Three-Finger -Neurotoxins and the Nicotinic Acetylcholine

1

Journal of Pharmacological Sciences

©2004 The Japanese Pharmacological Society

Critical Review

J Pharmacol Sci 94, 1 – 17 (2004)

Three-Finger -Neurotoxins and the Nicotinic Acetylcholine Receptor,

Forty Years On

Selvanayagam Nirthanan1,2,* and Matthew C.E. Gwee1

1Department of Pharmacology, Faculty of Medicine, National University of Singapore, Singapore 1192602Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, USA

Received October 30, 2003

Abstract. The discovery, about forty years ago, of � -bungarotoxin, a three-finger � -neuro-toxin from Bungarus multicinctus venom, enabled the isolation of the nicotinic acetylcholinereceptor (nAChR), making it one of the most thoroughly characterized receptors today. Sincethen, the sites of interaction between � -neurotoxins and nAChRs have largely been delineated,revealing the remarkable plasticity of the three-finger toxin fold that has optimally evolved toutilize different combinations of functional groups to generate a panoply of target specificitiesto discern subtle differences between nAChR subtypes. New facets in toxinology have nowbroadened the scope for the use of �-neurotoxins in scientific discovery. For instance, thedevelopment of short, combinatorial library-derived, synthetic peptides that bind with sub-nanomolar affinity to � -bungarotoxin and prevent its interaction with muscle nAChRs has ledto the in vivo neutralization of experimental � -bungarotoxin envenomation, while the successfulintroduction of pharmatopes bearing “� -bungarotoxin-sensitive sites” into toxin-insensitivenAChRs has permitted the use of various � -neurotoxin tags to localize and characterize newreceptor subtypes. More ambitious strategies can now be envisaged for engineering rationallydesigned novel activities on three-finger toxin scaffolds to generate lead peptides of therapeuticvalue that target the nicotinic pharmacopoeia. This review details the progress made towardsachieving this goal.

Keywords: nicotinic acetylcholine receptor, �-neurotoxin, three-finger toxin, neuromuscular block, pharmacological probe

1. Animal toxins: key players in science and medicine .. 22. Snake envenomation: nefarious role of neurotoxins... 23. Snake toxins that affect cholinergic

neurotransmission................................................... 24. Curaremimetic � -neurotoxins: more than just

a case of mimicry.................................................... 35. Nicotinic acetylcholine receptors: on a roll ................ 46. The three-finger toxin scaffold: three fingers

in many pies............................................................ 47. Three-finger � -neurotoxins: a closer look

at new angles .......................................................... 5i. Short-chain and long-chain neurotoxins

ii. Atypical long-chain neurotoxinsiii. Non-conventional neurotoxins

8. Pharmacology of three-finger � -neurotoxins ............. 7i. In vitro assays for � -neurotoxin bioactivity

ii. Reversibility of �-neurotoxin neuromuscular blockade

iii. Site-selectivity of � -neurotoxins9. Structure-function relationships of � -neurotoxins:

what makes them tick?............................................ 9i. Structurally invariant residues in � -neurotoxins

ii. Conformational determinants of structure-function of � -neurotoxins

10. Molecular interactions: a toxin perspective................. 1011. Molecular interactions: a receptor perspective............ 10

i. Insights from the molluscan acetylcholine binding protein

ii. Insights from nicotinic acetylcholine receptors of � -neurotoxin-resistant species

iii. Insights from synthetic peptides derived from the nicotinic acetylcholine receptor

12. Three-finger � -neurotoxins as pharmacological probes: old dogs, new tricks.................................... 13

13. Conclusions: not the last word yet .............................. 14

*Corresponding author (affiliation #2). FAX: +1-617-7347557E-mail: [email protected]

Invited article

S Nirthanan & MCE Gwee2

1. Animal toxins: key players in science and medicine

It will be an interesting paradox if it transpires that

the venomous creatures that have so long been reviled

by the human race turn out to provide key lessons for

revolutions in medicine (and science), which prolong

and enhance human existence.

Mark J. Dufton (1993) (Ref. 1).

In the course of their long evolutionary history, snakesand other venomous animals have created innovativeand intricate protein structural motifs to generate a vastresource of pharmacologically novel peptide toxins thattarget a wide variety of receptors and ion channels withhigh affinity and specificity. The specificity of sometoxins is so exquisite that they are capable of discri-minating subtle differences in binding sites on a singlereceptor population, like the binding sites located at theinterfaces of the �1 /� or �1 /� subunits in the muscle(�1)2�1�� nicotinic acetylcholine receptor (nAChR)(2). Although early venom research was motivated byour desire for satisfactory cures for snake envenomation,our perspectives on animal toxins have changed drama-tically due to accumulating data that has revealed a farwider scope for these natural biomolecules, which haveassumed great significance as molecular probes andpharmacological tools to investigate the functionalbiology of receptors and ion channels as well as provid-ing lead compounds for the design of clinically usefuldrugs (3 – 5).

There is perhaps no better example to highlight thesignificant contributions made by venom peptides toscience and medicine than the discovery, about fortyyears ago, of the curaremimetic neurotoxin, � -bungaro-toxin, from the venom of the Taiwanese many-bandedkrait (Bungarus multicinctus) (6), which spawned thefield of molecular pharmacology (7) by enabling theisolation and study of the nAChR from the electric organof the electric eel and other sources (8, 9). The discoveryof � -bungarotoxin and orthologues from other speciesalso enabled the localization of the nAChR in situ andidentification of subtypes of the receptor in variousmuscle and neural tissues (2), making the nAChR one ofthe most thoroughly characterized receptors today (10).

2. Snake envenomation: nefarious role of neurotoxins

Snake venoms are a cocktail of toxins and enzymesthat have optimally evolved as a lethal weapon for pre-dation as well as defense against predators. Dependingon the species, snake envenomation in humans mayresult in peripheral neurotoxicity, coagulative disorders,myotoxicity, renal failure as well as severe necrosis atthe site of the bite, all of which can be potentially fatal

(11). Snake envenomation is a major clinical problemwith over 2.5 million cases of snake bites reportedworldwide, particularly in parts of the Asian region andAfrica where the annual mortality is estimated to be100,000 and 20,000, respectively (12). One of theprimary targets of snake venom is the peripheral nervoussystem, the skeletal muscle neuromuscular junction inparticular, where neurotransmission is inhibited, leadingto paralysis of skeletal muscles including those ofrespiration (11). Clearly therefore, the study of theinteraction of snake venom toxins with the skeletalmuscle neuromuscular junction is also of great clinicalsignificance.

3. Snake toxins that affect cholinergic neurotrans-

mission

The arsenal of snake neurotoxins that interfere withcholinergic neurotransmission consists of a wide varietyof toxins that target multiple subtypes of both nicotinicand muscarinic acetylcholine receptors at peripheral aswell as central sites. Often, a combination of many typesof neurotoxins may be present together in the venom ofone species. The principal neurotoxic components ofElapid (cobras, kraits, mambas, coral snakes, andAustralian elapids) and Hydrophiid (sea snakes) snakevenoms are neurotoxins that bind to postsynapticnAChRs at the skeletal muscle neuromuscular junctionto produce blockade of neuromuscular transmission (9,13, 14). These neurotoxins, referred to as curaremimeticor �-neurotoxins, will be the subject of this review.

