Alpha conotoxins

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The International Journal of Biochemistry & Cell Biology 32 (2000) 10171028

Review

-Conotoxins

Hugo R. Arias *, Michael P. Blanton

Departments of Pharmacology and Anesthesiology, School of Medicine, Texas Tech Uniersity Health Sciences Center, Lubbock

TX79430, USA

Received 16 May 2000; accepted 2 August 2000

Abstract

-Conotoxins (-CgTxs) are a family of Cys-enriched peptides found in several marine snails from the genus Conu

These small peptides behave pharmacologically as competitive antagonists of the nicotinic acetylcholine recept

(AChR). The data indicate that (1) -CgTxs are able to discriminate between muscle- and neuronal-type AChRs an

even among distinct AChR subtypes; (2) the binding sites for -CgTxs are located, like other cholinergic ligands,

the interface of and non-subunits (, , and for the muscle-type AChR, and for several neuronal-type AChR

(3) some -CgTxs differentiate the high- from the low-affinity binding site found on either /non- subunit interfac

and that (4) specific residues in the cholinergic binding site are energetically coupled with their corresponding pai

in the toxin stabilizing the -CgTx-AChR complex. The-CgTxs have proven to be excellent probes for studying th

structure and function of the AChR family. 2000 Elsevier Science Ltd. All rights reserved.

Keywords:-Conotoxins; Competitive antagonists; Nicotinic acetylcholine receptors

Contents

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

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

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

www.elsevier.com/locate/ijb

Abbreiations: AChR, nicotinic acetylcholine receptor; 5-HT, 5-hydroxytryptamine; 5-HT3R, 5-hydroxytryptamine type 3 rece

tor; NMDA, N-methyl-D-aspartate; -BTx,-bungarotoxin; -CgTx, -conotoxin; A-CgTx,A-conotoxin; -CgTx, -conotoxi

-CgTx, -conotoxin; -CgTx, -conotoxin; -CgTx, -conotoxin; O-CgTx,O-conotoxin; -CgTx, -conotoxin.

* Corresponding author. Tel.: +1-806-7432425, ext.: 244; fax: +1-806-7432744.

E-mail address:[email protected] (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


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H.R. Arias, M.P. Blanton/The International Journal of Biochemistry & Cell Biology 32 (2000) 101710281018

4. Biological function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1023

5. P ossible medical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025

1. Introduction

From an evolutionary point of view, snails

from the genus Conus are among the most suc-

cessful 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 (1050 amino acids in length)

with specific pharmacological activities. Due to

the remarkably fast interspecific divergence ofpeptide sequences, eachConusspecies has a reper-

toire of 50 200 different peptides (reviewed in

[2]). These peptides, called conotoxins, affect the

functioning of different voltage-gated ion chan-

nels and neurotransmitter-gated receptors (re-

viewed 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. -conotoxins (-CgTxs), A-, and A-

CgTxs], M- (e.g. - and -CgTxs), O- (e.g. -, -,

-, and O-CgTxs), P- (e.g. Spasmodic peptide;[5]), R-, S- (e.g. -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. -CgTx and A-CgTxs) which specifi-

cally bind to several nicotinic acetylcholine recep-

tors (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 -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 muscle-

type AChR is a pentamer comprised of two 1

subunits, one 1, one and, one or subunit,

depending on whether the receptor is in an embry-

onic or adult stage, respectively. The 1 subuni

contain two adjacent cysteines at position 192 an

193 (sequence number from Torpedo AChR

which are involved in the recognition and bindin

of cholinergic agonists and competitive antag

nists. Based on the presence or the absence

these two cysteines, the neuronal-type AChR sub

unit classes are designated (when they conta

both cysteines) or (when they do not). To dat

eight subunits (2 9) and three subuni

(2 4) have been identified. The 7, 8, and polypeptides are the only subunits capable

forming homo-oligomeric ion channels. Interes

ingly, the two subunits display non-equivalenc

for the binding of several cholinergic agonists an

competitive antagonists in both muscle- and neu

ronal-type AChRs. In this regard, -CgTxs hav

become one of the most powerful tools to suppo

this non-equivalence (reviewed in [8,9]).

