of them exhibit four Cys residues in the
tors (AChRs) (reviewed in [4,8]). following conserved arrangements; CCX3CX5C
The AChR is the prototype of a superfamily of (the a3/5 subfamily), CCX4CX3C (the a4/3 subfam-
ion channel-coupled receptors which are gated by ily), and CCX4CX7C (the a4/7 subfamily). Each
specific neurotransmitters. This superfamily also alternate Cys pair forms a disulfide loop (i.e.
includes the type A g-aminobutyric acid, glycine, loops I and II, respectively). In each case, the X
and type 3 5-hydroxytryptamine (5-HT) receptors represents the number of amino acids between the
(reviewed in [8,9]). There are two main AChR Cys residues. The only representative of the a3/5/3
types, the muscle- and neuronal-type. The muscle- subfamily, which has three Cys bonds, is a-CgTx
type AChR is a pentamer comprised of two a1 SII. The spacing between disulfide bonds is an
subunits, one b1, one d and, one g or o subunit, important determinant of backbone structure.
depending on whether the receptor is in an embry- Additionally, a conserved Pro residue is found
H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 1019

between the second and the third Cys in almost all subfamilies [11–13]. Table 1 shows the primary
a-CgTxs that have been characterized to date. and secondary structural features of some a- and
The exception is the aA-CgTx subfamily (e.g. aA-CgTxs as examples of each subfamily.
aA-PIVA, aA-EIVA, and aA-EIVB), which has a The three-dimensional structures of several a-
core sequence that is very different than the other CgTxs 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]. ing Tyr residue on the surface of the a-CgTx
These studies provide support for the idea that PnIA structure (panel C, in purple) contrasts with
these small polypeptides achieve their conforma- the flat hydrophobic surface of a-CgTx MII
tional stability by means of disulfide bonding. (panel D, residues in purple). Furthermore,
Although a-CgTxs are apparently very rigid struc- charged residues are exposed on the a-CgTx MII
tures, two or more interconvertible conformers surface (panel D, negative residues in blue and
may exist in solution (reviewed in [4]). The overall positive residues in red), whereas they are not
shapes of the different a-CgTx subfamilies are as exposed on the a-CgTx PnIA surface (panel C).
follow: the a4/7 subfamily is more rectangular, the In addition, both the backbone and the surface of
a3/5 subfamily is more triangular, whereas the aA a-CgTx GI (panel E), which is specific for muscle-
subfamily has been described as an iron. The type AChRs (see Table 2), diverge significantly
structural comparison of several a-CgTxs specific from the other PnIA and MII a-CgTxs.
for neuronal-type AChRs, such as a-CgTx ImI,
PnIA, PnIB, MII, and EpI (Table 3), exhibit
remarkable similarity in local backbone confor- 3. Synthesis
mations and relative solvent-accessible surface ar-
eas [21]. A model of the tertiary structure of The Conus venom is biosynthesized and stored
a-CgTxs PnIA, MII, and GI is shown in Fig. 1 in the venom duct of the venom apparatus (re-
(taken from [16]). The backbones of the a-CgTxs viewed in [27]). When the snail is hunting prey,
PnIA and MII structures (panels C and D, respec- the venom, by contraction of the muscular venom
tively) are very similar. Nevertheless, the surfaces bulb, is delivered through a harpoon-like radula
of both the polypeptides are unique. The protrud- tooth located in the proboscis.

