Native forms with disulfides and C-terminal amide, calculated with the residue masses and pKa set used on this site.
Hunting with peptides
Cone snails (genus Conus and related genera) are marine gastropods found mostly in tropical reefs. Several hundred species are known. They are slow-moving predators of worms, other molluscs and, in some species, fish, and they catch their prey by injecting venom through a hollow, harpoon-like modified tooth.
Each species produces a venom containing from around a hundred to over a thousand different peptides, and the venoms of different species overlap very little. Across the genus this adds up to a chemical library estimated at hundreds of thousands of distinct peptides. The field was established largely by Baldomero Olivera and colleagues at the University of Utah from the 1970s onwards. A few fish-hunting species, notably Conus geographus, have caused fatal human envenomations.
What a conotoxin looks like
Most conotoxins share a recognisable design:
- Small size: typically 10–40 residues.
- Several disulfide bonds: usually two or three, sometimes more, packed into a very short chain. The arrangement of cysteines in the sequence is called the cysteine framework, and it largely determines the fold.
- Extensive modification: C-terminal amidation, hydroxyproline, γ-carboxyglutamate, bromotryptophan, sulfotyrosine, glycosylation and D-amino acids all occur.
- A hypervariable active region between the conserved cysteines, which differs from peptide to peptide and determines the target.
This combination produces molecules that are rigid, protease-resistant and extremely selective. The disulfides hold the hypervariable loops in a precise shape, and the loops then fit their targets with high affinity. The chemistry of this arrangement, including the problem of forming the right disulfide pairs out of many possibilities, is described in the guide to disulfide bonds.
The precursors are made on the ribosome with a signal sequence, a pro-region and the mature toxin at the C-terminus. Conotoxins are grouped into gene superfamilies (A, M, O, T and many others) by the similarity of their signal sequences, and into pharmacological families by their target, labelled with Greek letters. A single superfamily can contain several pharmacological families, and vice versa.
Targets
Conotoxins act mainly on ion channels and receptors of the nervous system. The major pharmacological families are:
| Family | Target | Effect |
|---|---|---|
| α-Conotoxins | Nicotinic acetylcholine receptors | Block neuromuscular or neuronal nicotinic receptors |
| μ-Conotoxins | Voltage-gated sodium channels | Block sodium current; muscle paralysis |
| δ-Conotoxins | Voltage-gated sodium channels | Delay channel inactivation; excitation |
| κ-Conotoxins | Voltage-gated potassium channels | Block potassium current |
| ω-Conotoxins | Voltage-gated calcium channels | Block neurotransmitter release |
| Conantokins | NMDA receptors | Antagonism; no disulfides, rich in γ-carboxyglutamate |
Fish-hunting species combine several of these into what Olivera called toxin cabals. One group of peptides acts within seconds to cause rigid paralysis by over-exciting nerves, a "lightning-strike" effect; another blocks neuromuscular transmission to cause flaccid paralysis. The combination immobilises a fish far faster than any single toxin could.
Some species also use a strategy that is not neurotoxic at all. Conus geographus releases a fast-acting insulin into the water around a school of fish, inducing hypoglycaemic shock and making them easy to engulf. Its insulin lacks the regions that allow human insulin to form dimers and hexamers, which is precisely what makes it act fast. The insulin profile explains why self-association slows the action of insulin.
Ziconotide: a conotoxin as a medicine
The best-known conotoxin is ω-conotoxin MVIIA from the fish-hunting Conus magus. It is a 25-residue peptide with three disulfide bonds and a C-terminal amide, and it blocks N-type (Cav2.2) voltage-gated calcium channels, which are involved in transmitting pain signals in the spinal cord.
Its synthetic form, ziconotide, was approved in the United States in 2004 and in the European Union in 2005 for severe chronic pain. Because it does not cross the blood–brain barrier and would act on channels throughout the body if given systemically, it is delivered directly into the fluid around the spinal cord through an implanted pump. Its use is limited by this route of administration and by neurological side effects, but it established the principle that a venom peptide can be developed into an analgesic acting by a mechanism different from opioids.