2. Structure

The initial purification and chemical character

zation of an -CgTx was performed by Olive

and co-workers in 1981 [10]. Since then, a grea

deal of structural information has been obtaine

Most of them exhibit four Cys residues in th

following conserved arrangements; CCX3CX5(the 3/5 subfamily), CCX4CX3C (the 4/3subfam

ily), and CCX4CX7C (the 4/7 subfamily). Eac

alternate Cys pair forms a disulfide loop (i.

loops I and II, respectively). In each case, the represents the number of amino acids between th

Cys residues. The only representative of the 3/5subfamily, which has three Cys bonds, is -CgT

SII. The spacing between disulfide bonds is a

important determinant of backbone structur

Additionally, a conserved Pro residue is foun


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H.R. Arias, M.P. Blanton/The International Journal of Biochemistry & Cell Biology 32 (2000) 10171028 10

between the second and the third Cys in almost all

-CgTxs that have been characterized to date.

The exception is the A-CgTx subfamily (e.g.

A-PIVA, A-EIVA, and A-EIVB), which has a

core sequence that is very different than the other

subfamilies [11 13]. Table 1 shows the prima

and secondary structural features of some - an

A-CgTxs as examples of each subfamily.

The three-dimensional structures of several

CgTxs have been determined using either X-ra

Fig. 1. Surface models of-CgTxs PnIA (A) and MII (B) and backbone representations of-CgTxs PnIA (C), MII (D), and GI (

(taken from [16], with permission). Hydrophobic, polar, positive-charged, and negative-charged residues are displayed in purp

yellow, red, and blue, respectively. Backbone conformations of -CgTxs PnIA (C), MII (D), and GI (E) are shown in ribbon

Surface distributions in dots with the same color codes. Arrows indicate carboxyl (blue) and amino (yellow) terminal residues.


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crystallography or NMR spectroscopy [11,1426].

These studies provide support for the idea that

these small polypeptides achieve their conforma-

tional stability by means of disulfide bonding.

Although-CgTxs are apparently very rigid struc-

tures, two or more interconvertible conformers

may exist in solution (reviewed in [4]). The overall

shapes of the different -CgTx subfamilies are as

follow: the 4/7subfamily is more rectangular, the

3/5 subfamily is more triangular, whereas the A

subfamily has been described as an iron. The

structural comparison of several -CgTxs specific

for neuronal-type AChRs, such as -CgTx ImI,

PnIA, PnIB, MII, and EpI (Table 3), exhibit

remarkable similarity in local backbone confor-

mations and relative solvent-accessible surface ar-

eas [21]. A model of the tertiary structure of

-CgTxs PnIA, MII, and GI is shown in Fig. 1

(taken from [16]). The backbones of the -CgTxs

PnIA and MII structures (panels C and D, respec-tively) are very similar. Nevertheless, the surfaces

of both the polypeptides are unique. The protrud-

ing Tyr residue on the surface of the -CgT

PnIA structure (panel C, in purple) contrasts wit

the flat hydrophobic surface of -CgTx M

(panel D, residues in purple). Furthermor

charged residues are exposed on the -CgTx M

surface (panel D, negative residues in blue an

positive residues in red), whereas they are n

exposed on the -CgTx PnIA surface (panel C

In addition, both the backbone and the surface o

-CgTx GI (panel E), which is specific for muscl

type AChRs (see Table 2), diverge significant

from the other PnIA and MII -CgTxs.

3. Synthesis

The Conus venom is biosynthesized and store

in the venom duct of the venom apparatus (r

viewed in [27]). When the snail is hunting pre

the venom, by contraction of the muscular venobulb, is delivered through a harpoon-like radu

tooth located in the proboscis.