Table 1
Primary and secondary structure of each conotoxin subfamily

a-Conotoxin Conus species Subfamily Primarya and secondaryb structure References

? ?
MI Conus magus a3/5 GRCCHPACGKNYSCc [30]
? ?
? ?
PnIA Conus pennaceus a4/7 GCCSLPPCAANNPDYdCc [55]
? ?
? ?
ImI Conus imperialis a4/3 GCCSDPRCAWRCc [56]
? ?
? ?
SII Conus striatus a3/5/3 GCCCNPACGPNYGCCGTSCS [57]
? ?
? ?
? ? ? ?
The sequence alignment of the different a-CgTxs is shown in the standard one-letter amino acid code. The letter O represents
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.
Amidated COOH-terminus. Instead, a-CgTx SII presents a free COOH-terminus.
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)

High-affinity binding site Low-affinity binding site


MI BC3H-1 1.5090.24 (ad) 22.0 9 1.1 (ag) [30]

0.429 0.15 (ad) 23.09 4.1 (ag) [31]
0.409 0.01b (a2bgd) [12]
0.409 0.17b (abd) 1895 (abg) [12]
1.4 9 0.1b (kinetic) [12]
Mouse 0.55 9 0.06 (a2bgd) 18.390.93 (a2bgd) [42]
1.34 (abd) 11.7 (abg) [42]
5.3 (a2bd) 1391.3 (a2bgd) [39]
1.9 (ad) 15 (ag) [32]
12b (ad) [58]
2.89 1.3 (ad) 7.8 9 1.3 (ag) [33]
Torpedo 2.691.0 (ag) 2.39 0.7 (ad) [30]
1209 10 (ag) 219 1 (ad) [45]
4.5091.60 (ag) 0.489 0.10 (ad) [31]
1.69 1.4 (a2bgd) 1.09 0.3 (a2bgd) [40]
329 2 (abg) 1.290.1 (abd) [40]
Human (a2bod) 99 1 (ad) 0.099 0.01 (ao) [48]
GI BC3H-1 4.991.9 (ad) 58912 (ag) [30]
1.3 90.3 (ad) 609 1 (ag) [43]
Mouse 20b (ad) [58]
Torpedo 360940 (ag) 2492 (ad) [45]
4.59 1.3 (ag) 0.099 0.02 (ad) [43]
41.39 8.2c (ag) [59]
Human (a2bod) 29 1 (ad) 0.149 0.01 (ao) [48]
Frog 2-4b [56]
Fish (Eigenmannia) 50-100b [56]
CnIA Torpedo 190 [19]
SI BC3H-1 6809 160 (ad) 2209 45 (ag) [30]
13009 430 (ad) 2909 110 (ag) [43]
Human (a2bod) 809 14 (ao) 8.3191.2 (ad) [48]
Torpedo 0.17 90.04 (ag or ad) [44]
16 91 (ag or ad) [45]
1 [57]
SIA BC3H-1 7.709 0.14 (ad) 2009 8.6 (ag) [30]
Torpedo 5909 40 (ag) \260 (ad) [45]
EI BC3H-1 9.4091.20 (ad) 0.28 9 0.03 (ag) [31]
Torpedo 0.41 90.09 (ad) 0.199 0.02 (ag) [31]
SII BC3H-1 18 9 6.6 [30]
Torpedo 8 [57]
aA-EIVA Torpedo 17b [12]
Mouse 11b (a2bgd) [12]
11b (abg)
32b (kinetic)
15b (abd)
37b (kinetic)
BC3H-1 1509 10 [12]
aA-EIVB Torpedo 18b [12]
aA-PIVA Torpedo 1 [13]

These values were obtained by inhibition of [125I]a-BTx binding except those determined by the following: b
or fluorescence spectroscopy.
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 a

IC50 nM References

CnIA a7 14 800 b [19]