The skeletal muscle neuromuscular junction is alsosusceptible to neurotoxin action at presynaptic sites.Presynaptic neurotoxins (e.g., �-bungarotoxin (Bun-

garus multicinctus)) are either phospholipase A2

enzymes or contain these enzymes as an integral part ofthe neurotoxin complex and, essentially, mediate theirneurotoxicity by inhibiting the release of acetylcholine(13, 15, 16). Other neurotoxins that interfere withcholinergic neurotransmission include fasciculins frommamba (Dendroaspis spp.) venoms that inhibit theactivity of acetylcholinesterase present at the neuro-muscular junction as well as at central synapses (17) and�-neurotoxins (Bungarus spp.), which bind to neuronal�3�2 as well as other nAChRs composed of �2 subunits(18, 19). Several muscarinic toxins have also beenisolated from mamba venoms (Dendroaspis spp.), whichare antagonists or agonists at various subsets of mus-carinic receptors in the brain as well as at peripheral sites(20 – 23).

�-Neurotoxins and the nAChR 3

4. Curaremimetic a-neurotoxins: more than just a

case of mimicry

“The designation of � - and �-bungarotoxin were simply

based on the moving velocity of the toxins through a

potato starch electrophoresis preparation during their

purification and it was not my intention to use the suffix

� - to designate a postsynaptic site of action. However,

I found it amazing that, as if to follow conformity, toxins

isolated thereafter and found to manifest postsynaptic

effects were also named with this suffix”

C.C. Chang (1999) (Ref. 24)

Curaremimetic or � -neurotoxins interfere with cho-linergic transmission at the skeletal muscle neuro-muscular junction by binding with high affinity andselectivity to postsynaptic nAChRs to produce blockadeof neuromuscular transmission (9, 13, 14, 24, 25). Thesetoxins, which mimic the neuromuscular blocking effectsof the plant alkaloid (+)-tubocurarine, but with approxi-mately 15 – 20-fold greater affinity and poor revers-ibility of action, are referred to as curaremimetic neuro-toxins or postsynaptic neurotoxins to reflect their post-junctional site of action at the neuromuscular junction

(13) or simply as � -neurotoxins, a suffix of historicalsignificance (24). Most � -neurotoxins are derivedfrom Elapidae or Hydrophiidae snake venom andbelong to the three-finger toxin family. Well-knownexamples of such toxins include � -bungarotoxin(Bungarus multicinctus) and erabutoxin-b (Laticauda

semifasciata) (Fig. 1). The only interesting exceptionare the waglerins, isolated from the venom of theWagler’s pit viper (Tropidolaemus wagleri (Viperidae)),which are 22 – 24-residues-long and contain a singledisulfide bridge and in addition, show remarkableselectivity for the �1 /� over the �1 /� and �1 /� inter-faces of the mouse muscle nAChR (7).

It must be emphasised that snake venoms are not theexclusive source of � -neurotoxins. The venoms ofmarine cone snails also represent a rich combinatorial-like library of evolutionarily selected, neuropharmaco-logically active peptides called conotoxins that target awide variety of receptors and ion-channels. In particular,�-conotoxins are short (approximately 12 – 30-residues-long) disulfide-rich peptides that are capable of dis-criminating between the different ligand-binding inter-faces of the muscle nAChR as well as some that are

Fig. 1. The 3D-structures of three-finger neurotoxins from snake venoms that interact with nicotinic acetylcholine receptors.The three-dimensional structures are shown in similar orientation and in line ribbon representation. Disulfide bridges are shown inblack. The species names and the Protein Data Bank accession codes for structures are as follows: A: Erabutoxin-a (Laticauda

semifasciata) 5EBX, B: Toxin-� (Naja nigricollis) 1NEA, C: Candoxin (Bungarus candidus) 1JGK, D: Bucandin (Bungarus

candidus) 1F94, E: �-Cobratoxin (Naja kaouthia) 2CTX, F: �-Bungarotoxin (Bungarus multicinctus) 1IK8, G: LSIII (Laticauda

semifasciata) 1LSI, and H: � -Bungarotoxin (Bungarus multicintus) 2NBT. All are averaged NMR structures except erabutoxin-a,2.0-Å crystal structure and �-cobratoxin, 2.4-Å-crystal structure.


selective for various subtypes of neuronal nAChRs; thisis a remarkable feat given their small and compact dis-position (reviewed in 26 – 30).

�-Neurotoxins bear the imprint of a region of thenAChR that is likely to be in proximity to, and perhapseven overlap, the binding site for the natural neuro-transmitter acetylcholine (25). The pharmacological andstructural characterization of this important region ofthe nAChR will greatly enhance our understanding ofhow these, and perhaps other ligand-gated ion-channels,work.

5. Nicotinic acetylcholine receptors: on a roll

“The nicotinic acetylcholine receptor, as a result of the

combined contributions of electrogenic fish, Elapid

snakes and many talented scientists, became the first

neurotransmitter receptor to be characterized, purified

and cloned”

Brian Molles and Palmer Taylor (2002) (Ref. 7)

The nAChR is perhaps one of the best characterizedion-channel to date, due in part to the discovery of � -bungarotoxin and a rich and accessible source of recep-tor from the electric ray and eel. Consequently, themammalian neuromuscular junction is also the moststudied and best understood synapse (31 – 33). ThenAChRs are transmembrane allosteric proteins of MWapproximately 290 kDa that are involved in fast ionicresponses to acetylcholine (10, 34, 35). They arepentamers formed by the association of five subunitsarranged symmetrically around the ionic pore in a planeperpendicular to the membrane (Fig. 2: A and B) (35,36). Each subunit is composed of a large amino-terminaldomain that contributes to the formation of the ligandbinding pocket; four membrane-spanning domains (MI,MII, MIII, MIV); a large and variable cytoplasmic loopbetween MIII and MIV; and a small extracellularcarboxyl terminal. The MII domains of all five subunitscontribute to the formation of the cation channel pore(Fig. 2: A and B). In vertebrates, the combinatorialassembly of various nicotinic receptor subunits (�1 –�10, �1 – �4, � , � , or �) generates a wide diversity ofreceptors, with various electrical and binding properties.Very broadly, nicotinic receptors can be divided into twomain families: the muscle and neuronal nAChRs (34).The well-characterized muscle receptor consists of acombination of �1, �1, � , and � or � subunits in thestoichiometry of (�1)2�1�� or (�1)2�1�� in theembryonic or adult receptor, respectively. These aredensely distributed on the postsynaptic membrane ofthe neuromuscular junction and mediate intercellularcommunication between the nerve ending and skeletalmuscle. The muscle-type receptor (�1)2�1�� is also

found in abundance in the electric organ of the Torpedo

ray. Neuronal nicotinic receptors are composed of �2 –�10 and �2 – �4 subunits.