Table 1

Primary and secondary structure of each conotoxin subfamily

Conus species-Conotoxin Subfamily Primarya and secondaryb structure References

[30]Conus magus GRCCHPACGKNYSCcMI 3/5

Conus pennaceus [55]PnIA GCCSLPPCAANNPDYdCc4/7

Conus imperialis 4/3ImI GCCSDPRCAWRCc [56]

GCCCNPACGPNYGCCGTSCSSII [57]Conus striatus 3/5/3

A-PIVA A GCCGSYONAACHOCSCKDROSYCGc [13]Conus purpurascens

a The sequence alignment of the different -CgTxs is shown in the standard one-letter amino acid code. The letter O represen

trans-4-hydroxyproline.b -Conotoxin subfamilies3/5, 4/7, and 4/3 are formed by two disulfide bonds, while the A-CgTx subfamily and -CgTx S

(3/5/3 subfamily) have three disulfide bonds.c Amidated COOH-terminus. Instead, -CgTx SII presents a free COOH-terminus.d Sulfated residue.

? ?

? ?? ?

? ?

? ?

? ?

? ?

? ?

? ?

? ? ? ?


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Table 2

-Conotoxin specificity for different muscle-type nicotinic acetylcholine receptors

AChR Source-ConotoxinReferencesIC

a

50

(subunit interface)

High-affinity binding site Low-affinity binding siteNM M

1.500.24 ()MI 22.0 1.1 ()BC3H-1 [30]0.420.15 () 23.04.1 () [31]0.400.01b (2) [12]0.400.17b () 185 () [12]1.40.1b (kinetic) [12]

Mouse 0.550.06 (2) 18.30.93 (2) [42]1.34 () 11.7 () [42]5.3 (2) 131.3 (2) [39]1.9 () 15 () [32]12b () [58]2.81.3 () 7.8 1.3 () [33]2.61.0 () 2.30.7 ()Torpedo [30]12010 () 211 () [45]4.501.60 () 0.480.10 () [31]

1.61.4 (2) 1.00.3 (2) [40]322 () 1.20.1 () [40]91 () 0.090.01 () [48]Human (2)4.91.9 () 5812 ()BC3H-1 [30]GI1.30.3 () 601 () [43]20b ()Mouse [58]

Torpedo 36040 () 242 () [45]4.51.3 () 0.090.02 () [43]41.38.2c () [59]21 () 0.140.01 () [48]Human (2)

2-4bFrog [56]50-100b [56]Fish (Eigenmannia)190Torpedo [19]CnIA680160 () 22045 ()SI [30]BC3H-11300430 () 290110 () [43]

Human (2) 8014 () 8.311.2 () [48]0.170.04 ( or )Torpedo [44]161 ( or ) [45]1 [57]

BC3H-1SIA 7.700.14 () 2008.6 () [30]59040 () 260 ()Torpedo [45]420

EI 9.401.20 ()BC3H-1 0.280.03 () [31]0.410.09 () 0.190.02 ()Torpedo [31]

186.6SII [30]BC3H-18Torpedo [57]

TorpedoA-EIVA 17b [12]11b (2)Mouse [12]11b ()32b (kinetic)

15b

()37b (kinetic)15010BC3H-1 [12]

A-EIVB 18bTorpedo [12]1 [13]TorpedoA-PIVA

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

or c fluorescence spectroscopy.


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Table 3

-Conotoxin specificity for different neuronal-type nicotinic acetylcholine receptors

-Conotoxin AChR SubtypeReferencesIC

a

50 nM

CnIA 14 800 b7 [19]

3.5 [46]MII 32

8.01.1 [36]

24.32.9c (synaptosomes) [36]

17.30.1c (slices) [36]

40042 [46]

34 3000d (noradrenaline) [62]

8419d (adrenaline) [54]EpI 34

21030d (noradrenaline) [54]

32/34 1.6 [54]

14PnIA [50]7/?