MII a3b2 3.5 [46]
8.0 9 1.1 [36]
24.3 9 2.9c (synaptosomes) [36]
17.3 90.1c (slices) [36]
a4b2 400 [46]
a3b4 3000d (noradrenaline) [62]
EpI a3b4 84 919d (adrenaline) [54]
210 9 30d (noradrenaline) [54]
a3b2/a3b4 1.6 [54]
PnIA a7/? 14 [50]
a7 252 [51]
176 (Kd) [51]
a7 (human)/5-HT3R (rat) 61 200 9 1 100b [53]
a3b2 9.56 [51]
a3b4 21 000–28 000d (noradrenaline) [52]
AuIB a3b4 500 9140 (Kd) [37]
750 [37]
20 000d (noradrenaline) [52]
a7 \7000 [37]
PnIB a7/? 33 [50]
a7 61.3 [51]
84.9 (Kd) [51]
a7 (human)/5-HT3R (rat) 29 600 9600b [53]
a3b2 1970 [51]
a3b4 700b [30]
700d (noradrenaline) [52]
1000d (adrenaline) [52]
ImI a7 (rat) 220 [58]
a7 100 [60]
a7 86.2 9 1.2 [61]
a7 (human) 2450 9 100b [48]
a9 1800 [58]
a3b4/a3b4a5 2500 9 1200e [34]
a3b4 \3000 [60]
Aplysia 47 (desensitizing Cl− response) [35]
\20 000 (sustained Cl− response)
150 922 (cationic response)

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 se- conotoxins have been considered to be initially
quences by amino acid hypermutation (reviewed translated as larger prepropeptide precursors 70–
in [2,7]). The synthesis of conotoxins can be com- 120 amino acids in length with a single copy of
pared with a combinatorial library search strat- the toxin present at the C-terminal end. Other
egy. From studies with the O-superfamily, neuropeptide precursors encode either multiple
H.R. Arias, M.P. Blanton / The International Journal of Biochemistry & Cell Biology 32 (2000) 1017–1028 1023