The extracellular amino-terminal domains of nAChRsare approximately 210 amino-acid-residues-long andcontain binding sites for agonists and competitiveantagonists. The ligand-binding site is located at theinterface between two subunits with contributions fromboth counterparts (34, 37). The principal part of thebinding pocket is formed by the �1-subunit residuescontributing to loops A, B, and C, whereas the neighbor-ing subunit (� , � , or � in muscle nAChR) residuescontribute to loops D, E and F that form the comple-mentary part of the binding pocket (34, 37, 38). Thus,the muscle nAChR contains two different ligand-bindingsites (�1 /� and �1 /� or �1 /�) with distinct affinitiesfor nicotinic antagonists (34).

An excellent insight into the structure of nAChRs,and ligand-gated ion channels in general, was madepossible by the discovery and characterization of anacetylcholine-binding protein from the snail Lymnaea

stagnalis (Fig. 2: B and C) (39, 40) which is a remark-able homologue of the amino-terminal extracellulardomain of the nAChR. The crystal structure of theacetylcholine binding protein at 2.7 Å resolutionrevealed that each ligand-binding site is located in acleft at the subunit interface, formed by a series ofloops (A, B, and C) from the principal face of onesubunit and another series of loops (D, E, and F) fromthe complementary face of an adjacent subunit (40),conforming to existing biochemical and mutationaldata on nAChRs. Based on the structure of the AChBP,nearly all the residues of the agonist-binding site of thenAChR that were previously identified by photoaffinitylabeling and mutagenesis experiments are located in asmall cavity of about 10 – 12-Å diameter that is pri-marily formed by aromatic residues contributed by theparticipating subunits (10, 35, 41).

6. The three-finger toxin scaffold: three fingers in

many pies

�-Neurotoxins from Elapid and Hydrophiid snakevenoms belong to the three-finger toxin superfamily ofnon-enzymatic polypeptides containing 60 – 74 aminoacid residues. The characteristic feature of all three-finger toxins is their distinctive structure formed bythree adjacent loops that emerge from a small, globular,hydrophobic core that is cross-linked by four conserveddisulfide bridges (25, 42 – 45). The three loops thatproject from the core region resemble three outstretchedfingers of the hand (Fig. 1). The toxin is essentially aflat “leaf-like” molecule with a slight concavity, the


plane being determined by the extensive multi-stranded� -structure that is the predominant feature (25, 44).

In addition to the structural plasticity of the threefingers, the three-finger fold is also amenable to a vari-ety of overt and subtle deviations, such as the number of� -strands present, size of the loops, and C-terminal tailas well as twists and turns of various loops, all of whichmay have great significance with respect to functionaldiversity and selectivity of molecular targets (25, 45).Hence, despite the similar overall fold, three-fingertoxins demonstrate an assorted range of pharmacologicalactivities including, but not limited to, peripheral andcentral neurotoxicity, cyotoxicity, cardiotoxicity, inhibi-tion of enzymes such as acetylcholinesterase and pro-teinases, hypotensive effect, and platelet aggregation(43 – 46).

In many instances, the functional sites of interactionbetween these pharmacologically diverse toxins andtheir molecular targets have been fairly accuratelydelineated and found to involve one or more different

regions of the three-finger molecule (43 – 45). Henceit has been proposed that the “three-finger” scaffold isused by the snake to “hang” different combinations offunctional groups, generating a panoply of target speci-ficities (43, 47). It thus appears that snakes adhere toa policy of structural economy by utilizing a limitednumber of molecular molds to achieve remarkablefunctional diversity. Furthermore, three-finger toxinsare also among the simplest proteins that could beutilized to address essential questions about proteins ingeneral, including protein-protein interactions, antigenicand immunogenic properties of proteins, folding pro-cesses, and the dynamic characteristics of proteins aswell as offering an interesting molecular basis for thestudy of the evolution of protein folds (25).

7. Three-finger a-neurotoxins: a closer look at new

angles

To date, more than 100 three-finger �-neurotoxins

Fig. 2. The structure of the muscle nicotinic acetylcholine receptor. A: The nicotinic acetylcholine receptor is a pentamercomposed of five homologous subunits. The muscle receptor of the stoichiometry (�1)2�1�� is represented in this model. Thereceptor is depicted perpendicular to the axis of the ion channel pore. For clarity, the � subunit is not shown. Each subunit iscomposed of four helical transmembrane domains (M1, MII, MIII, MIV). The MII domain of all five subunits lines the channelpore. B: Top view of the pentameric receptor, viewed along the five-fold axis, showing the association of the five subunits.The extracellular amino-terminal domain of the �1-subunit and the adjacent subunit (� or �) cooperate to form two distinctbinding pockets for acetylcholine (or other agonists and competitive antagonists) at the interface between the subunits. C, D: Theacetylcholine binding protein from snail brain, a structural homologue of the nicotinic acetylcholine receptor ligand-bindingdomain. The 2.7-Å resolution crystal structure of the acetylcholine binding protein homopentamer from the snail (Lymnaea

stagnalis) glial cells (Protein Data Base accession #119B) (40). C: As viewed from the top, along the five-fold axis. Each subunit(depicted in different colors) is a single domain protein. The cavity or pocket at each interface likely constitutes the ligand-binding site, and these five equivalent dimer interfaces serve as the only links between the subunits. D: As viewed perpendicularto the five-fold axis. It forms a 62-Å-high cylinder with a diameter of 80 Å. The N- and C-termini are located at the ‘top’ and‘bottom’ of the pentamer, respectively. With the exception of the amino-terminal region of each subunit which consists of a short�-helix segment, � -sheet sandwich structure predominates. The ligand-binding pockets are roughly equatorially positioned,about 30 Å away from the C-termini. The structure is colored to show the five subunits constituting the pentameric structure.


have been isolated and sequenced from Elapidae andHydrophiidae snakes (42). Depending on their aminoacid sequence and /or tertiary structures, �-neurotoxinscan be classified into short-chain � -neurotoxins, long-chain �-neurotoxins, atypical long-chain � -neurotoxins,

and non-conventional three-finger neurotoxins (Table 1and Fig. 3) (25, 48). Although the primary moleculartarget of all these categories of three-finger neurotoxinsappears to be the muscle-type nAChR, some toxins arealso known to interact with other subtypes of nAChRsand in the case of some poorly characterized non-conventional toxins, it is possible that other unknownmolecular targets may exist. Hence, while a classifi-cation of three-finger neurotoxins that interact with themuscle nAChR may be feasible from a purely structuralstand-point, it must be remembered that �-neurotoxinsare not a functionally homogenous group.

