Abstract
Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson's disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.
Similar content being viewed by others
Introduction
Marine organisms belonging to the genus Conus are a rich source of pharmacological agents that act on diverse ion channels1, 2, 3, 4, 5. These agents, known as conopeptides, have evolved as selective tools for capture of prey or deterrence of predators. The ∼500 known different Conus species each contain greater than 100 different components in their venom, leading to an estimated >50 000 different pharmacologically active conopeptides.
The best characterized biologically-active venom components are conotoxins, small, disulfide-rich peptides, most of which are specifically targeted to different voltage- or ligand-gated ion channels3, 6. One rationale for the vast diversity of different conotoxins found in Conus venom is that this is an evolutionary consequence of the remarkable diversity of molecular isoforms of ion channels. Each Conus species uses the equivalent of several drug combinations to rapidly capture its prey. This review focuses on one family of compounds in Conus venom referred to as α-conotoxins, which are nicotinic acetylcholine receptor (nAChR) antagonists.
All Conus species characterized thus far have at least one nAChR antagonist in their venom7. The largest family of such antagonists are the α-conotoxins, small, disulfide rich peptides generally 12–19 amino acids in length8, 9. The α-conotoxins are classified based on their cysteine pattern CC-C-C, with a disulfide connectivity of Cys1-Cys3 and Cys2-Cys4. There is a further sub-classification of α-conotoxins based on the number of residues in their inter-cysteine loops (see Figure 1). The α3/5-conotoxins (3 residues in the first and 5 in the second loop) are selective blockers of the muscle nAChR, whereas the α4/7, α4/4, and α4/3 subfamilies are generally blockers of neuronal nAChRs3, 7, 8. Although this review focuses primarily on neuronally targeted α-conotoxins, the currently known muscle nAChR targeted α-conotoxins are briefly discussed below (also see Table 1).
Muscle nAChR-targeted α-conotoxins
The first α-conotoxins to be purified from Conus venom were α-conotoxins GI, GIA, and GII from the fish-hunting cone snail Conus geographus10. The best characterized is α-CTx GI, which blocks neuromuscular transmission both in- vitro11, 12, 13 and in vivo14, but does not block any of the neuronal nAChR subtypes15, 16. α-CTx MI, from Conus magus, was subsequently isolated and characterized as another blocker of the ACh responses at the neuromuscular junction17. Subsequently, a series of muscle nAChR blocking α-conotoxins were isolated from Conus striatus, α-CTx SI, α-CTx SIA, and α-CTx SII18, 19, 20, Conus consors, α-CTx CnIA, and α-CTx CnIB21 and Conus achatinus, α-CTx Ac1.1a and α-CTx Ac1.1b22.
Binding data on mouse muscle receptors demonstrate that α-CTx GI, α-CTx MI, and α-CTx SIA are preferentially targeted to the α/δ interface of the muscle nAChR (with an affinity >10 000 fold higher than for the α/γ interface23, 24, 25. In contrast, for the Torpedo nAChR, all three peptides display much higher affinity for the α/γ vs the α/δ interface24. Two newly discovered toxins from Conus achatinus, α-CTx Ac1.1a, and α-CTx Ac1.1b, block the mouse α/δ interface with a potency that is >50 000 fold higher than α/γ22. In contrast, α-CTx SI has low affinity for both the α/γ and α/δ interfaces of mouse muscle nAChR, and does not discriminate between the two sites as well as either α-CTx GI or α-CTx MI24. α-CTx SI has less toxicity in vivo than either α-CTx GI or α-CTx MI18. In addition, α-CTx SI does not discriminate between the two binding sites on the Torpedo nAChR26, 27. Structure-activity studies have indicated that Arg at position 9 of α-CTx GI is responsible for the differential affinity of this toxin for the two binding sites of the Torpedo nAChR28; this residue also confers high affinity for the α/γ interface of mouse muscle nAChR27. Instead of Arg, α-CTx SI has a Pro in the homologous position, which may account for its differential pharmacology27, 28. Comparison of solution structures of α-CTx SI with α-CTx GI suggests that it is the loss of the basic charge, rather than differences in backbone structure, that underlies the pharmacological differences between the two toxins29. The residues on the mouse δ subunit that confer high binding affinity to α-CTx MI have been determined and include Ser36, Tyr113, and Ile17830. The α-CTx MI residues that interact with the receptor binding pocket have also been determined30, 31, 32. In addition, a recent study has indicated the importance of a positively charged residue (either an Arg or Lys) at the C-terminus of α-CTx GI, α-CTX SI, and α-CTX SIA in enhancing affinity for both binding sites on the Torpedo nAChR33.
Although all of the “classical” muscle nAChR blocking α-conotoxins belong to the α3/5-conotoxin subfamily, several muscle nAChR blocking α-conotoxins belong to other branches of the α-conotoxin family (Figure 1). α-CTx EI, isolated from the fish-hunting Atlantic species Conus ermineus34, is an α4/7-conotoxin, a subfamily that is usually associated with blocking neuronal nAChRs. The solution structure of α-CTx EI is similar to other α4/7 conotoxins (Figure 1)35. The backbone structure of α4/7 conopeptides is illustrated in Figure 1. Although α-CTx EI does block some neuronal nAChRs36, unlike other α4/7 conotoxins it's also a potent blocker of the muscle nAChR34, 36. Unlike the α3/5 conotoxins that preferentially block the mammalian α/δ interface (see above), α-CTx EI blocks both the α/δ and α/γ interface with similiar affinity34. Moreover, α-CTx EI has higher affinity for the α/δ interface of the Torpedo nAChR, in contrast to the α/γ-interface preferring α3/5-conotoxins34. Recently, two other α4/7-conotoxins that block the muscle nAChR, and to a lesser extent neuronal nAChRs, have been discovered, α-conotoxins SrIA and SrIB from Conus spurius36. Similar to α-CTx EI, α-CTx SrIA and α-CTx SrIB contain post-translational modifications in their native sequences. Unlike other α4/7 conotoxins, however, α-CTx SrIA, and SrIB are reported to have nAChR potentiating activity on the α4β2 subtype36.
Another unusual muscle-specific α-conotoxin is α-CTx PIB, which has an uncommon 4/4 intercysteine loop spacing37. It was purified from the venom of Conus purpurascens and found to block both adult and fetal mouse muscle nAChRs expressed in oocytes with nanomolar potency, with little effect on any neuronal subtypes37.
Neuronal nAChR-targeted α-conotoxins
The α-conotoxins targeting neuronal nAChRs are numerous and have even more exquisite subtype selectivity, probably due to the large diversity of isoforms in this subfamily of receptors. The members of this family of toxins that have been discovered thus far are listed below, in no particular order (also see Tables 2 and 3).