copies of a specific peptide or several distinct toxins. were used for photoaffinity labeling of the AChR
The high number of polypeptide structures ob- [29]. These studies showed that depending on the
served in different cone snails that inhibit a a-CgTx derivative used, the specifically labeled
specific target are thought to have evolved by AChR subunits were b and g, or d and g. However,
hypermutation of the amino acids located closer to labeling of detergent-solubilized AChR was exclu-
the C-terminal. Exceptions are the Cys residues sive for residues 121 and 183 of the g subunit. The
located in the toxin proper. The rest of the precur- p-benzoylphenylalanine derivative of a-CgTx GI
sor sequence remains highly conserved. The signal also labeled the a subunits [38].
sequence is the polypeptide region with the highest In order to identify the determinants of a-CgTx
level of sequence conservation. Between the signal MI selectivity, Dr Steven Sine’s laboratory used
sequence and the mature toxin there exists an subunit chimeras and site-directed mutagenesis
intervening pro-region 40 amino acids in length [33,39]. From these studies, it was found that the
which exhibits a low mutation rate. 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,
4. Biological function while the low affinity for the ag interface is deter-
mined by residues K34, S111, Y117, L119, and F172 of
The pioneering studies done in Dr Baldomero the g subunit. Since dY113 and gS111 are exchanged
Olivera’s laboratory provide the initial methodol- for Arg and Tyr in the Torpedo AChR, these two
ogy for determining the biological activity of a- natural differences may account for the observed
site-specificity between the two species. This idea is
CgTxs. Initial pharmacological data demonstrated
corroborated by the fact that the mutation dR113Y
that, in general, the family of a-CgTxs behave as
in the a2bd2 or the mutation gY111R in a2bg2
competitive antagonists of the AChR (reviewed in
Torpedo AChR results either in an enhancement of
[7,8]). The a-CgTxs compete for the binding sites
or a decrease in a-CgTx MI affinity, respectively
of acetylcholine and cholinergic agonists. However,
[40]. The pairs gK34/dS36 and gF172/dI178, as pri-
a distinct conotoxin from Conus purpurascens,
mary determinants for a-CgTx MI selectivity in
called c-CgTx PIIIE, inhibits the functional activ-
mouse AChR, coincide with that for the selectivity
ity of the AChR in a non-competitive manner [28].
of carbamylcholine [41]. In contrast, neither the
The earliest studies showed that a-CgTxs inhibited gS111Y nor dY113S mutation affected carbamyl-
muscle-type AChRs. Table 2 shows the a-CTx choline affinity, suggesting that agonists and at
specificities for muscle-type AChRs from different least the a-CgTx MI subtype do not have identical
species. Nonequivalent binding of some a-CgTxs at selectivity determinants. Residues gK34, gS111, and
the two agonist/competitive antagonist binding gF172 contribute to loops D, F, and G, respectively,
domains in Torpedo AChR [29 – 31] is also summa- of the agonist/competitive antagonist binding site
rized in Table 1. Although early studies focused on (reviewed in [8]). Since other residues from the a
a-CgTxs action in muscle-type AChRs, more recent subunit are considered to be involved in the ago-
efforts have identified pharmacological effects of nist/competitive antagonist binding sites (reviewed
these compounds in neuronal-type AChRs. Table in [8,9]), Sugiyama et al. [42] examined the contri-
3 shows the a-CgTx specificities for different neu- bution of some of these residues to the binding of
ronal-type AChRs. a-CgTx MI. Mutations aY190F and aY189F do
In order to determine which subunits of the not affect a-CgTx MI affinity, whereas removal of
Torpedo AChR are involved in the a-CgTx binding aromaticity by exchanging these residues for Thr
site, purified a-CgTx MI was crosslinked to the has a marked influence on a-CgTx association. This
AChR with bivalent succinimide reagents of differ- suggests that aromaticity may be required to stabi-
ent lengths [29]. With a 12-atom crosslinker, all the lize the cationic peptide. The effect elicited by
four subunits were labeled, whereas a 4-atom substitutions of Y93 (loop A; see [8]) and D152
crosslinker labeled the b and g subunits. In addi- residue (loop B; see [8]) indicate that both peptide
tion, two azidosalicylate a-CgTx GIA derivatives 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 mologous residue of A9, resulted in a loss of
amino acids contribute to the binding domain. In selectivity [45].
addition, substitution of Y12A on the MI toxin Taking into account that neuronal receptors
dramatically reduces its affinity for the high- containing the subunit composition a4b2, a2b2,
affinity site (ad) with little effect on toxin potency or a3b4 are more than 200-fold less sensitive to
at the low-affinity site (ag) [43]. This and addi- a-CgTx MII than a3b2 AChRs, the determinants
tional data suggest that the orientation of residue of a-CgTx selectivity were identified using
Y12 is important in the formation of the a-CgTx chimeric subunits and subunits with single residue
MI-AChR complex. substitutions [46]. Residues b2T59, a3K185, and
In order to determine the existence of a linkage a3I188 were identified as specific determinants for
relationship between mutations in a and d/g sub- a-CgTx MII sensitivity. The amino acid a3K185
units, a mutant cycle analysis was employed [42]. may electrostatically interact with E11 from a-
From this study, a high coupling energy between CgTx MII.
S36 and I178 of the d subunit was demonstrated. In Regarding a-CgTx ImI specificity, the pairs
contrast, a relatively low linkage between residues a7W55/a1R55, a7S59/a1Q59, and a7T77/a1K77 have
aY93/V188 and pairs gK34/dS36, gS111/dY113, and been considered as components conferring high
gF172/dI178 is evident. Taking into account that affinity binding to a7/5-HT3R compared with a1/
the energetic contributions of amino acids in the a 5-HT3R homooligomeric chimeras [47] (reviewed
chain to a-CgTx MI association with the AChR in [8]). The third pair (a7T77/a1K77) may be con-
seem to be independent from the ones at the d/g sidered as a new loop or an allosterically coupled
subunits, it is postulated that one of the surfaces loop. Experiments performed in parallel show
of the neurotoxin molecule interacts with the a that two regions in the a-CgTx ImI molecule are
subunit, whereas the other surface interacts with essential for binding to the a7/5-HT3R chimera
the d or the g subunit. In this regard, recent [48]: a region comprising residues D5-P6-R7 in the
conformational sudies using [H1]-NMR spec- first loop and a second region in loop II formed
troscopy suggest that both the faces of the a- by W10. The fact that D5 functions as an N-termi-
CgTx GI are involved in the orientation of the nal cap and P6 as a helix-initiator suggests that
molecule within the ad subunit interface [25]. The the contribution of both the amino acids to bind-
binding face of a-CgTx GI, a toxin closely related ing may be due to their structural roles rather
to the MI subtype in structure, interacts by means than due to direct interaction with the AChR [21].
of residues C2, N4, P5, A6, and C7 (from loop I) The structural role of P6 was recently corrobo-
with the a1 subunit, whereas the selectivity face rated by mutagenesis studies [49]. Subsequent
comprising amino acids R9 and H10 (from loop II) thermodynamic mutant cycle analyses demon-
is oriented towards the d subunit (the subunit strated the existence of a dominant pairwise inter-
forming the high-affinity a-CgTx GI locus in action between a-CgTx ImI R7 and a7Y195
mammalian AChRs). Residues R9 and H10 were (located in loop C; reviewed in [8]), and multiple
found to be responsible for the high differential weak interactions between a-CgTx ImI D5 and
selectivity and affinity between both the choliner- W149, Y151, and G153 of a7 (which are all located
gic ligand binding sites [44,45]. The lack of effect in loop B; reviewed in [8]), and between a-CgTx
of the mutation P9 to the neutral residue Ala in ImI W10 and a7T77 (which is probably located in
the a-CgTx SI suggests that the cationic group of a new loop) and a7N111 (located in loop F; re-
A9 in the a-CgTx GI plays a major role in ag viewed in [8]) [49].
selectivity in the Torpedo receptor. The critical Although a-CgTxs PnIA and PnIB differ in
difference between a-CgTx GI and SI has been only two amino acids (A10L and N11S, which are
ascribed to position 9 (reviewed in [24]). The located on the helix face exposed to the solvent
importance of a cationic group for high selectivity residues), the PnIA subtype preferentially inhibits
is further substantiated by the fact that mutations the a3b2 AChR, whereas a-CgTx PnIB is selective
on the a-CgTx MI at position K10, the ho- 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 1025

