Venomous Snakes, Spiders and Snails

The next time you cringe at the sight of a snake or an eight-legged creepy crawlie or smirk at a sluggish snail, think again! In fact we owe some gratitude to these creatures as well as to the biologists who have dedicated their lives working with them and learning more about them. In fact, research into venoms and how they target the nervous system so precisely has proved vital to us. Not only has it helped us better understand Myasthenia Gravis (MG), Lambert-Eaton syndrome (LEMS) and other related conditions, for example Neuromyotonia: we can also apply the science of venoms to help us in diagnosing and treating these conditions better. Here we give just a few examples relevant to you but many others are in use in other specialities.

Figure 1: The neuromuscular junction. The nerve ending (of the ‘motor neuron’) contacts the muscle fibre closely; they are separated only by a thin cleft. The green “synaptic vesicles” within the nerve ending are filled with ACh. When released, it darts across from the active zones to latch into the AChR–rich surface of the muscle fibre (thicker black lines).

First of all, a quick reminder about normal nerve to muscle transmission – the 'ignition system' – and how it can go wrong. The neuromuscular junction is elegantly constructed (see Figure 1). When an electrical impulse arrives from the brain, the nerve endings release a shower of a chemical transmitter – acetylcholine (ACh) – the 'ignition keys'. These travel across a narrow gap and latch into the tailor-made 'locks' on the muscle surface – the acetylcholine receptors (AChRs). They open a central channel in the AChR, which allows electrical current to flow through and this in turn triggers the muscle to contract (via complicated mechanisms). The surplus ACh is destroyed by a special protein – AChE (acetylcholinesterase) – to allow the muscle to relax again. In myasthenia gravis (MG), an immune attack by antibodies destroys many AChRs on the muscle, reducing the chances of a successful contraction. In Lambert-Eaton myasthenic sydrome (LEMS), different antibodies damage the nerve endings, and less ACh is released than normal, so that triggering is inefficient. Giving Mestinon to myasthenic and LEMS patients prevents the breakdown of the ACh, so that it lasts longer and has a better chance of triggering.

Cobras and other snakes have a complicated mix of toxins in their venoms. Among the most dangerous are the 'alpha-neurotoxins' which block the AChR almost irreversibly, thus causing paralysis. In 1963, two Taiwanese scientists – Drs Chang and Lee – found that, in their local Banded Krait called Bungarus multicinctus (see Figure 2), the ‘alpha-bungarotoxin’ was so potent that it could be tagged with colours (fluorescent dyes) or radioactivity, and used to 'label' the AChRs in microscope sections and to measure their numbers. In fact, it’s other toxins include beta- and gamma- bungarotoxins, which both reduce the release of ACh from the nerve endings as well as blocking other nearby potassium channels.

Fig 2 Bungarus multicinctus

Other biochemists were trying to understand how these receptors work and realised that the electric organs of electric fish (eels, skates and rays) are like muscles working backwards, converting chemical into electrical energy. These organs are packed with AChRs (and are a much richer source than muscle). Their AChRs can be dissolved with detergents, and then purified using alpha-bungarotoxin. In 1973, Drs Jon Lindstrom and Jim Patrick (in California) wanted to make antibodies (to help in their research), and immunised some rabbits with this pure AChR. To their surprise, the bunnies soon became flopsy (myasthenic), and, when treated with Mestinon, they recovered dramatically.

That was a crucial breakthrough, partly because it proved that myasthenia could indeed be caused by 'autoantibodies', just as our distinguished Vice-President, Prof Ian Simpson, had predicted twelve years previously. It also led to a very useful blood test for these antibodies, which is now a vital standard diagnostic tool. It uses radioactive alpha-bungarotoxin to tag AChR (from human muscle tumour cells grown in the lab), and quickly detects these antibodies in at least 85% of typical MG patients (though not in the 10% of patients who have antibodies to MuSK instead). Unlike many such tests, it almost never gives 'false positive' results. The alpha-bungarotoxin is also very useful in testing patients with inherited faults (the congenital myasthenic syndromes), eg to show directly whether they have too few AChRs on the muscle surface. Alpha-bungarotoxin is mass-produced by the Miami Serpentarium (in Florida), so many thanks to the Banded Krait.

Fig3. Conus magus

In 1981, John Newsom-Davis, Angela Vincent and Bethan Lang in our team showed that the LEMS is also caused by autoantibodies. They worked out by electrical studies that the likeliest targets on the nerve endings were their calcium channels. The first available toxins that label these came from American Funnel-web spiders (~1987). Using them to label calcium channels enabled Bethan to detect antibodies in some of our LEMS patients (though less than half). But then, in 1992, she found a new toxin from a fish-eating Cone snail (Conus magus) (see Figure 3) that gave much better results. Using this ‘conotoxin’, she now finds these antibodies to calcium channels in nearly every LEMS patient; again, it is a highly specific and efficient test. You guessed – this vicious snail also produces several other toxins, some of which act on the AChR and others on the sodium channels.

Some patients have uncontrollable muscle spasms (dystonia) or 'tics' that distort their faces, necks or limbs, for example blepharospasm (eyelids go into spasm) and torticollis (neck dystonia). They can be greatly helped by another toxin, this time from bacteria rather like those that cause tetanus (instead called Clostridium botulinum). At high doses, Botulinum toxin causes another dangerous paralysis, botulism. But, if injected in minute amounts into the right muscle every two to three months, it can bring great relief and help the patients back to a normal life. It interferes with the protein required for the release of ACh, and so causes a reversible paralysis that helps to reduce the dystonia or spasms. In fact, these bacteria produce several types of toxin (types A to G). Botulinum is now also being used for cosmetic purposes to get rid of facial wrinkling……so there’s an after- thought! Other toxins from kraits and rattlesnakes behave similarly, but they are less well localised and don’t last so long, for example the crotoxin (from the South American rattlesnake).

Fig 4. African green mamba (Deudroaspis angusticeps)

Finally, in neuromyotonia (NMT) there are involuntary twitches and ripples – sometimes with such muscle overgrowth that the patients are accused of taking anabolic steroids. Again, autoantibodies are often responsible, recognizing yet another target, this time the potassium channels. Normally, nerve impulses are stopped, and excessive ones controlled, by these potassium channels. If they are lost or destroyed (eg by antibodies), the muscles fail to relax properly and go on twitching. These channels can be labelled by yet another toxin – this time from the African Green Mamba (see Figure 4). Using this labelled ‘dendrotoxin’, Angela Vincent has so far detected antibodies in only some of these patients, so she is still looking for the ideal toxin. No doubt, research will continue to unravel more about the science of these toxins that are such a favourite among those of us who work in Neurosciences and on Myasthenia!

MGA NEWS Autumn 2002