i. Short-chain and long-chain neurotoxins

Based on the length of their polypeptide chains, � -neurotoxins were initially classified as short-chain � -neurotoxins (e.g., erabutoxin-b; toxin-� (Naja nigri-

collis)) that have 60 – 62 residues and four conserveddisulfide bonds and long-chain � -neurotoxins (e.g., � -bungarotoxin and � -cobratoxin (Naja kaouthia)) with66 – 75 residues and five disulfide bonds, with theadditional disulfide bridge located in the middle loop(loop II) (42). Notwithstanding their classification asshort-chain and long-chain neurotoxins, both types of � -neurotoxins bind with high affinity (Kd approximately10�9 – 10�11 M) to the Torpedo or muscle (�1)2�1��

nAChRs (25). Nonetheless, it has been reported thatshort-chain � -neurotoxins tend to associate with thenAChR 6 – 7-fold faster and dissociate 5 – 9-fold fasterthan long-chain �-neurotoxins (49). Apart from differ-ences in structure, long-chain, but not short-chain, � -neurotoxins are also able to bind with high affinity(Kd approximately 10�8 – 10�9 M) to neuronal homo-pentameric �7, �8, and �9 nicotinic receptors and wellas to heteropentameric receptors composed of �7 – �10subunits (50 – 52).

ii. Atypical long-chain neurotoxins

Based on sequence homology to long-chain � -neuro-toxins, the 69-residues-long neurotoxins isolated fromthe sea snake Laticauda semifasciata (Lc-a and Lc-b)(53) were also classified as long-chain toxins (42). Likeother prototype � -neurotoxins, both Lc-a and Lc-b,which differed from each other at only five positions intheir sequences, showed high affinity for the Torpedo

nAChR (Kd approximately 10�11 M) (50). However,Lc-a and Lc-b are atypical in the sense that, unlike thetypical long-chain � -neurotoxins, they retain only thefour conserved disulfide bridges and lack the fifthdisulfide bridge at the tip of loop II (53). Consequently,Lc-a and Lc-b bind poorly to neuronal �7 nAChRs(25, 50).

iii. Non-conventional neurotoxins

The non-conventional toxins (48) constitute anotherclass of three-finger toxins that consist of 62 – 68 amino-acid residues and five disulfide bridges (e.g., candoxin

Table 1. Structural classification of three-finger neurotoxins from snake venoms that interact with muscle nicotinic acetylcholine receptors

Structural characteristics Primary molecular target Other molecular targets Source

Short-chain

neurotoxins

Three-fingered monomers

of 60 – 62 amino-acid residues

with four conserved disulfide

bonds.

High affinity for muscle

or Torpedo (�1)2�1�� nAChR

(Kd approximately 10�9 – 10�11 M).

Elapidae and Hydrophidae

species. Typical examples

include erabutoxin-a

(Laticauda semifasciata),

toxin-� (Naja nigricolis).

Long-chain

neurotoxins




bonds and an additional

disulfide bond at the tip of loop II.

High affinity for muscle

or Torpedo (�1)2�1�� nAChR

(Kd approximately 10�9 – 10�11 M).

High affinity

(Kd approximately 10�8 – 10�9 M)

antagonists of the neuronal

�7 nAChR.

Isolated from Elapidae


include �-bungarotoxin

(Bungarus multicinctus),

� -cobratoxin (Naja kaouthia).

A typical long-chain

neurotoxins


of 69 amino-acid residues

and four conserved disulfide bonds.

High affinity for Torpedo (�1)2�1��

nAChR (Kd approximately 10�11 M).

Toxins Lc-a and Lc-b, isolated

exclusively from Laticauda

colubrina (Hydrophidae).

Non-conventional

neurotoxins




bonds and an additional

disulfide bond at the tip

of loop I.

Some (WTX, Naja kaouthia)

have low affinity

(Kd approximately �10�6 M)

and some (Candoxin, Bungarus

candidus) have high affinity

(Kd approximately 10 nM)

for muscle or Torpedo (�1)2�1��

nAChR.

Candoxin (Bungarus candidus)

is a high affinity antagonist

(Kd approximately 50 nM)

of the neuronal �7 nAChR.

Exclusively from Elapidae


include candoxin (Bungarus

candidus), WTX (Naja

kaouthia), Wntx-5 (Naja

sputatrix).


and bucandin (Bungarus candidus)). However, unlikein long-chain � -neurotoxins and �-neurotoxins, thefifth disulfide bridge in non-conventional toxins islocated in loop I (N-terminus loop) (Fig. 1). Some studies

have suggested that non-conventional toxins are typi-cally characterized by a lower order of toxicity (LD50

varying from approximately 5 – 80 mg /kg) (25, 54) asopposed to prototype � -neurotoxins (LD50 approxi-mately 0.04 – 0.3 mg /kg) (55), and because of this, theyare also referred to as weak toxins (54). However, whilesome non-conventional toxins from cobra venoms (e.g.,WTX (Naja kaouthia) (54) and Wntx-5 (Naja sputatrix)(56)) produced a weak inhibition of muscle (�1)2�1��nAChRs in micromolar inhibitory concentrations, andcandoxin from a krait venom (Bungarus candidus) wasa potent inhibitor of muscle (�1)2�1�� nAChRs inlow nanomolar (IC50 approximately 10 nM) inhibitoryconcentrations (57). In contrast to WTX and Wntx andin congruence with long-chain � -neurotoxins, candoxinwas also found to inhibit neuronal �7 nAChRs at lownanomolar (IC50 approximately 50 nM) inhibitory con-centrations (58). Hence, it may be possible that some

non-conventional toxins that bind weakly to musclenAChRs may have other, yet unidentified, moleculartargets.

8. Pharmacology of three-finger a-neurotoxins

i. In vitro assays for a-neurotoxin bioactivity

�-Neurotoxins are potent competitive antagonists ofthe nAChRs at the skeletal muscle neuromuscular junc-tion. Because the simultaneous occupation of bothligand-binding sites of the nAChR by agonists such asacetylcholine is required to elicit the conformationalchange that opens the gated ion-channel, the occupationof a single site by an antagonist � -neurotoxin isadequate to produce a non-functional receptor (2, 34,35). Skeletal muscle preparations from the chick (chickbiventer cervicis muscle (59)) and mouse (mousephrenic nerve-hemidiaphragm (60)) provide simple butreliable in vitro organ bath assays for the examination ofthe effects of venoms and toxins on neuromusculartransmission (46, 61). The chick biventer muscle inparticular is useful to discriminate between presynaptic,

Fig. 3. Amino acid sequences of three-finger neurotoxins that interact with that interact with muscle nicotinic acetylcholinereceptors. The cysteine residues are shaded in grey and the disulfide linkages and the segments contributing to the three loops areoutlined. The number of amino acid residues in each sequence is indicated at the end of the respective sequence. The speciesnames are as follows: toxin � (Naja nigricollis), NmmI (Naja mossambica mossambica), erabutoxin b (Laticauda semifasciata),�-cobratoxin (Naja kaouthia), �-bungarotoxin (Bungarus multicinctus), Lc-a and Lc-b (Laticauda colubrina), candoxin(Bungarus candidus), and WTX (Naja kaouthia). The Protein Data Bank accession numbers are also stated for each toxin. TheInternational Union of Pure and Applied Chemistry one-letter notation for amino acids is used (J Biol Chem. 1968;243:3557–3559).


postsynaptic, or myotoxic effects of neurotoxins since itcontains both, focally and multiply innervated musclefibers that, respectively, mediate twitch responsesevoked by electrical nerve stimulation and contractileresponses evoked by exogenous nAChR agonists suchas acetylcholine and carbachol (57, 61, 62). Whereas apure presynaptically active neurotoxin would abolishnerve-evoked twitches without affecting contractileresponses to exogenous nAChR agonists or directmuscle stimulation mediated by potassium chloride-induced depolarization, postsynaptically acting � -neuro-

toxins would block the responses to nAChR agonistsas well as nerve stimulation, but would not block thecontractile responses produced by direct muscle stimu-lation (Fig. 4).