the a-CgTx PnIA A10L mutant binds with higher nels containing the a1B subunit, is being used in
affinity to the a7 receptor suggests that position clinical trials for the treatment of certain chronic
10 is important for the observed selectivity [50,51]. pain syndromes (e.g. intractable pain resulting
These studies also suggest that both A10 and N11 from cancer, traumatic nerve demage, or amputa-
in a-CgTx PnIA independently interact with the tion) and it has also proved to be useful as a
a3b2 AChR whereas L10 in a-CgTx PnIB seems neuroprotector of cerebral ischemia provoked by
to be the only structural requirement for the stroke, cardiac arrest, or head trauma (reviewed
binding to either a7 or a3b2 subtype [51], as well in [65]). Finally, an immunoprecipitation assay
as for the inhibition of the nicotine-evoked cate- with [125I]v-CTx is used to diagnose the Lam-
cholamine release [52]. Subsequent thermody- bert–Eaton myastenic syndrome (reviewed in
namic mutant cycle analyses demonstrated the [65]), an autoimmune disease in which antibodies
existence of a dominant interaction between a- recognize endogenous calcium channels.
CgTx 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 Acknowledgements
loop A; reviewed in [8]) [53]. The evidence from
mutational experiments also suggests that the This work was supported in part by NINDS
binding site for a-CgTx PnIA [50] or for a-CgTx Grant R29 NS35786 from the National Institutes
PnIB [53] on the a7 receptor is different from the of Health (to M.P. Blanton). We thank Dr Tina
a-CgTx ImI site [49]. Machu for her critical reading of the manuscript.

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