2527 [51]

176 (Kd) [51]

61 2001 100b [53]7 (human)/5-HT3R (rat)

9.5632 [51]

34 21 00028 000d (noradrenaline) [52]

500140 (Kd)AuIB [37]34

750 [37]20 000d (noradrenaline) [52]

7000 [37]7

337/? [50]PnIB

7 61.3 [51]

84.9 (Kd) [51]

29 600600b7 (human)/5-HT3R (rat) [53]

32 1970 [51]

700b [30]34

700d (noradrenaline) [52]

1000d (adrenaline) [52]

220ImI [58]7 (rat)

1007 [60]

300d

86.21.2 [61]7

2450100b7 (human) [48]

9 1800 [58]

25001200e [34]34/345

300034 [60]

Aplysia 47 (desensitizing Cl response) [35]

20 000 (sustained Cl response)

15022 (cationic response)

a These values were obtained by electrophysiological techniques except those determined by the following: b inhibition

[125I]-BTx binding, inhibition of agonistinduced, c dopamine, d catecholamine (e.g. adrenaline or noradrenaline), e 5-HT relea

Cone snails generate novel polypeptide se-

quences by amino acid hypermutation (reviewed

in [2,7]). The synthesis of conotoxins can be com-

pared with a combinatorial library search strat-

egy. From studies with the O-superfamily,

conotoxins have been considered to be initial

translated as larger prepropeptide precursors 70

120 amino acids in length with a single copy o

the toxin present at the C-terminal end. Oth

neuropeptide precursors encode either multip


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copies of a specific peptide or several distinct toxins.

The high number of polypeptide structures ob-

served 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 precur-

sor 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

Oliveras laboratory provide the initial methodol-

ogy for determining the biological activity of -CgTxs. Initial pharmacological data demonstrated

that, in general, the family of-CgTxs behave as

competitive antagonists of the AChR (reviewed in

[7,8]). The -CgTxs compete for the binding sites

of acetylcholine and cholinergic agonists. However,

a distinct conotoxin from Conus purpurascens,

called-CgTx PIIIE, inhibits the functional activ-

ity of the AChR in a non-competitive manner [28].

The earliest studies showed that -CgTxs inhibited

muscle-type AChRs. Table 2 shows the -CTx

specificities for muscle-type AChRs from differentspecies. Nonequivalent binding of some -CgTxs at

the two agonist/competitive antagonist binding

domains inTorpedoAChR [2931] is also summa-

rized in Table 1. Although early studies focused on

-CgTxs action in muscle-type AChRs, more recent

efforts have identified pharmacological effects of

these compounds in neuronal-type AChRs. Table

3 shows the -CgTx specificities for different neu-

ronal-type AChRs.

In order to determine which subunits of the

TorpedoAChR are involved in the-CgTx bindingsite, purified -CgTx MI was crosslinked to the

AChR with bivalent succinimide reagents of differ-

ent lengths [29]. With a 12-atom crosslinker, all the

four subunits were labeled, whereas a 4-atom

crosslinker labeled the and subunits. In addi-

tion, two azidosalicylate -CgTx GIA derivatives

were used for photoaffinity labeling of the ACh

[29]. These studies showed that depending on th

-CgTx derivative used, the specifically labeleAChR subunits wereand, or and. Howeve

labeling of detergent-solubilized AChR was exclsive for residues 121 and 183 of the subunit. Th

p-benzoylphenylalanine derivative of -CgTx Galso labeled the subunits [38].