Other screening methods for � -neurotoxin bioactivityinclude conventional binding experiments in which thecompetitive binding of a radio-labelled toxin to nAChR-rich membranes from Torpedo electric organs is assayedand electrophysiological experiments where the abilityof the toxin to inhibit currents or ion fluxes inducedby agonists in nAChRs expressed in oocyte expressionsystems is studied (63).

ii. Reversibility of a-neurotoxin neuromuscular

blockade

Although commonly referred to as “curaremimetictoxins”, �-neurotoxins differ from (+)-tubocurarine inthat the majority of � -neurotoxins, especially the long-chain toxins, undergo almost irreversible binding toskeletal muscle nAChRs (9, 42, 49). However, many� -conotoxins from marine cone-snails that interactwith the nAChR (e.g., � -conotoxin MI and GI) are

well known to produce reversible postsynaptic neuro-muscular blockade in vitro and in vivo (64). In sharpcontrast to the poorly reversible nature of the � -neuro-toxin-induced neuromuscular blockade, candoxin (Bun-

garus candidus) (57) and some weak neurotoxin-homologues (CM10, CM12 (Naja haje annulifera) andS5C10 (Dendroaspis jamesoni)) (65) as well as toxinLSIII (Laticauda semifasciata) (66, 67) have been foundto produce neuromuscular blockade that is rapidly andcompletely reversible.

It could be argued that the reversibility or irrever-sibility of neuromuscular blockade induced by toxinsmay just result from their weak or high binding affinityto the nAChRs. However, electrophysiological studieshave revealed that � -bungarotoxin (IC50 approximately5 nM) produced an irreversible block of muscle(�1)2�1�� receptors (68), whereas candoxin (IC50

approximately 10 nM) produced a reversible blockadeof the same receptor (58). Furthermore, WTX (Naja

kaouthia), a non-conventional toxin that is structurallysimilar to candoxin but a 1000-fold weaker antagonist ofmuscle nAChRs, is almost irreversible in its action (54).Therefore, the reversibility of � -neurotoxin action atthe neuromuscular junction is not always a reflectionof their binding affinity to the receptor and may perhapsbe associated with a specific area of interaction on thetoxin molecule, distinct from the receptor recognitionsite (67). For instance, in contrast to most � -neuro-toxins, an aspartate at position 31 is absent in theneurotoxin-homologues CM10 and CM12, toxin LSIIIas well as in candoxin, all of which were reported to bereversible in their action. It is possible that the absenceof Asp31 may be associated with easy reversibility of

Fig. 4. In vitro pharmacological bioassay for screening �-neurotoxin effects at the skeletal muscle neuromuscular junction.Segments of the tracing of a typical experiment on the chick biventer cervicis muscle showing the neuromuscular blockingeffects of mikatoxin, an �-neurotoxin isolated from the venom of the New Guinean small-eyed snake Micropechis ikaheka (62).The isolated chick biventer cervicis muscle was mounted in an organ bath in physiological salt solution and maximal twitchresponses evoked by stimulating the motor nerve by electrical field stimulation (7 – 10 V, 0.2 Hz, in supramaximal rectangularpulses of 0.1-ms duration). Submaximal contractures to exogenously applied acetylcholine (ACh, 300 �M for 30 s), carbachol(CCh, 8 �M for 90 s), or potassium chloride (KCl, 30 mM for 60 s) were also obtained in the absence of electrical fieldstimulation, before and after the addition of mikatoxin. Vertical bar represents the muscle contractile response in gram tension andthe horizontal bar represents the time-scale in minutes. Mikatoxin at 10 �g �ml�1 (added at the point marked �-neurotoxin)produced a rapid blockade of the uninterrupted twitch responses of the muscle to nerve stimulation as well as a block of thecontractile responses evoked by ACh and CCh, but not to KCl. This pattern of neuromuscular blockade is characteristic ofpostsynaptic blockade produced by snake venom �-neurotoxins.


neuromuscular blockade produced by these toxins(57, 65)

iii. Site-selectivity of a-neurotoxins

The existence of differences in the two binding sitesof the nAChR has been attributed to the influence ofthe non-equivalent subunits (i.e., � , � , or �) on theconformation of the binding sites at their respectiveinterfaces with the �1-subunit (69, 70). Many agonistsand antagonists that interact with the muscle nAChRshow some subunit selectivity that results in preferencesfor the �1 /� or �1 /� or �1 /� interfaces (2, 71, 72).Conotoxin MI (Conus magus) that shows a 10,000-foldpreference for the �1 /� over the �1 /� subunit interfacein the mouse muscle nAChR (73, 74) and waglerin(Tropidolaemus wagleri) that shows a 2000-fold greateraffinity for the �1 /� site over the �1 /� or �1 /� sites inthe mouse nAChR (7, 75) are excellent examples of theremarkable selectivity of peptide toxins.

Although most � -neurotoxins from snake venomshave not been found to exhibit ligand-binding sitepreference in the muscle nAChR, a short-chain � -neuro-toxin from Naja mossambica mossambica (NmmI)shows an order of magnitude higher affinity for the �1 /�

or �1 /� interfaces (Kd 140 pM) than for the �1 /� site(Kd 130 nM), and this selectivity has been attributed totwo residues (Pro175 and Glu176) in the � subunit of thenAChR (76). Pharmacological studies on the non-conventional toxin candoxin (Bungarus candidus) alsosuggest that it may have differential affinity for the �1 /�

or �1 /� interfaces of the muscle nAChR (57). Further-more, site-directed mutagenesis studies on � -cobratoxinrevealed that the mutation of Lys23 and Lys49 to Glu23

and Glu49, respectively, caused a differential lowering ofbinding affinity at the two binding sites of the muscle(�1)2�1�� nAChRs (52). This observation was verifiedfor NmmI, whereby the mutation of Lys27 to Glu27

affected binding at the �1 /� site more than the �1 /�

site (77). Interestingly, position 29 in candoxin (homo-logous to Lys23 in �-cobratoxin and Lys27 in NmmI) isoccupied by a glutamic acid instead of a lysine, suggest-ing a possible role for Glu29 in conferring differentialsubunit selectivity (57).