α-Conotoxins ImI and ImII
The first α-conotoxin targeting neuronal nAChRs was α-CTx ImI. It was also the first α-conotoxin isolated from the venom of a worm hunting species, Conus imperialis38. This α-conotoxin has the unusual inter-cysteine loop spacing 4/3 (Figure 1A). This peptide was found to be inactive when injected interaperitoneally, whereas intracereberal injection caused seizures and death38. In addition, this toxin blocks nicotinic responses of B cells on frog sympathetic ganglia but not mammalian neuromuscular nAChRs38, 39. Subsequent pharmacological characterization on heterologously expressed nAChRs showed an IC50 of 220 nmol/L on homomeric α7 nAChRs and 1.8 μmol/L on homomeric α9 nAChRs16. Ellison et al, (2004) reported an IC50 of 595 nmol/L on homomeric α7 nAChR, but they also showed potent inhibition of heteromeric α3β2 nAChR (IC50 41 nmol/L). The seizure inducing effects of this toxin are presumably due to its block of α-bungarotoxin-sensitive α7 nAChRs on hippocampal neurons40. Structure-activity studies have suggested a role for residues in the first loop of the toxin, Asp5, Pro6, and Arg7, as well as Trp10 in the second loop, in interacting with the α7 subunit41. Homology modeling using Aplysia AChBP, a structural homolog of the N-terminal binding region of nAChRs, confirms a key role for Arg7 and Trp10 in interaction with residues at the ligand binding site42, 43. Co-crystallization of α-CTx ImI with Aplysia AChBP reveals a more open C-loop to accommodate the α-conotoxin44. The α7 subunit residues that interact with the toxin have also been determined45, 46.
In recent years, attempts have been made to improve the biological stability of α-conotoxins by protecting the disulfide bonds against reduction or scrambling due to exposure to intra- or extracellular environments, such as blood. One of these studies utilized selenocysteines in place of cysteines to yield several selenoconotoxin analogs of α-CTx ImI47. Although similar to wildtype α-CTx ImI in both structure and activity against α7 nAChRs, the selenoconotoxin analogs were more stable than wildtype α-CTx ImI under a variety of chemical and biological reducing conditions47. A second method of improving conformational stability is to replace the cystine bridges with non-reducible dicarba linkages. The dicarba analog of α-CTx ImI had a similar structure to wildtype α-CTx ImI, with a slight difference in the geometry of disulfide vs dicarba bridges. The activity of the analog against α7 nAChR was similar to wildtype α-CTx ImI48.
Another α4/3 conotoxin from the venom of Conus imperialis, α-CTx ImII, was identified using a PCR-based discovery strategy49. Although highly homologous to α-CTx ImI in sequence (9 out of 12 residues are shared), α-CTx ImII, unlike α-CTx ImI, does not compete with α-bungarotoxin for binding to heterologously expressed α7 nAChRs, suggesting a distinct and perhaps novel binding site on the α7 nAChR for α-CTx ImII50. The difference in binding of the two toxins is due to the presence of a Pro at position 6 of α-CTx ImI, which has been shown to be important for interaction of this toxin with α7 nAChRs41. α-CTx ImII has an Arg at this position; mutation of this Arg to a Pro creates an analog that competes with α-bungarotoxin binding49.
α-Conotoxins MII, PIA, OmIA
A second α-conotoxin isolated from Conus magus was α-CTx MII. It belongs to the α4/7 subfamily (Figure 1). α-CTx MII blocks the α3β2 nAChR subtype with a potency 2–4 orders of magnitude higher than most other nicotinic receptors, including the muscle subtype51. However, binding studies in knock-out animals52 as well as functional studies53, have shown that α-CTx MII also potently acts at α6-containing nAChRs. Subsequently, a series of α-CTx MII analogs were made that selectively target the chimeric α6/α3β2β354 vs α3β2 nAChR53. These toxin analogs are the most selective α6* nAChR antagonists reported to date. The wildtype α-CTx MII, as well as a number of α6* (the asterisk indicates the presence of additional subunits) selective α-CTx MII analogs, have been used to characterize nAChR subtypes that modulate dopamine release in rat55, 56, 57, 58, 59, 60, 61, mice62, 63, 64, 65, and monkey striatum66, 67. These studies indicate a role for α6β2* and α6α4β2* nAChRs in the modulation of dopamine release in the striatum and the nucleus accumbens. The contribution of both the α4 and the β3 subunits to α-CTx MII binding sites in dopaminergic neurons has been confirmed in studies using knock-out mice64, 68, 69. Some studies also suggest a selective downregulation of α6* nAChRs upon chronic nicotine exposure70, 71. α-CTx MII has also been used to characterize distinct nAChRs that modulate [3H]NE release in rat72 vs mouse hippocampus73. Table 4 summarizes some of the nAChR-mediated physiological functions characterized using the α-conotoxins.
Structure-activity studies have identified the amino acid residues on the α3 and the β2 nAChR subunits that interact with α-CTx MII. These include Lys185 and Ile188 on the α3 subunit and Thr59, Val109, Phe117 and Leu119 on the β2 subunit74, 75. In addition, α-CTx MII[S4A; E11A; L15A] has been used to identify the nAChR subunit amino acid residues that interact with, and confer selectivity, for the α6 vs the α3 subunit76. Notably, these nAChR subunit residues are distinct from those that interact with wildtype α-CTx MII74.
Radiolabeling of α-CTx MII has allowed direct measurement of the binding sites for this α-conotoxin within the brain77. Fluorescently-labeled analogs of α-CTx MII have also recently been synthesized78. Using the radiolabeled α-CTx MII, Quik and co-workers have found a selective downregulation of α-CTx MII binding sites within rodent and monkey striatum after nigorstriatal damage79, 80, 81, 82 as well as in humans with Parkinson's82. Direct measurement of α-CTx MII sites also shows preferential recovery of these sites in monkeys allowed to recover from nigrostriatal damage66. Binding studies using an analog of α-CTx MII, α-CTx MII[E11A], have shown a selective loss of a specific subtype of nAChR (α6α4β2*) in rodent and monkey models of Parkinson's disease. These studies were replicated in post-mortem tissue from humans with Parkinson's83. Thus, this α-CTx MII analog has a nAChR subtype selectivity that has potential in the early diagnosis of Parkinson's disease.
A number of attempts have been made to enhance the stability of α-CTx MII against proteolysis as well to improve its lipophilicity, with the aim of increasing the oral bioavailability of this toxin. Backbone cyclization by use of linker placement and joining of the N- and C-termini resulted in several cyclic α-CTx MII analogs. Two of these analogs had structures similar to native α-CTx MII, although only one of these analogs-the one with the longest linker-retained activity similar to native α-CTx MII. The cyclic analog was much more stable than the native toxin in human plasma84. To enhance the lipophilicity of α-CTx MII, the toxin was conjugated with 2-amino-D,L-dodecanoic acid (Laa) at the N-terminus. This terminally-conjugated α-CTx MII analog had similar structure and activity compared to the parent peptide85, but displayed significantly improved permeability across Caco-2 cell monolayers85. Although the Laa-conjugated analog did not cross the blood brain barrier to any great extent, its absorption through the GI tract after oral administration was greater than the parent α-CTx MII86.
A number of other α-conotoxins have selectivity either towards α3β2 or α6β2β3 nAChRs. α-CTx PIA, discovered through PCR-based cloning of cDNA from venom duct of Conus purpurascens, has about 70-fold lower IC50 for chimeric α6/α3β2β3 vs α3β2 nAChRs87. In contrast, α-CTx OmIA, purified from venom of Conus omaria, has a preferential (20-fold) selectivity for α3β2 vs α6/α3b2b3 nAChRs88, 89. It is also a potent inhibitor of α7 nAChR89.
