AVOIDING A DOSE OF YOUR OWN MEDICINE

Abstract

How do venomous animals or plants protect themselves from their own poisons? This question must have occurred to everyone at some time or another. Of course, there are lots of ways; many plant and fungal poisons target animal-specific proteins; others are stored in inactive forms until release. But a whole host of exquisitely deadly animal toxins have the potential to act against their hosts, and here it may be necessary to invoke co-evolution of resistance. A perfect example of this is found in the pufferfish genera Takifugu and Tetraodon. Their poison, tetrodotoxin (TTX),binds very specifically to the mouth of the voltage-gated sodium channel, the key protein of nerve and muscle that is essential for action potentials and thus the functioning of the nervous system. We know that TTX is fantastically deadly: this is because pufferfish is a prized sushi delicacy in Japan. Chefs licensed to serve fugu need detailed knowledge of fish anatomy, as some parts(particularly the liver and ovaries) are particularly poisonous. This in turn makes `official' fugu sushi so expensive that many Japanese try the do-it-yourself option, resulting in several fatalities per year.Given that the toxicity of TTX is established by human misadventure, and that its target is a very highly conserved protein family, how do pufferfish escape their own poison?Previous work in one species had identified an unusual aromatic amino acid substitution in the first of the four transmembrane domains of a particular sodium channel, Nav1.4, but had not demonstrated that this actually made the protein resistant to the toxin. Byrappa Venkatesh and co-workers from the newly opened Singapore biotech company `Biopolis' took these findings further, with parallel physiological and comparative genetic approaches.The results were simple, but clear; they data-mined the homologous channel genes from the genome projects of related pufferfish representatives of both genera, Takifugu rubiripes and Tetraodon nigroviridis, and compared them with zebrafish and human. Compared with humans, there were two orthologous Nav1.4 channels, consistent with a whole-genome duplication event early in the evolution of ray-finned fishes. If the pufferfish were protected by sodium channel mutations, they were likely to be in one or both of these channels. Again, in both pufferfish species, mutations were found in the highly conserved pore loop of domain I (which forms the entrance to the sodium channel) in both copies of Nav1.4.However, not all changes are functionally significant, so it was necessary to prove that these alterations really did confer resistance to TTX. Accordingly, the corresponding amino acids were altered in a mammalian (rat)channel gene, expressed in cultured cells and their electrophysiological properties compared with the normal channel by whole-cell patch clamp analysis. Whereas sodium current through the rat channel was completely blocked by 1 μmol l–1 TTX, even 100 μmol l–1 TTX achieved only a 55% block of the mutant channels. In fact, altering tyrosine 401 to either cysteine or asparagine (as found in pufferfish) increased the Kd for TTX binding by 2000–2500-fold.So pufferfish sodium channels are resistant to TTX – but why? This is a necessary adaptation, because pufferfish do not make their own TTX but obtain it from microorganisms in their diet, and so must resist ingestion better than humans. The authors outline three intriguing possibilities. Firstly, resistant channels allow pufferfish to eat diets that are toxic to other animals, thus giving them a unique ecological niche. Secondly,accumulating this TTX protects pufferfish from predation. Thirdly, consistent with very high levels of TTX in the ovary, males are actually attracted by very low concentrations of TTX in seawater. So TTX may actually act as a mating pheromone for pufferfish!