9. Structure-function relationships of a-neurotoxins:

what makes them tick?

i. Structurally invariant residues in a-neurotoxins

The structures of almost all three-finger toxins includ-ing � -neurotoxins have conserved structurally invariantresidues that are not involved in direct interactionwith the respective target receptors of these toxins but,rather, contribute to the proper polypeptide chain folding

and structural integrity of the polypeptide (42). Theseinclude the eight cysteine residues that form the fourconserved disulfide bridges located in the core region ofthe three-finger scaffold and constrain the three loopsthat protrude from it (42). Tyr25 or a homologousaromatic residue (Phe27) is also conserved in most toxinsthat adopt a three-finger structure (Fig. 3) since it isrequired for proper folding of the polypeptide chain (78,79) and the stability of the anti-parallel �-sheet structureof three-finger toxins (80). The structurally invariantGly42 and Pro48 residues are essential for maintainingthe spatial conformation of � -neurotoxins (81). Interest-ingly, some charged amino acid residues (e.g., Arg39 inerabutoxin-a and Asp60 in � -cobratoxin) have also beenconserved in three-finger toxins for structural stabilityand have been reported to stabilize the native conforma-tion of the protein by forming a salt links with the C-or N-terminus of the toxin (42).

ii. Conformational determinants of structure-func-

tion of a-neurotoxins

The principal differences in the three-finger folds ofshort-chain and long-chain �-neurotoxins include thepresence of an extra disulfide bridge at the tip of loop II,a longer C-terminus tail, and a shorter loop I in long-chain toxins. These and other subtle structural deviationsthat exist between short-chain and long-chain � -neuro-toxins often reflect some functional significance (43).For instance, the high affinity binding of long-chain�-neurotoxins, but not short-chain � -neurotoxins, toneuronal �7 nAChRs has been clearly attributed tostructural variations in loop II of the scaffold (50, 82).Although the structure of three-finger � -neurotoxins isgenerally assumed to be devoid of � -helices (25, 44),the fifth disulfide bridge present in long-chain � -neuro-toxins (and �-neurotoxins) produces a cyclic “helix-like” conformation at the tip of loop II that consistsof two sequential turns held in place by the disulfidebridge (Fig. 1). This conformation has been reported tobe crucial for their binding to their respective neuronalnAChR targets (50, 52, 83, 84). Furthermore, NMRanalysis of free � -bungarotoxin and � -bungarotoxin incomplex with short cognate peptides of the nAChRtoxin-binding region revealed that the cyclic conforma-tion of the tip of loop II was a dynamic entity (85). Thisflexibility of loop II would enable long-chain � -neuro-toxins to accommodate substantial structural deviationsand adopt alternate conformations in order to bind todiverse receptor subtypes (47, 86).

Short-chain � -neurotoxins as well as atypical long-chain toxins (Lc-a and Lc-b from Laticauda colubrina),which lack this fifth disulfide bridge and non-conven-tional toxins WTX (Naja kaouthia) and Wntx-5 (Naja


sputatrix), both of which have the fifth disulfide bridgein loop I and lack the loop II helical conformation,have weak affinity, in micromolar concentrations (Kd

approximately 3 – 22 �M) at best, for the neuronal �7nAChR (50, 54, 56). Interestingly, candoxin (Bungarus

candidus) is the first known toxin that lacks this criticalhelix-like conformation cyclized by the fifth disulfidebridge in loop II and yet blocks neuronal �7 nAChRsin low nanomolar concentrations (58). It is likely thatcandoxin utilizes other, yet undetermined, functionaldeterminants in its interaction with the neuronal �7nAChR.

10. Molecular interactions: a toxin perspective

�-Neurotoxins utilize a common binding core toestablish contacts with key invariant residues on thenAChR and other toxin-specific residues to interact withsubtype-specific receptor residues (47). Evidence forthis came from exhaustive mutational analysis studieswhich revealed that short-chain and long-chain � -neuro-toxins recognize the Torpedo or muscle (�1)2�1��receptor by a common binding core consisting ofpositively charged and aromatic residues Lys27, Trp29,Asp31, Phe32, Arg33, and Lys47 (erabutoxin-a numbering)(Fig. 5: A and B) (25). In addition, each type of toxinalso utilizes specific residues for receptor-recognition(Fig. 5: C and D), which are in erabutoxin-a, His6,

Gln7, Ser8, Ser9, and Gln10 of loop I and Tyr25, Gly34, Ile36,and Glu38 of loop II (87, 88) and in � -cobratoxin,Arg36 of loop II and Phe65 of the C-terminus tail (79).The functional significance of Lys27, Arg33, and Lys47 ofloop II was independently verified by mutagenesisstudies on another short-chain � -neurotoxin NmmI(Naja mossambica mossambica) (77) and by structuralstudies on the long-chain � -bungarotoxin (85, 89).Furthermore, the functionally important residues for theTorpedo or muscle (�1)2�1�� receptor are all locatedon the concave surfaces of both short-chain and long-chain � -neurotoxins with the most critical residueArg33 located at the very tip of loop II (Fig. 5: D and F)(25, 88).

Significantly, none of the residues in loop I of thelong-chain toxin �-cobratoxin were found to be func-tionally important (52, 79) in contrast to the involvementof loop I in the short-chain toxin erabutoxin-a. Sinceloop I in �-cobratoxin is considerably shorter thanthat in erabutoxin-a, it is possible that this makes itinaccessible to the receptor surface and precludes itsinvolvement in any interaction with the receptor (51).Nonetheless, NMR studies have found that Thr8 andPro10 in loop I of another long-chain toxin, � -bungaro-toxin, appeared to interact with cognate peptides of the

Torpedo nAChR containing the putative neurotoxinbinding site (85, 90). While the question remains as towhether this data could be extrapolated to representtoxin interaction with the intact nAChR, it could beargued that loop I of � -bungarotoxin, which is longerthan that of � -cobratoxin by two residues, could indeedbe long enough to be involved in receptor interaction(85). Although residues in the C-terminus tails of � -cobratoxin (Phe65) and � -bungarotoxin (Lys70 and Arg72)were found to be important for receptor interaction(52, 85), the long-chain �-neurotoxin LSIII (Laticauda

semifasciata) which has a short C-terminal tail (Fig. 1:F) was also found to bind with high affinity to the musclenAChR, suggesting a minimal functional role for thecharacteristic long C-terminal of long-chain � -neuro-toxins (50).

The atypical long-chain � -neurotoxins Lc-a and Lc-b(Laticauda colubrina) retain most of the functionallyimportant residues required for binding to muscle(�1)2�1�� nAChRs (Fig. 6). However, of these keyresidues for the muscle receptor, only three (Lys27, Lys47,Arg37) are present in homologous positions in non-conventional three-finger toxins from cobras (e.g., WTX(Naja kaouthia) and Wntx-5 (Naja sputatrix)) thatshowed weak interaction with muscle nAChRs. Incontrast, the critical residues Trp29, Arg33, Arg36, Glu38,and Gly34 are present at homologous positions in theprimary sequence and in similar spatial disposition inthe tertiary structure of candoxin (Fig. 6) (58). Sinceconserved functionally important residues most likelyhave comparable binding function among neurotoxins(56), this may explain the nanomolar affinity ofcandoxin for muscle and neuronal �7 nAChRs, notpreviously reported for any other non-conventional toxin(57).