α-Conotoxins GIC and GID
Two α-conotoxins specific for neuronal nAChRs have been purified from the venom of Conus geographus. α-CTx GIC, an α4/7 conotoxin identified from the genomic DNA of Conus geographus, has low nanomolar potency for α3β2 and α6β2 nAChRs vs muscle, α3β4 and α4β2 nAChRs90, 91. α-CTx GID, although similar to α-CTx GIC in its inter-cysteine loop spacing (α4/7), is structurally different from α-CTx GIC and other neuronal nAChR targeted α-conotoxins in several ways. First, it has additional N-terminal residues compared to α-CTx GIC and most other previously identified α-conotoxins. Second, it has two posttranslational modifications: a γ-carboxyglutamic acid residue just before the first Cys residue and a hydroxyproline at position 16. Third, there is a positively charged residue, Arg, at position 12, whereas α-CTx GIC and most other α-conotoxins have either a hydrophobic (Ala or Phe) or uncharged (Asn) residue. Interestingly, a positive charge at this position seems to be responsible for α-CTx GID's relatively high affinity for α4β2 nAChRs compared to other α-conotoxins lacking this positive residue92, 93. It is worth noting here that the two muscle-specific α-conotoxins-SrIA and SrIB-that have potentiating effects on the α4β2 nAChR also have an Arg at the homologous position (Table 1). An 'Ala walk' of α-CTx GID also indicates important roles for most residues of the toxin, with the exception of Val13, in interaction with α4β2 nAChR93. Similar to α-CTx GIC, α-CTx GID also potently blocks α3β2 nAChR92, 93. In contrast to α-CTx GIC, however, α-CTX GID also blocks α7 nAChR92, 93. Structure-activity studies indicate the most important residue for interaction with both the α7 and the α3β2 nAChRs is Pro9, with smaller contribution by Asp3 and Arg12. Asn14 seems to be important for interaction with α7 nAChR only93. The β2 subunit residues conferring the high potency of α-CTx GID on α3β2 nAChR are the same residues that also confer high potency of α-CTx MII and α-CTx PnIA for this receptor subtype75.
α-Conotoxin EpI
α-CTx EpI, an α4/7-conotoxin purified from the venom of Conus episcopatus, is another example of a post-translationally modified conotoxin. This conotoxin is different from previously characterized α-conotoxins in that it has a sulfated Tyr at position 1594. However, both the native sulfated and synthetic non-sulfated peptide are similar in activity and inhibit α-bungarotoxin-resistant nAChR responses in adrenal chromaffin cells and rat intracardiac ganglia parasympathetic neurons, but do not inhibit muscle nAChR responses94. Therefore, this toxin was designated as a specific blocker of α3β2 and α3β4, but not α7, nAChRs. However, studies with heterologously expressed nAChRs in oocytes showed the inverse selectivity, potent inhibition of α7 nAChRs with little effect on α3β4 or α3β2 nAChRs95. The inhibition of α7 nAChRs by α-CTx EpI is not surprising considering that α-CTx EpI is identical to α-CTx ImI in its first loop, the region containing residues shown to be important for interaction of α-CTx ImI with α7 nAChRs41.
α-Conotoxins AnIA, AnIB, and AnIC
Three peptides isolated from the venom of Conus anemone add to the list of sulfated α-conotoxins96. α-CTx AnIA, α-CTx AnIB, and α-CTx AnIC all have a sulfotyrosine at position 16. The synthetic α-CTx AnIB had subnanomolar potency at α3β2 nAChRs, with about 200-fold lower potency at α7 nAChR, and little or no activity on muscle and other tested heteromeric nAChRs. The non-sulfated peptide retained activity at α3β2 nAChRs, whereas it was 10-fold less potent on α7 nAChR. Removal of the two N-terminal glycines (to yield the same toxin as α-CTx AnIA) negatively affected binding kinetics, and as a result potency, of the toxin on α3β2 nAChRs96.
α-Conotoxins AuIA, AuIB, and AuIC
Three α-conotoxins isolated from the venom of Conus aulicus are α-CTx AuIA, AuIB, and AuIC. Similar to most other neuronal nAChR blocking α-conotoxins, α-CTx AuIA and AuIC belong to the α4/7 family, whereas α-CTx AuIB has the unusual 4/6 inter-cysteine loop spacing72. All three are selective blockers of the neuronal subtype, α3β4, with α-CTx AuIB being the most potent72. This α-conotoxin has been used to show involvement of distinct nAChR subtypes in modulation of [3H]NE, [3H]ACh, and [3H]DA release from hippocampal, interpeduncular nucleus and striatal synaptosomes, respectively72, 97.
α-Conotoxins PnIA and PnIB
α-CTx PnIA and PnIB were purified from the venom of Conus pennaceous98. Both toxins have a post-translational modification, sulfotyrosine at position 1599; however, the functional characterizations were all performed with non-sulfated synthetic toxins. Although they only differ in two amino acids, α-CTx PnIA is a potent blocker of α3β2 nAChR, whereas α-CTx PnIB blocks α7 nAChR more potently100.
Substitution of Ala10 in α-CTx PnIA with Leu, found in the homologous position in α-CTx PnIB, shifts selectivity of α-CTx PnIA[A10L] towards α7, with a potency that is even greater than α-CTx PnIB100, 101. The systematic truncation of the second loop affects the potency of α-CTx PnIA[A10L] for both the α7 nAChR and AChBP due to the loss of several hydrogen bonds between toxin and receptor upon toxin truncation102. Replacing Asn11 with Ser in α-CTx PnIA, the other residue different between α-CTx PnIA and α-CTx PnIB, caused loss of potency for both α3β2 and α7 nAChRs100.
Structure-activity studies have located residues in the α3 subunit that confer affinity for α-CTx PnIA. These include Pro182, Ile188 (also found to interact with α-CTx MII) and Gln198103. All three residues were found to be located within the C-loop of the subunit. The β2 subunit residues that interact with α-CTx PnIA are the same residues that also interact with α-CTx MII and α-CTx GID75. Adding an additional positive charge to the C-terminus of α-CTx PnIA[A10L] to yield the analog α-CTX PnIA[A10L, D14K] enhanced the affinity of the toxin for Lymnaea and Aplysia AChBPs33, 104. A recent study presented the crystal structure of α-CTx PnIA[A10L;D14K] bound to Aplysia AChBP and indicated predominantly hydrophobic and hydrophobic/ aromatic interactions between the analog and the AChBP binding pocket104. Double cycle mutant analysis has also indicated pairwise hydrophobic and aromatic interactions between α-CTx PnIB and the α7 nAChR105.
α-Conotoxin BuIA
α-CTx BuIA was cloned from RNA extracted from the venom of Conus bullatus106. Similar to the muscle nAChR blocking α-CTx PIB, it possesses a 4/4 cysteine loop spacing. Unlike other α-conotoxins, α-CTx BuIA is not specific for a particular subtype of nAChR, blocking almost all neuronal subtypes with nanomolar potency, with exception of α4β2106. However, its differential kinetics can distinguish between nAChRs that contain either a β2 or a β4 subunit: block of β2* nAChRs is rapidly reversed whereas block of β4* nAChRs is only slowly reversed upon toxin washout106. The subunit residues that are critical to these off-rate differences have been determined and, interestingly, are the same residues that interact with a number of other α-conotoxins75, 107. Two studies have solved the structure of α-CTx BuIA108, 109. The latter study has shown that the native globular structure of α-CTx BuIA is highly flexible and maintains multiple conformations in solution, as opposed to some other α-conotoxins that have a rigid globular structure109. This multiple-conformation structural feature may underlie the toxin's promiscuous selectivity profile.