In order to identify the determinants of-CgTMI selectivity, Dr Steven Sines laboratory use

subunit chimeras and site-directed mutagenes[33,39]. From these studies, it was found that th

high affinity of subtype MI for the subuninterface of mammalian AChRs is determined b

amino acids S36, Y113, and I178 from the subunwhile the low affinity for the interface is dete

mined by residues K34, S111, Y117, L119, and F172

the subunit. SinceY113 andS111 are exchange

for Arg and Tyr in the Torpedo AChR, these tw

natural differences may account for the observesite-specificity between the two species. This idea corroborated by the fact that the mutation R113in the 22 or the mutation Y111R in 2

TorpedoAChR results either in an enhancement oor a decrease in -CgTx MI affinity, respective

[40]. The pairs K34/S36 and F172/I178, as prmary determinants for -CgTx MI selectivity

mouse AChR, coincide with that for the selectiviof carbamylcholine [41]. In contrast, neither th

S111Y nor Y113S mutation affected carbamycholine affinity, suggesting that agonists and

least the-CgTx MI subtype do not have identicselectivity determinants. Residues K34, S111, an

F172 contribute to loops D, F, and G, respectivelof the agonist/competitive antagonist binding si

(reviewed in [8]). Since other residues from thesubunit are considered to be involved in the ago

nist/competitive antagonist binding sites (reviewein [8,9]), Sugiyama et al. [42] examined the contrbution of some of these residues to the binding

-CgTx MI. Mutations Y190F and Y189F dnot affect-CgTx MI affinity, whereas removal o

aromaticity by exchanging these residues for Thhas a marked influence on-CgTx association. Th

suggests that aromaticity may be required to stablize the cationic peptide. The effect elicited b

substitutions of Y93 (loop A; see [8]) and D1

residue (loop B; see [8]) indicate that both peptid

and nonpeptidic ligands bind to the same site i


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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 high-

affinity site () with little effect on toxin potency

at the low-affinity site () [43]. This and addi-

tional data suggest that the orientation of residue

Y12 is important in the formation of the -CgTx

MI-AChR complex.

In order to determine the existence of a linkage

relationship between mutations in and / sub-

units, a mutant cycle analysis was employed [42].

From this study, a high coupling energy between

S36 and I178 of the subunit was demonstrated. In

contrast, a relatively low linkage between residues

Y93/V188 and pairs K34/S36, S111/Y113, and

F172/I178 is evident. Taking into account that

the energetic contributions of amino acids in the

chain to -CgTx MI association with the AChR

seem to be independent from the ones at the /subunits, it is postulated that one of the surfaces

of the neurotoxin molecule interacts with the

subunit, whereas the other surface interacts with

the or the subunit. In this regard, recent

conformational sudies using [H1]-NMR spec-

troscopy suggest that both the faces of the -

CgTx GI are involved in the orientation of the

molecule within the subunit interface [25]. The

binding face of-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 1 subunit, whereas the selectivity face

comprising amino acids R9 and H10 (from loop II)

is oriented towards the subunit (the subunit

forming the high-affinity -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 choliner-

gic ligand binding sites [44,45]. The lack of effect

of the mutation P9 to the neutral residue Ala in

the -CgTx SI suggests that the cationic group of

A9

in the -CgTx GI plays a major role in selectivity in the Torpedo receptor. The critical

difference between -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 -CgTx MI at position K10, the ho-

mologous residue of A9, resulted in a loss

selectivity [45].

Taking into account that neuronal recepto

containing the subunit composition 42, 2

or 34 are more than 200-fold less sensitive t

-CgTx MII than 32 AChRs, the determinan

of -CgTx selectivity were identified usin

chimeric subunits and subunits with single residu

substitutions [46]. Residues 2T59, 3K185, an

3I188 were identified as specific determinants fo

-CgTx MII sensitivity. The amino acid 3K1

may electrostatically interact with E11 from

CgTx MII.