11. Molecular interactions: a receptor perspective

i. Insights from the molluscan acetylcholine binding

protein

The remarkable structural and functional similaritiesbetween the molluscan acetylcholine binding proteinand the amino-terminal ligand-binding domain of thenAChR enabled the construction of models of � -neuro-toxins (� -cobratoxin or � -bungarotoxin) in complexwith the receptor, incorporating established biochemicaland mutagenesis data on � -neurotoxin-nAChR interac-tions (82, 85, 89, 91). The model complexes clearlyshowed that � -neurotoxins bind perpendicularly to thereceptor axis, with the tip of loop II plugging into thebinding site of the receptor at the interface betweentwo subunits and the C-terminal tails of � -cobratoxinand � -bungarotoxin as well as loop I of � -bungarotoxin


Fig. 5. A, B: Critical residues in erabutoxin-a and �-cobratoxin for the Torpedo (or muscle) nicotinic acetylcholine receptor.The 2.0-Å crystal structure of erabutoxin-a (5EBX) (A) and the 2.4-Å crystal structure of � -cobratoxin (2CTX) (B) showing theresidues by which they interact with the Torpedo receptor. The Protein Data Bank accession codes are stated in parenthesis.Models based on data for erabutoxin-a (87, 102) and �-cobratoxin (79). The concave surfaces of the toxins are shown. Theresidues constituting the common binding core in both toxins are shown in red. The residues specific for each toxin are shownin dark green. In erabutoxin-a, the specific residues that have supplementary roles in the toxin-receptor interaction are shown inlight green. The Corey-Pauling-Koltun space-filling molecular representation is presented. C, D: Critical residues in erabutoxin-afor the Torpedo (or muscle) nicotinic acetylcholine receptor. The concave surface (Corey-Pauling-Koltun representation) oferabutoxin-a (C) depicting the residues identified as playing crucial (red), important (green), and supplementary (blue) rolesin binding to the muscle or Torpedo receptor as defined by affinity decreases of �100-fold, 10 – 100-fold, and �10-fold,respectively. A side view of erabutoxin-a (� -carbon backbone structure) (D) showing the side chains of all the functionallyimportant residues for interaction with the Torpedo receptor facing the concave surface. E, F: Critical residues in �-cobratoxin forthe Torpedo (or muscle) nicotinic acetylcholine receptor. The residues in �-cobratoxin (Corey-Pauling-Koltun representation) (E)that constitute the common binding core (i.e., crucial for binding to both neuronal �7 and Torpedo receptors) are shown inred, whereas those specific for binding only to Torpedo receptors are shown in green. A side view of �-cobratoxin (�-carbonbackbone structure) (F) showing the side chains of all the functionally important residues for interaction with the Torpedo

receptor facing the concave surface.


making adjacent additional interactions at the receptorsurface (Fig. 7: A – C). The receptor was also shownto establish major contacts with the toxin principallyvia loop C, assisted by loops A and B of the principal(�1) subunits as well as loops D and F of the comple-mentary (� , � , or �) subunits (25, 41, 82, 86) (Fig. 7: D).Furthermore, the �-neurotoxin is postulated to competewith acetylcholine by introducing the positive charge ofArg33 located at the very tip of loop II into the ligand-binding pocket of the receptor, thereby simulating a“small ligand” that can plug into the cavity offered bythe receptor interface (41, 82, 88).

ii. Insights from nicotinic acetylcholine receptors of

a-neurotoxin-resistant species

Comparative studies on muscle nAChRs of differentanimal species, such as those from the mongoose andcobra that are naturally resistant to the effects of � -neurotoxins and those from the mouse and Torpedo

ray which are sensitive, have also provided valuableinformation on toxin-receptor interactions (92 – 94).Collectively, these studies led to the definition of twosubsites, an aromatic subsite that includes two aromaticresidues at positions 187 and 189 and a proline subsitethat includes Pro194 and Pro197, in loop C of the ligand-binding domain of the �1-subunit of the nAChR. Aminoacid substitutions at these key positions (94) as well asN-linked glycosylation of loop C residues (e.g., Phe189)(73, 95) are believed to confer � -neurotoxin resistanceto the muscle nAChR of species resistant to snakeenvenomation.

iii. Insights from synthetic peptides derived from the

nicotinic acetylcholine receptor

Synthetic peptides have also been used to localize the�-neurotoxin binding sequences of the �1-subunit ofnAChRs (86, 96). Collectively, these studies identifiedreceptor segments coincident with loop C of the �1-subunit of the nAChR as the primary � -neurotoxinbinding site. This region includes conserved aromaticresidues Tyr190 and Tyr198 and the unique Cys192 – Cys193

disulfide bridge (86). Synthetic peptide binding studiesalso position the �-neurotoxin binding site in a homo-logous region on the neuronal �7 nAChR (89, 97).Further important information about the structure ofthe loop C region of the nAChR has been gleaned fromNMR and X-ray crystallography studies involvingcomplexes between � -bungarotoxin and receptor pep-tides (86, 96). These data found that the overall three-finger fold of � -bungarotoxin was preserved in thetoxin-receptor peptide complex with conformationalrearrangements occurring at the tips of loops I and IIand the C-terminus tail. The complexes also revealed asnug fit of the peptide to the toxin, with the boundpeptide adopting a �-hairpin conformation that resem-bled the corresponding loop region in the molluscanacetylcholine binding protein (41, 91). This permittedthe superimposition of the toxin-peptide complex ontoa model of the acetylcholine binding protein and, byanalogy, visualizes the interaction between � -neuro-toxins and the nAChR. The receptor peptides werefound to bind between toxin loops I and II with the tipof loop I interfacing with the receptor peptide residuehomologous to the aromatic residue at position 189 ofthe �1-subunit loop C (86). Furthermore, four positivelycharged residues in the toxin (Arg36 and Lys38 (loop II)

Fig. 6. Functionally invariant residues in three-finger neurotoxins that interact with muscle nicotinic acetylcholine receptors.Amino-acid sequences of representative short-chain �-neurotoxins, long-chain �-neurotoxins, atypical long-chain neurotoxins,and non-conventional three-finger toxins showing the distribution of residues critical for binding to muscle (�1)2�1�� nicotinicacetylcholine receptors (shaded in grey). Figure is based on mutational experimental data for erabutoxin-a (87, 102) and �-cobratoxin (79) and structural data for �-bungarotoxin (85, 89). Since conserved functionally important residues are likelyto have comparable binding function among neurotoxins, the putative functional residues in candoxin, WTX, Lc-a, and Lc-b arealso shown. The species names and Protein Data Bank accession numbers are as for Fig. 3. The cysteine residues are in boldlettering and the disulfide linkages and the segments contributing to the three loops and C-terminus tail are outlined.


and Lys70 and Arg72 (C-terminus tail)) are in closeapposition with highly conserved aromatic residues(Trp187, Tyr189, Tyr190) in the �1-subunit, suggesting thatcation-� interactions may be involved in the formationof the receptor-toxin complex (85).