The differential kinetics of α-CTx BuIA were utilized to determine the extent of the participation of the β2 and the β4 subunits in nAChRs on rat and mouse hippocampal noradrenergic terminals. These studies indicated the presence of the β4 subunit in all nAChRs on rat terminals, but its presence in only 60% of mouse terminal nAChRs73.
α-Conotoxins PeIA, RgIA and Vc1.1
Three newly discovered α-conotoxins have been shown to target the α9α10 nAChR, a subtype with highly unusual pharmacology as compared to other nAChRs110, 111. α-CTx PeIA, cloned from the venom of Conus pergrandis, blocks heterologously expressed α9α10 nAChRs, as well as native α9α10 nAChRs in cochlear hair cells with IC50s of about 7 and 4 nmol/L, respectively112. However, this toxin is also a potent blocker of α3β2 and chimeric α6/α3β2β3 nAChRs, with IC50s of about 23 and 30 nmol/L, respectively112.
α-CTx RgIA, cloned from the venom of marine worm-hunting species Conus regius, is another α4/3 conotoxin similar to α-CTx ImI (Figure 1); however, unlike α-CTx ImI, it's a much more potent blocker of α9α10 than α7 nAChRs113 and the most selective α9α10 antagonist reported to date. α-CTx Vc1.1, cloned from the venom of Conus victoriae, is an α4/7 conotoxin originally shown to block nAChRs found in adrenal chromaffin cells114, 115. In addition, this peptide blocks vascular inflammatory responses evoked by electrical stimulation of unmyelinated sensory nerves114. Further pharmacological characterization of this α-conotoxin indicated that it is a potent inhibitor of the α9α10 nAChR116, 117. Unlike α-CTx RgIA, however, native α-CTx Vc1.1 (referred to as Vc1a) contains three post-translational modifications: a hydroxyproline at position 6, a γ-carboxyglutamate at position 14 and an amidated C-terminus117. Both α-CTx RgIA and α-CTx Vc1.1 were shown to be effective analgesic agents in a rat model of nerve injury116, 117, 118 and α-CTx Vc1.1 (drug name ACV1) entered human phase II clinical trials for treatment of neuropathic pain119. However, the role of the α9α10 nAChRs in mediating this analgesic effect has been challenged117, 120.
Structure-activity data with α-CTx RgIA have indicated that this toxin binds to the ACh binding site on the receptor and that toxin residues Asp5, Pro6, Arg7, and Arg9 are important for interaction with α9α10 nAChRs121. The recently published three-dimensional structure of α-CTx RgIA also confirms an important role for Arg9 in interacting with negatively charged residues in the α9α10 nAChR122. Saturation transfer difference NMR studies examining the binding of α-CTx Vc1.1 to Lymnaea stagnalis AChBP, a close structural homolog of the α7 nAChR binding site, indicates a role for Tyr10 in binding to the AChBP123. Interestingly, this residue in α-CTx RgIA does not seem to be important for interaction with α9α10 nAChRs121.
α-Conotoxin TxIA
α-CTx TxIA, from the venom of Conus textile, was recently discovered using a novel approach for identification of new α-conotoxins from crude venom124. In this approach, the venom of Conus textile was screened against the Lymnaea AChBP in a competition binding assay with 125I-α-bungarotoxin. Biochemical characterization indicated that α-CTx TxIA belongs to the α4/7 conotoxin family and has the same cysteine arrangement and disulfide connectivity common to other α-conotoxins in this family. Binding and functional assays indicated that the affinity of this toxin for Lymnaea AChBP (1.7 nmol/L) was higher than other previously identified α-conotoxins, and that α-CTx TxIA had high potency for α3β2 nAChRs. Structure-activity studies, together with co-crystallization of an analog of α-CTxIA, α-CTx TxIA[A10L], with Aplysia AChBP indicated an important role for a long chain hydrophobic residue at position 9 or 10 and the Arg at position 5 for toxin affinity for AChBP and α7, but not α3β2, nAChRs124
α-Conotoxin Lp1.1
α-CTx Lp1.1 was cloned from both the genomic DNA and cDNA of Conus leopardus125. Although it belongs to the α4/7 conotoxin family, its primary sequence is unique in that it lacks the conserved Ser and Pro that is found in the first loop of all known neuronally active α-conotoxins (Table 2). This toxin caused uncoordinated swimming when injected intramuscularly in fish. At higher concentrations, it causes seizure and paralysis126. Interestingly, another α4/7 conotoxin (LeD2) isolated from a different Conus species, Conus litteratus, has the identical sequence to Lp1.1127.
α-Conotoxins ArIA and ArIB
We recently identified two new α-conotoxins from the venom of Conus arenatus, α-CTx ArIA and α-CTx ArIB128. Both belong to the α4/7 conotoxin family and are potent blockers of the α7 nAChR. However, both toxins also block the α3β2 nAChR with nanomolar potency128. Structure-function analysis was used to create two analogs of α-CTx ArIB, α-CTx ArIB[V11L; V16A] and α-CTx ArIB[V11L; V16D], which have high affinity for α7 nAChRs but have comparatively low activity on α3β2 nAChRs128. Compared to α-bungarotoxin, however, the faster off-rate kinetics of the α-CTx ArIB analogs make them useful ligands in equilibrium binding experiments. α-CTx ArIB[V11L; V16D] blocks rat, mouse and human nAChRs128, 129. A radiolabeled version, 125I-α-CTx ArIB[V11L; V16A], has also been developed130.
Concluding remarks
The last quarter of the century has witnessed the discovery of variety of different α-conotoxins targeting various isoforms of nAChRs. The next few years promise even more groundbreaking progress thanks to the recent advancements in phylogeny and exogenomic discovery of novel conotoxins6, 131. In addition, structure-activity studies in combination with homology modeling will lead to better understanding of interactions between α-conotoxins and nAChR ligand binding site, allowing the creation of analogs with improved potency and/or selectivity towards particular subtypes of nAChRs. In view of the important physiological role of nAChRs in pain, inflammation, nicotine addiction, Alzheimer's and Parkinson's disease, specific targeting of the relevant nAChR subtypes is an attractive pharmaceutical strategy, with the α-conotoxins being among the most promising drug development leads.
References
Gray WR, Olivera BM, Cruz LJ . Peptide toxins from venomous Conus snails. Annu Rev Biochem 1988; 57: 665–700.
McIntosh JM, Jones RM . Cone venom — from accidental stings to deliberate injection. Toxicon 2001; 39: 1447–51.
Terlau H, Olivera BM . Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol Rev 2004; 84: 41–68.
Armishaw CJ, Alewood PF . Conotoxins as research tools and drug leads. Curr Protein Pept Sci 2005; 6: 221–40.
Norton RS, Olivera BM . Conotoxins down under. Toxicon 2006; 48: 780–98.
Olivera BM, Teichert RW . Diversity of the neurotoxic Conus peptides: a model for concerted pharmacological discovery. Mol Interv 2007; 7: 251–60.
McIntosh JM, Olivera BM, Cruz LJ . Conus peptides as probes for ion channels. Methods Enzymol 1999; 294: 605–24.
Janes RW . alpha-Conotoxins as selective probes for nicotinic acetylcholine receptor subclasses. Curr Opin Pharmacol 2005; 5: 280–92.