The disulfide arrangement of ω-MVIIA, Cys1–Cys16, Cys8–Cys20 and Cys15–Cys25, forms an inhibitor cystine knot, the motif described in the guide to disulfide bonds, in which one disulfide threads through a ring formed by the other two and the backbone. This is a large part of its stability.
α-Conotoxins and disulfide isomers
α-Conotoxins are among the smallest conotoxins, typically 12–20 residues with four cysteines and two disulfides. α-Conotoxin GI from Conus geographus, ECCNPACGRHYSC-NH₂, has 13 residues.
With four cysteines there are three possible disulfide connectivities, called globular, ribbon and beads. Native α-conotoxins use the globular arrangement, Cys1–Cys3 and Cys2–Cys4. Synthetic material can fold into any of the three, and the non-native isomers usually have much lower activity. Separating and identifying them by HPLC after oxidative folding is a standard problem in conotoxin chemistry, discussed in the guides to disulfide bonds and peptide HPLC.
Some α-conotoxins block nicotinic receptor subtypes involved in pain and have been studied as potential analgesics. One of them, Vc1.1, was the subject of an influential study in which its termini were joined through a short linker to form a head-to-tail cyclic peptide. The cyclic version was more stable and showed analgesic activity when given orally in animal models, a notable result for a peptide.
Conotoxins as research tools
Their exquisite selectivity makes conotoxins valuable tools, independent of any therapeutic use. Many ion channel and receptor subtypes can be distinguished pharmacologically only because a conotoxin binds one and not the others. Radiolabelled or fluorescent conotoxins map where specific channels are located in tissues, and ω-conotoxin GVIA was instrumental in defining the N-type calcium channel. The same principle underlies the use of other venom peptides, from spiders, scorpions and snakes, throughout neuroscience.
Conotoxins with the tools on this site
Entering the ziconotide sequence CKGKGAKCSRLMYDCCTGSCRSGKC into the MW calculator gives the mass of the reduced free acid. For the native peptide, subtract 3 × 2.016 Da for the three disulfides and 0.984 Da for the amide, giving the values in the fact sheet. With four lysines, two arginines and only a single aspartate, ziconotide is strongly basic: it carries a net charge of about +6 at pH 7.4 and has a calculated pI of about 11.7.
Because conotoxins often carry unusual modifications, calculated values from the sequence alone can be misleading. Hydroxyproline adds 15.995 Da per residue, γ-carboxyglutamate adds 43.990 Da and an extra negative charge, and D-residues change nothing in mass while changing the structure. The mass shifts are listed in the guide to post-translational modifications.
Frequently asked questions
Why do cone snails need so many different toxins?
Rapid evolution of venom genes allows each species to specialise on its prey, and combinations of toxins acting on different targets immobilise prey faster than any single toxin. The diversity also appears to arise from high rates of gene duplication and mutation in the regions between the conserved cysteines.
Are conotoxins dangerous to handle in the lab?
Some are highly toxic if they enter the body, and they are handled under institutional safety procedures. Several are subject to export controls in some countries because of their potency.
Why is ziconotide not given by mouth or injection into a vein?
As a peptide it would be digested in the gut, and it does not cross the blood–brain barrier. Given into the blood it would also act on calcium channels throughout the body. Delivering it into the spinal fluid places it close to its target.
How are conotoxins made for research?
Almost always by chemical synthesis, followed by oxidative folding or directed disulfide formation. Isolation from venom yields only tiny amounts, and synthesis allows analogues to be made.
References
- Olivera BM, Rivier J, Clark C, et al. (1990) Diversity of Conus neuropeptides. Science 249:257–263.
- Terlau H, Olivera BM (2004) Conus venoms: a rich source of novel ion channel-targeted peptides. Physiological Reviews 84:41–68.
- Miljanich GP (2004) Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Current Medicinal Chemistry 11:3029–3040.
- Clark RJ, Jensen J, Nevin ST, et al. (2010) The engineering of an orally active conotoxin for the treatment of neuropathic pain. Angewandte Chemie International Edition 49:6545–6548.
- Safavi-Hemami H, Gajewiak J, Karanth S, et al. (2015) Specialized insulin is used for chemical warfare by fish-hunting cone snails. PNAS 112:1743–1748.