12. Three-finger a-neurotoxins as pharmacological

probes: old dogs, new tricks

Since the advent of �-bungarotoxin as a pharmaco-logical probe to dissect the biology of the nAChR, awide array of three-finger � -neurotoxin derivativesincluding radioactive, fluorescent, and biotinylatedconjugates have been developed and are commercially

available (47). In addition, the functional blockadeproduced by � -neurotoxins is also useful in characteriz-ing nAChR ligands in pharmacological and electro-physiological studies. Apart from these well-definedand much exploited roles, � -neurotoxins from snakevenoms have recently been the focus of novel experi-mental strategies.

A new facet in toxinology evolved with efforts toobtain synthetic peptides that bind � -neurotoxins withhigh affinity. This venture began with the identificationof a 13-residues-long lead-peptide from a combinatorialphage-display library that inhibited the binding of � -bungarotoxin with low-micromolar affinity (98). A newpeptide library was then designed based on systematic

Fig. 7. �-Neurotoxin binding residues of the functional loops constituting the ligand-binding site of nicotinic acetylcholinereceptors and a model of an � -neurotoxin – nicotinic acetylcholine receptor complex. A molecular model of �-cobratoxinbinding to the neuronal �7 nicotinic acetylcholine receptor (Ligand-Gated Ion Channel Database (103) accession #ACHa7gaga)as viewed from the side (A), top (B), and bottom (C). The homology model of the neuronal �7 receptor is based on the crystalstructure of the molluscan acetylcholine-binding protein (Protein Data Bank accession #119B) with only two of the fivesubunits depicted in this model. The first, second, and third loops and the carboxy terminal tail of �-cobratoxin are labelled as 1,2, 3, and CT, respectively, in A: �-Cobratoxin is shown bound, perpendicularly to the receptor axis, with the tip of loop IIplugging into the binding site of the receptor at the interface between two subunits and the C-terminal tail making additionalinteractions at the adjacent receptor surface. See also Ref. 82. D: Sequence alignment of the functional loops of �1, � , � , and �subunits of the muscle nicotinic acetylcholine receptor and the �7 subunit of the neuronal �7 nicotinic receptor showing theamino acid residues involved in binding �-neurotoxins. In the muscle receptor, loops A, B, and C belong to the �1 subunit,whereas loops D, E, and F belong to the adjacent complementary � , � , or � subunit. In the neuronal �7 receptor, loops A, B, andC belong to the principal binding face and loops D, E, and F belong to the complementary binding face of the �7 subunit.Residues that interact with short-chain (bold), long-chain (bold and underlined), or both types of �-neurotoxins (bold andshaded in grey) are shown (numbered as in the mouse muscle nAChR). Data based on Refs. 10, 35, and 82.


single residue replacement followed by rational multipleresidue replacement of amino acids in the library-lead-peptide (96). Some of these high-affinity peptides werebetter inhibitors (IC50 approximately 2 nM) of � -bungarotoxin binding to muscle nAChR than analogouspeptides derived from natural amino acid sequences ofthe muscle nAChR. Interestingly, in preliminary in vivostudies, the high-affinity peptides injected into micewere capable of neutralizing the toxicity of � -bungaro-toxin, suggesting that this approach may eventuallyyield potentially useful antidotes for neurotoxin enveno-mation (99). The approach of creating high affinitypeptides by “systematic residue replacement” may alsobe applied as a general method for the design andsynthesis of high-affinity peptides in any biochemicalsystem that involves a ligand and a receptor molecule(96).

The scope of � -neurotoxins as molecular probeswas enhanced by the demonstration that a neuronal �3subunit of the nAChR, normally insensitive to � -neuro-toxin blockade, could be converted to an � -neurotoxin-sensitive receptor (IC50 approximately 20 – 40 nM) bythe exchange of just five residues in its loop C withthe corresponding residues from the � -bungarotoxin-sensitive neuronal �7 nAChR (100). More recently,this procedure has also been carried out with success onnon-� subunits of the nAChR (86). Likewise, and moregenerally, it may be possible to introduce � -bungaro-toxin sensitive sites (“pharmatopes”) into other non-acetylcholine receptors and membrane proteins, therebypermitting the use of various � -neurotoxin tags tolocalize and isolate and characterize new receptor sub-types (86). The development of synthetic mimotypepeptides that bind �-bungarotoxin with greater affinitiesthan peptides derived from native nAChR sequences(96) may provide even more sensitive “pharmatopes”that could be introduced into proteins of interest withthe aim of creating high affinity toxin-binding sitesthat allow novel experimental access to � -neurotoxinprobes (86).

The ingenuity of snakes in utilizing a common three-finger protein scaffold to produce toxins with a widearray of pharmacological functions offers an exemplaryexample for protein engineering (43, 45). Using thesame principle, toxins with new pharmacologicalproperties can be engineered by providing the three-finger scaffold with a novel, customized bindingproperty (47). For example, the functional residues offasciculin 2, an anti-acetylcholinesterase toxin with athree-finger structure (17), have been transferred tohomologous positions of a host short-chain � -neuro-toxin creating a chimera that lost its nAChR bindingproperties but acquired anti-acetylcholinesterase activity

comparable to the donor toxin (101). With this success,it is now possible to generate rationally designed novelactivities on toxin scaffolds, ab initio, opening the doorsto the development of mini-proteins of therapeutic value(47).

13. Conclusions: not the last word yet

The past forty years or so, with the molecular andgenomic revolutions to its credit, have seen remarkableprogress being made in our scientific prowess. Alongwith the flow, our understanding of the structure andfunction of both � -neurotoxins and the nAChR, as wellas the interaction between the two, has vastly improvedto the atomic level. The amino acid residues by whichthe toxin molecule binds to the receptor and the sites bywhich the receptor interacts with the toxin have largelybeen delineated. These data reveal the intricacy andremarkable plasticity of the three-finger toxin fold thathave optimally evolved to interact with specific mole-cular targets with precision capable of discerning subtledifferences in just a few amino acid residues betweenreceptor subtypes and high affinity that is often manyfold greater than the affinity of the natural ligand forthat receptor. Three-finger � -neurotoxins are also idealcandidates to address essential questions about proteinsin general, including protein-protein interactions, as wellas offering an interesting molecular basis about theevolution of protein scaffolds. Data gleaned from the�-neurotoxin – nAChR interaction can also be extrapo-lated to other members of the ligand-gated ion channel

superfamily including GABAA, serotonin 5-HT3, andglycine receptors, making this field of study all the moreimportant for neurobiology in general. � -Neurotoxinsand other biogenic molecules from animal venoms havealready established themselves as key players in scien-tific discovery and medicine as ultra-selective pharma-cological probes as well as lead compounds for drugdiscovery. With the advent of novel technologies anddiscovery of new toxins, it is not presumptuous to envis-age more ambitious strategies for protein engineeringand design of scientific probes and therapeutic mole-cules by exploiting the natural potential of animal toxins.

Acknowledgments

We dedicate this review to the memory of Dr. JimmyCheah Li-Sam of the Department of Pharmacology,National University of Singapore, a dear friend, valuablecolleague, and skilled “classical” pharmacologist, whopassed away most unexpectedly in December, 2002.His contribution and support to our work had beenimmeasurable.


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Three-Finger -Neurotoxins and the Nicotinic Acetylcholine