Olivera BM, Quik M, Vincler M, McIntosh JM . Subtype-selective conopeptides targeted to nicotinic receptors: concerted discovery and biomedical applications. Channels (Austin) 2008; 2(2): 143–52.
Gray WR, Luque A, Olivera BM, Barrett J, Cruz LJ . Peptide toxins from Conus geographus venom. J Biol Chem 1981; 256: 4734–40.
McManus OB, Musick JR, Gonzalez C . Peptides isolated from the venom of Conus geographus block neuromuscular transmission. Neurosci Lett 1981; 25: 57–62.
McManus OB, Musick JR . Postsynaptic block of frog neuromuscular transmission by conotoxin GI. J Neurosci 1985; 5: 110–6.
Blount K, Johnson A, Prior C, Marshall IG . alpha-Conotoxin GI produces tetanic fade at the rat neuromuscular junction. Toxicon 1992; 30: 835–42.
Marshall IG, Harvey AL . Selective neuromuscular blocking properties of alpha-conotoxins in vivo. Toxicon 1990; 28: 231–4.
Luetje CW, Wada K, Rogers S, Abramson SN, Tsuji K, Heinemann S, et al. Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptor subunit combinations. J Neurochem 1990; 55: 632–40.
Johnson DS, Martinez J, Elgoyhen AB, Heinemann SF, McIntosh JM . alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors. Mol Pharmacol 1995; 48: 194–9.
McIntosh M, Cruz LJ, Hunkapiller MW, Gray WR, Olivera BM . Isolation and structure of a peptide toxin from the marine snail Conus magus. Arch Biochem Biophys 1982; 218: 329–34.
Zafaralla GC, Ramilo C, Gray WR, Karlstrom R, Olivera BM, Cruz LJ . Phylogenetic specificity of cholinergic ligands: alpha-conotoxin SI. Biochemistry 1988; 27: 7102–5.
Myers RA, Zafaralla GC, Gray WR, Abbott J, Cruz LJ, Olivera BM . alpha-Conotoxins small peptide probes of nicotinic acetylcholine receptors. Biochemistry 1991; 30: 9370–7.
Ramilo CA, Zafaralla GC, Nadasdi L, Hammerland LG, Yoshikami D, Gray WR, et al. Novel alpha- and omega-conotoxins from Conus striatus venom. Biochemistry 1992; 31: 9919–26.
Favreau P, Krimm I, Le Gall F, Bobenrieth MJ, Lamthanh H, Bouet F, et al. Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors. Biochemistry 1999; 38: 6317–26.
Liu L, Chew G, Hawrot E, Chi C, Wang C . Two potent alpha3/5 conotoxins from piscivorous Conus achatinus. Acta Biochim Biophys Sin (Shanghai) 2007; 39: 438–44.
Kreienkamp HJ, Sine SM, Maeda RK, Taylor P . Glycosylation sites selectively interfere with alpha-toxin binding to the nicotinic acetylcholine receptor. J Biol Chem 1994; 269: 8108–14.
Groebe DR, Dumm JM, Levitan ES, Abramson SN . alpha-Conotoxins selectively inhibit one of the two acetylcholine binding sites of nicotinic receptors. Mol Pharmacol 1995; 48: 105–11.
Luo S, McIntosh JM . Iodo-alpha-conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors. Biochemistry 2004; 43: 6656–62.
Hann RM, Pagan OR, Eterovic VA . The alpha-conotoxins GI and MI distinguish between the nicotinic acetylcholine receptor agonist sites while SI does not. Biochemistry 1994; 33: 14058–63.
Groebe DR, Gray WR, Abramson SN . Determinants involved in the affinity of alpha-conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors. Biochemistry 1997; 36: 6469–74.
Hann RM, Pagan OR, Gregory LM, Jacome T, Eterovic VA . The 9-arginine residue of alpha-conotoxin GI is responsible for its selective high affinity for the alphagamma agonist site on the electric organ acetylcholine receptor. Biochemistry 1997; 36: 9051–6.
Benie AJ, Whitford D, Hargittai B, Barany G, Janes RW . Solution structure of alpha-conotoxin SI. FEBS Lett 2000; 476: 287–95.
Sine SM, Kreienkamp HJ, Bren N, Maeda R, Taylor P . Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity. Neuron 1995; 15: 205–11.
Bren N, Sine SM . Hydrophobic pairwise interactions stabilize alpha-conotoxin MI in the muscle acetylcholine receptor binding site. J Biol Chem 2000; 275: 12692–700.
Jacobsen RB, DelaCruz RG, Grose JH, McIntosh JM, Yoshikami D, Olivera BM . Critical residues influence the affinity and selectivity of alpha-conotoxin MI for nicotinic acetylcholine receptors. Biochemistry 1999; 38: 13310–5.
Kasheverov IE, Zhmak MN, Vulfius CA, Gorbacheva EV, Mordvintsev DY, Utkin YN, et al. Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors. Febs J 2006; 273: 4470–81.
Martinez JS, Olivera BM, Gray WR, Craig AG, Groebe DR, Abramson SN, et al. alpha-Conotoxin EI a new nicotinic acetylcholine receptor antagonist with novel selectivity. Biochemistry 1995; 34: 14519–26.
Park KH, Suk JE, Jacobsen R, Gray WR, McIntosh JM, Han KH . Solution conformation of alpha-conotoxin EI a neuromuscular toxin specific for the alpha 1/delta subunit interface of torpedo nicotinic acetylcholine receptor. J Biol Chem 2001; 276: 49028–33.
Lopez-Vera E, Aguilar MB, Schiavon E, Marinzi C, Ortiz E, Restano Cassulini R, et al. Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors. Febs J 2007; 274: 3972–85.
Lopez-Vera E, Jacobsen RB, Ellison M, Olivera BM, Teichert RW . A novel alpha conotoxin (alpha-PIB) isolated from C, purpurascens is selective for skeletal muscle nicotinic acetylcholine receptors. Toxicon 2007; 49: 1193–9.
McIntosh JM, Yoshikami D, Mahe E, Nielsen DB, Rivier JE, Gray WR, et al. A nicotinic acetylcholine receptor ligand of unique specificity alpha-conotoxin ImI. J Biol Chem 1994; 269: 16733–9.
Tavazoie SF, Tavazoie MF, McIntosh JM, Olivera BM, Yoshikami D . Differential block of nicotinic synapses on B versus C neurones in sympathetic ganglia of frog by alpha-conotoxins MII and ImI. Br J Pharmacol 1997; 120: 995–1000.
Pereira EF, Alkondon M, McIntosh JM, Albuquerque EX . Alpha-conotoxin-ImI: a competitive antagonist at alpha-bungarotoxin-sensitive neuronal nicotinic receptors in hippocampal neurons. J Pharmacol Exp Ther 1996; 278: 1472–83.
Quiram PA, Sine SM . Structural elements in alpha-conotoxin ImI essential for binding to neuronal alpha7 receptors. J Biol Chem 1998; 273: 11007–11.
Hansen SB, Talley TT, Radic Z, Taylor P . Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica. J Biol Chem 2004; 279: 24197–202.
Ulens C, Hogg RC, Celie PH, Bertrand D, Tsetlin V, Smit AB, et al. Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP. Proc Natl Acad Sci USA 2006; 103: 3615–20.
Hansen SB, Sulzenbacher G, Huxford T, Marchot P, Taylor P, Bourne Y . Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. Embo J 2005; 24: 3635–46.