Regarding -CgTx ImI specificity, the pai

7W55/1R55, 7S59/1Q59, and 7T77/1K77 hav

been considered as components conferring hig

affinity binding to 7/5-HT3R compared with

5-HT3R homooligomeric chimeras [47] (reviewe

in [8]). The third pair (7T77/1K77) may be con

sidered as a new loop or an allosterically coupleloop. Experiments performed in parallel sho

that two regions in the -CgTx ImI molecule a

essential for binding to the 7/5-HT3R chime

[48]: a region comprising residues D5-P6-R7 in th

first loop and a second region in loop II forme

by W10. The fact that D5 functions as an N-term

nal cap and P6 as a helix-initiator suggests th

the contribution of both the amino acids to bind

ing may be due to their structural roles rath

than due to direct interaction with the AChR [21

The structural role of P6 was recently corrobrated by mutagenesis studies [49]. Subsequen

thermodynamic mutant cycle analyses demo

strated the existence of a dominant pairwise inte

action between -CgTx ImI R7 and 7Y1

(located in loop C; reviewed in [8]), and multip

weak interactions between -CgTx ImI D5 an

W149, Y151, and G153 of7 (which are all locate

in loop B; reviewed in [8]), and between -CgT

ImI W10 and 7T77 (which is probably located

a new loop) and 7N111 (located in loop F; r

viewed in [8]) [49].Although -CgTxs PnIA and PnIB differ

only two amino acids (A10L and N11S, which ar

located on the helix face exposed to the solven

residues), the PnIA subtype preferentially inhibi

the 32 AChR, whereas-CgTx PnIB is selectiv

for the 7 receptor [50] (Table 3). The fact th


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the -CgTx PnIA A10L mutant binds with higher

affinity to the 7 receptor suggests that position

10 is important for the observed selectivity [50,51].

These studies also suggest that both A10 and N11

in -CgTx PnIA independently interact with the

32 AChR whereas L10 in -CgTx PnIB seems

to be the only structural requirement for the

binding to either 7 or 32 subtype [51], as well

as for the inhibition of the nicotine-evoked cate-

cholamine release [52]. Subsequent thermody-

namic mutant cycle analyses demonstrated the

existence of a dominant interaction between -

CgTx PnIB L10 and 7W149 (located in loop B;

reviewed in [8]), and weaker interactions between

-CgTx PnIB P6 and 7W149, and between both

P6 and P7 of-CgTx PnIB and 7Y93 (located in

loop A; reviewed in [8]) [53]. The evidence from

mutational experiments also suggests that the

binding site for -CgTx PnIA [50] or for -CgTx

PnIB [53] on the 7 receptor is different from the-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 mem-

ory. In addition, this receptor family has been

implicated in the pathophysiology of several neu-

ropsychiatric disorders including Alzheimers andParkinsons disease, schizophrenia, Tourettes

syndrome, nocturnal frontal lobe epilepsy, as well

as nicotine addiction, myasthenia gravis, and vari-

ous 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, -CTxs

might be used as therapeutic agents in the treat-

ment of some of the above mentioned diseases. In

this regard, additional Conus toxins not discussedin 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 anticon-

vulsant agent [64]. The -CTx MVIIA, which is

highly specific for the voltage-gated calcium chan-

nels containing the 1B subunit, is being used

clinical trials for the treatment of certain chron

pain syndromes (e.g. intractable pain resultin

from cancer, traumatic nerve demage, or amput

tion) and it has also proved to be useful as

neuroprotector of cerebral ischemia provoked b

stroke, cardiac arrest, or head trauma (reviewe

in [65]). Finally, an immunoprecipitation assa

with [125I]-CTx is used to diagnose the Lam

bert Eaton myastenic syndrome (reviewed

[65]), an autoimmune disease in which antibodi

recognize endogenous calcium channels.

Acknowledgements

This work was supported in part by NIND

Grant R29 NS35786 from the National Institut

of Health (to M.P. Blanton). We thank Dr TinMachu for her critical reading of the manuscrip

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channel targets, and drug design: 50 million years

neuropharmacology, Mol. Biol. Cell 8 (1997) 2101 210[3] R.A. Myers, L.J. Cruz, J.E. Rivier, B.M. Olivera, Con

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