Quiram PA Sine SM . Identification of residues in the neuronal alpha7 acetylcholine receptor that confer selectivity for conotoxin ImI. J Biol Chem 1998; 273: 11001–6.
Quiram PA, Jones JJ, Sine SM . Pairwise interactions between neuronal alpha7 acetylcholine receptors and alpha-conotoxin ImI. J Biol Chem 1999; 274: 19517–24.
Armishaw CJ, Daly NL, Nevin ST, Adams DJ, Craik DJ, Alewood PF . Alpha-selenoconotoxins a new class of potent alpha7 neuronal nicotinic receptor antagonists. J Biol Chem 2006; 281: 14136–14143.
MacRaild CA, Illesinghe J, van Lierop BJ, Townsend AL, Chebib M, Livett BG, et al. Structure and activity of (2,8)-dicarba-(3,12)-cystino alpha-ImI an alpha-conotoxin containing a nonreducible cystine analogue. J Med Chem 2009; 52: 755–62.
Ellison M, McIntosh JM, Olivera BM . Alpha-conotoxins ImI and ImII, similar alpha 7 nicotinic receptor antagonists act at different sites. J Biol Chem 2003; 278: 757–64.
Ellison M, Gao F, Wang HL, Sine SM, McIntosh JM, Olivera BM . Alpha-conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor subtypes. Biochemistry 2004; 43: 16019–26.
Cartier GE, Yoshikami D, Gray WR, Luo S, Olivera BM, McIntosh JM . A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J Biol Chem 1996; 271: 7522–8.
Champtiaux N, Han ZY, Bessis A, Rossi FM, Zoli M, Marubio L, et al. Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice. J Neurosci 2002; 22: 1208–17.
McIntosh JM, Azam L, Staheli S, Dowell C, Lindstrom JM, Kuryatov A, et al. Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors. Mol Pharmacol 2004; 65: 944–52.
Kuryatov A, Olale F, Cooper J, Choi C, Lindstrom J . Human alpha6 AChR subtypes: subunit composition assembly and pharmacological responses. Neuropharmacology 2000; 39: 2570–90.
Kulak JM, Nguyen TA, Olivera BM, McIntosh JM . Alpha-conotoxin MII blocks nicotine-stimulated dopamine release in rat striatal synaptosomes. J Neurosci 1997; 17: 5263–70.
Kaiser SA, Soliakov L, Harvey SC, Luetje CW, Wonnacott S . Differential inhibition by alpha-conotoxin-MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices. J Neurochem 1998; 70: 1069–76.
Azam L, McIntosh JM . Effect of novel alpha-conotoxins on nicotine-stimulated [3H]dopamine release from rat striatal synaptosomes. J Pharmacol Exp Ther 2005; 312: 231–7.
Cao YJ, Surowy CS, Puttfarcken PS . Different nicotinic acetylcholine receptor subtypes mediating striatal and prefrontal cortical [3H]dopamine release. Neuropharmacology 2005; 48: 72–9.
Dwoskin LP, Wooters TE, Sumithran SP, Siripurapu KB, Joyce BM, Lockman PR, et al. N,N′-Alkane-diyl-bis-3-picoliniums as nicotinic receptor antagonists: inhibition of nicotine-evoked dopamine release and hyperactivity. J Pharmacol Exp Ther 2008; 326: 563–76.
Exley R, Clements MA, Hartung H, McIntosh JM, Cragg SJ . Alpha6-containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens. Neuropsychopharmacology 2008; 33: 2158–66.
Perez XA, Bordia T, McIntosh JM, Grady SR, Quik M . Long–term nicotine treatment differentially regulates striatal alpha6alpha4beta2* and alpha6(nonalpha4)beta2* nAChR expression and function. Mol Pharmacol 2008; 74: 844–53.
Salminen O, Murphy KL, McIntosh JM, Drago J, Marks MJ, Collins AC, et al. Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice. Mol Pharmacol 2004; 65: 1526–35.
Grady SR, Salminen O, Laverty DC, Whiteaker P, McIntosh JM, Collins AC, et al. The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum. Biochem Pharmacol 2007; 74: 1235–46.
Salminen O, Drapeau JA, McIntosh JM, Collins AC, Marks MJ, Grady SR . Pharmacology of alpha-conotoxin MII-sensitive subtypes of nicotinic acetylcholine receptors isolated by breeding of null mutant mice. Mol Pharmacol 2007; 71: 1563–71.
Meyer EL, Yoshikami D, McIntosh JM . The neuronal nicotinic acetylcholine receptors alpha 4* and alpha 6* differentially modulate dopamine release in mouse striatal slices. J Neurochem 2008; 105: 1761–9.
Lai A, Sum J, Fan H, McIntosh JM, Quik M . Selective recovery of striatal 125I-alpha-conotoxin MII nicotinic receptors after nigrostriatal damage in monkeys. Neuroscience 2004; 127: 399–408.
McCallum SE, Parameswaran N, Bordia T, McIntosh JM, Grady SR, Quik M . Decrease in alpha3*/alpha6* nicotinic receptors but not nicotine-evoked dopamine release in monkey brain after nigrostriatal damage. Mol Pharmacol 2005; 68: 737–46.
Cui C, Booker TK, Allen RS, Grady SR, Whiteaker P, Marks MJ, et al. The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors. J Neurosci 2003; 23: 11045–53.
Gotti C, Moretti M, Clementi F, Riganti L, McIntosh JM, Collins AC, et al. Expression of nigrostriatal alpha 6-containing nicotinic acetylcholine receptors is selectively reduced but not eliminated by beta 3 subunit gene deletion. Mol Pharmacol 2005; 67: 2007–15.
Lai A, Parameswaran N, Khwaja M, Whiteaker P, Lindstrom JM, Fan H, et al. Long-term nicotine treatment decreases striatal alpha 6* nicotinic acetylcholine receptor sites and function in mice. Mol Pharmacol 2005; 67: 1639–47.
Perry DC, Mao D, Gold AB, McIntosh JM, Pezzullo JC, Kellar KJ . Chronic nicotine differentially regulates alpha6- and beta3-containing nicotinic cholinergic receptors in rat brain. J Pharmacol Exp Ther 2007; 322: 306–15.
Luo S, Kulak JM, Cartier GE, Jacobsen RB, Yoshikami D, Olivera BM, et al. alpha-conotoxin AuIB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. J Neurosci 1998; 18: 8571–8579.
Azam L, McIntosh JM . Characterization of nicotinic acetylcholine receptors that modulate nicotine-evoked [3H]norepinephrine release from mouse hippocampal synaptosomes. Mol Pharmacol 2006; 70: 967–76.
Harvey SC, McIntosh JM, Cartier GE, Maddox FN, Luetje CW . Determinants of specificity for alpha-conotoxin MII on alpha3beta2 neuronal nicotinic receptors. Mol Pharmacol 1997; 51: 336–42.
Dutertre S, Nicke A, Lewis RJ . Beta2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor. J Biol Chem 2005; 280: 30460–8.
Azam L, Yoshikami D, McIntosh JM . Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A, E11A, L15A]. J Biol Chem 2008; 283: 11625–32.
Whiteaker P, McIntosh JM, Luo S, Collins AC, Marks MJ . 125I-alpha-conotoxin MII identifies a novel nicotinic acetylcholine receptor population in mouse brain. Mol Pharmacol 2000; 57: 913–25.
Vishwanath VA, McIntosh JM . Synthesis of fluorescent analogs of alpha-conotoxin MII. Bioconjug Chem 2006; 17: 1612–7.
Quik M, Polonskaya Y, Kulak JM, McIntosh JM . Vulnerability of 125I-alpha-conotoxin MII binding sites to nigrostriatal damage in monkey. J Neurosci 2001; 21: 5494–500.
Kulak JM, McIntosh JM, Quik M . Loss of nicotinic receptors in monkey striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment is due to a decline in alpha-conotoxin MII sites. Mol Pharmacol 2002; 61: 230–8.
Quik M, Sum JD, Whiteaker P, McCallum SE, Marks MJ, Musachio J, et al. Differential declines in striatal nicotinic receptor subtype function after nigrostriatal damage in mice. Mol Pharmacol 2003; 63: 1169–79.
Quik M, Bordia T, Forno L, McIntosh JM . Loss of alpha-conotoxinMII- and A85380-sensitive nicotinic receptors in Parkinsons disease striatum. J Neurochem 2004; 88: 668–79.
Bordia T, Grady SR, McIntosh JM, Quik M . Nigrostriatal damage preferentially decreases a subpopulation of alpha6beta2* nAChRs in mouse monkey and Parkinson's disease striatum. Mol Pharmacol 2007; 72: 52–61.
Clark RJ, Fischer H, Dempster L, Daly NL, Rosengren KJ, Nevin ST, et al. Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII. Proc Natl Acad Sci USA 2005; 102: 13767–72.
Blanchfield JT, Dutton JL, Hogg RC, Gallagher OP, Craik DJ, Jones A, et al. Synthesis structure elucidation in vitro biological activity toxicity and Caco-2 cell permeability of lipophilic analogues of alpha-conotoxin MII. J Med Chem 2003; 46: 1266–72.
Blanchfield JT, Gallagher OP, Cros C, Lewis RJ, Alewood PF, Toth I . Oral absorption and in vivo biodistribution of alpha-conotoxin MII and a lipidic analogue. Biochem Biophys Res Commun 2007; 361: 97–102.
Dowell C, Olivera BM, Garrett JE, Staheli ST, Watkins M, Kuryatov A, et al. Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. J Neurosci 2003; 23: 8445–52.
Chi SW, Kim DH, Olivera BM, McIntosh JM, Han KH . Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist alpha-conotoxin OmIA that discriminates alpha3 vs, alpha6 nAChR subtypes. Biochem Biophys Res Commun 2006; 345: 248–54.
Talley TT, Olivera BM, Han KH, Christensen SB, Dowell C, Tsigelny I, et al. Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors. J Biol Chem 2006; 281: 24678–86.
McIntosh JM, Dowell C, Watkins M, Garrett JE, Yoshikami D, Olivera BM . Alpha-conotoxin GIC from Conus geographus a novel peptide antagonist of nicotinic acetylcholine receptors. J Biol Chem 2002; 277: 33610–5.
Chi SW, Kim DH, Olivera BM, McIntosh JM, Han KH . Solution conformation of alpha-conotoxin GIC a novel potent antagonist of alpha3beta2 nicotinic acetylcholine receptors. Biochem J 2004; 380: 347–52.
Nicke A, Loughnan ML, Millard EL, Alewood PF, Adams DJ, Daly NL, et al. Isolation structure and activity of GID a novel alpha 4/7-conotoxin with an extended N-terminal sequence. J Biol Chem 2003; 278: 3137–44.
Millard EL, Nevin ST, Loughnan ML, Nicke A, Clark RJ, Alewood PF, et al. Inhibition of neuronal nicotinic acetylcholine receptor subtypes by alpha-conotoxin GID and analogues. J Biol Chem 2009; 284: 4944–51.
Loughnan M, Bond T, Atkins A, Cuevas J, Adams DJ, Broxton NM, et al. alpha-conotoxin EpI a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors. J Biol Chem 1998; 273: 15667–74.
Nicke A, Samochocki M, Loughnan ML, Bansal PS, Maelicke A, Lewis RJ . Alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors. FEBS Lett 2003; 554: 219–23.
Loughnan ML, Nicke A, Jones A, Adams DJ, Alewood PF, Lewis RJ . Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone. J Med Chem 2004; 47: 1234–41.
Grady SR, Meinerz NM, Cao J, Reynolds AM, Picciotto MR, Changeux JP, et al. Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum. J Neurochem 2001; 76: 258–68.
Fainzilber M, Hasson A, Oren R, Burlingame AL, Gordon D, Spira ME, et al. New mollusc-specific alpha-conotoxins block Aplysia neuronal acetylcholine receptors. Biochemistry 1994; 33: 9523–9.
Wolfender JL, Chu F, Ball H, Wolfender F, Fainzilber M, Baldwin MA, et al. Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB: further investigation of labile sulfo- and phosphopeptides by electrospray matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry. J Mass Spectrom 1999; 34: 447–54.
Luo S, Nguyen TA, Cartier GE, Olivera BM, Yoshikami D, McIntosh JM . Single-residue alteration in alpha-conotoxin PnIA switches its nAChR subtype selectivity. Biochemistry 1999; 38: 14542–8.
Hogg RC, Miranda LP, Craik DJ, Lewis RJ, Alewood PF, Adams DJ . Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor. J Biol Chem 1999; 274: 36559–64.
Jin AH, Daly NL, Nevin ST, Wang CI, Dutertre S, Lewis RJ, et al. Molecular engineering of conotoxins: the importance of loop size to alpha-conotoxin structure and function. J Med Chem 2008; 51: 5575–84.
Everhart D, Reiller E, Mirzoian A, McIntosh JM, Malhotra A, Luetje CW . Identification of residues that confer alpha-conotoxin–PnIA sensitivity on the alpha 3 subunit of neuronal nicotinic acetylcholine receptors. J Pharmacol Exp Ther 2003; 306: 664–70.
Celie PH, Kasheverov IE, Mordvintsev DY, Hogg RC, van Nierop P, van Elk R, et al. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat Struct Mol Biol 2005; 12: 582–8.
Quiram PA, McIntosh JM, Sine SM . Pairwise interactions between neuronal alpha(7) acetylcholine receptors and alpha–conotoxin PnIB. J Biol Chem 2000; 275: 4889–96.
Azam L, Dowell C, Watkins M, Stitzel JA, Olivera BM, McIntosh JM . Alpha-conotoxin BuIA, a novel peptide from Conus bullatus distinguishes among neuronal nicotinic acetylcholine receptors. J Biol Chem 2005; 280: 80–7.
Shiembob DL, Roberts RL, Luetje CW, McIntosh JM . Determinants of alpha-conotoxin BuIA selectivity on the nicotinic acetylcholine receptor beta subunit. Biochemistry 2006; 45: 11200–7.
Chi SW, Kim DH, Olivera BM, McIntosh JM, Han KH . NMR structure determination of alpha-conotoxin BuIA a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold. Biochem Biophys Res Commun 2006; 349: 1228–34.
Jin AH, Brandstaetter H, Nevin ST, Tan CC, Clark RJ, Adams DJ, et al. Structure of alpha-conotoxin BuIA: influences of disulfide connectivity on structural dynamics. BMC Struct Biol 2007; 7: 28.
Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J . alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci USA 2001; 98: 3501–6.
Baker ER, Zwart R, Sher E, Millar NS . Pharmacological properties of alpha 9 alpha 10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras. Mol Pharmacol 2004; 65: 453–60.
McIntosh JM, Plazas PV, Watkins M, Gomez-Casati ME, Olivera BM, Elgoyhen AB . A novel alpha-conotoxin PeIA cloned from Conus pergrandis discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors. J Biol Chem 2005; 280: 30107–12.
Ellison M, Haberlandt C, Gomez-Casati ME, Watkins M, Elgoyhen AB, McIntosh JM, et al. Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR. Biochemistry 2006; 45: 1511–7.
Sandall DW, Satkunanathan N, Keays DA, Polidano MA, Liping X, Pham V, et al. A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo. Biochemistry 2003; 42: 6904–11.
Clark RJ, Fischer H, Nevin ST, Adams DJ, Craik DJ . The synthesis structural characterization and receptor specificity of the alpha–conotoxin Vc1.1. J Biol Chem 2006; 281: 23254–63.
Vincler M, Wittenauer S, Parker R, Ellison M, Olivera BM, McIntosh JM . Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. Proc Natl Acad Sci USA 2006; 103: 17880–4.
Nevin ST, Clark RJ, Klimis H, Christie MJ, Craik DJ, Adams DJ . Are alpha9alpha10 nicotinic acetylcholine receptors a pain target for alpha-conotoxins? Mol Pharmacol 2007; 72: 1406–10.
Satkunanathan N, Livett B, Gayler K, Sandall D, Down J, Khalil Z . Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain Res 2005; 1059(2): 149–58.
Livett BG, Sandall DW, Keays D, Down J, Gayler KR, Satkunanathan N, et al. Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor. Toxicon 2006; 48: 810–29.
Callaghan B, Haythornthwaite A, Berecki G, Clark RJ, Craik DJ, Adams DJ . Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N–type calcium channels in rat sensory neurons via GABAB receptor activation. J Neurosci 2008; 28: 10943–51.
Ellison M, Feng ZP, Park AJ, Zhang X, Olivera BM, McIntosh JM, et al. Alpha-RgIA a novel conotoxin that blocks the alpha9alpha10 nAChR: structure and identification of key receptor-binding residues. J Mol Biol 2008; 377: 1216–27.
Clark RJ, Daly NL, Halai R, Nevin ST, Adams DJ, Craik DJ . The three-dimensional structure of the analgesic alpha-conotoxin RgIA. FEBS Lett 2008; 582: 597–602.
Westermann JC, Clark RJ, Craik DJ . Binding mode of alpha–conotoxins to an acetylcholine binding protein determined by saturation transfer difference NMR. Protein Pept Lett 2008; 15: 910–4.
Dutertre S, Ulens C, Buttner R, Fish A, van Elk R, Kendel Y, et al. AChBP-targeted alpha-conotoxin correlates distinct binding orientations with nAChR subtype selectivity. Embo J 2007; 26: 3858–67.
Yuan DD, Han YH, Wang CG, Chi CW . From the identification of gene organization of alpha conotoxins to the cloning of novel toxins. Toxicon 2007; 49: 1135–49.
Peng C, Han Y, Sanders T, Chew G, Liu J, Hawrot E, et al. alpha4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors. Peptides 2008; 29: 1700–7.
Luo S, Zhangsun D, Zhang B, Quan Y, Wu Y . Novel alpha–conotoxins identified by gene sequencing from cone snails native to Hainan and their sequence diversity. J Pept Sci 2006; 12: 693–704.
Whiteaker P, Christensen S, Yoshikami D, Dowell C, Watkins M, Gulyas J, et al. Discovery synthesis and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist. Biochemistry 2007; 46: 6628–38.
Innocent N, Livingstone PD, Hone A, Kimura A, Young T, Whiteaker P, et al. Alpha-conotoxin Arenatus IB[V11L, V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors. J Pharmacol Exp Ther 2008; 327: 529–37.
Whiteaker P, Marks MJ, Christensen S, Dowell C, Collins AC, McIntosh JM . Synthesis and characterization of 125I-alpha-conotoxin ArIB[V11L; V16A] a selective alpha7 nicotinic acetylcholine receptor antagonist. J Pharmacol Exp Ther 2008; 325: 910–9.
Olivera BM . Conus peptides: biodiversity-based discovery and exogenomics. J Biol Chem 2006; 281: 31173–7.
Kuzmin A, Jerlhag E, Liljequist S, Engel J . Effects of subunit selective nACh receptors on operant ethanol self-administration and relapse-like ethanol-drinking behavior. Psychopharmacology (Berl) 2009; 203: 99–108.
Jerlhag E, Egecioglu E, Dickson SL, Svensson L, Engel JA . Alpha–conotoxin MII-sensitive nicotinic acetylcholine receptors are involved in mediating the ghrelin-induced locomotor stimulation and dopamine overflow in nucleus accumbens. Eur Neuropsychopharmacol 2008; 18: 508–18.
Jerlhag E, Grotli M, Luthman K, Svensson L, Engel JA . Role of the subunit composition of central nicotinic acetylcholine receptors for the stimulatory and dopamine-enhancing effects of ethanol. Alcohol Alcohol 2006; 41: 486–93.
Larsson A, Jerlhag E, Svensson L, Soderpalm B, Engel JA . Is an alpha–conotoxin MII-sensitive mechanism involved in the neurochemical stimulatory and rewarding effects of ethanol? Alcohol 2004; 34: 239–50.
Maslennikov IV, Shenkarev ZO, Zhmak MN, Ivanov VT, Methfessel C, Tsetlin VI, et al. NMR spatial structure of alpha-conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors. FEBS Lett 1999; 444: 275–80.
Chi SW, Lee SH, Kim DH, Kim JS, Olivera BM, McIntosh JM, et al. Solution structure of alpha-conotoxin PIA a novel antagonist of alpha6 subunit containing nicotinic acetylcholine receptors. Biochem Biophys Res Commun 2005; 338: 1990–7.
Acknowledgements
We thank Dr Baldomero M OLIVERA for providing comments on the manuscript. Support provided by Kirschstein-National Research Service Award Postdoctoral Fellowship DA 016835 (to Layla AZAM) and by National Institutes of Health Grant MH53631 (to J Michael MCINTOSH).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Azam, L., McIntosh, J. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors. Acta Pharmacol Sin 30, 771–783 (2009). https://doi.org/10.1038/aps.2009.47
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/aps.2009.47
Keywords
This article is cited by
-
Mechanism of interactions between α-conotoxin RegIIA and carbohydrates at the human α3β4 nicotinic acetylcholine receptor
Marine Life Science & Technology (2022)
-
Conformational dynamics of \(\alpha \)-conotoxin PnIB in complex solvent systems
Molecular Diversity (2020)
-
Synthesis and Functional Identification of Oligopeptides Derived from the α3/5-Conotoxins
International Journal of Peptide Research and Therapeutics (2018)
-
Effects of α-conotoxin ImI on TNF-α, IL-8 and TGF-β expression by human macrophage-like cells derived from THP-1 pre-monocytic leukemic cells
Scientific Reports (2017)
-
The Nicotinic α6-Subunit Selective Antagonist bPiDI Reduces Alcohol Self-Administration in Alcohol-Preferring Rats
Neurochemical Research (2016)