Tetrodotoxin (TTX) is a potent neurotoxin. Its name derives from Tetraodontiformes, an order that includes pufferfish, porcupinefish, ocean sunfish, and triggerfish; several of these species carry the toxin. Although tetrodotoxin was discovered in these fish, it is found in several other animals (e.g., in blue-ringed octopuses, rough-skinned newts, and moon snails). It is also produced by certain infectious or symbiotic bacteria like Pseudoalteromonas, Pseudomonas, and Vibrio as well as other species found in symbiotic relationships with animals and plants. Although it produces thousands of intoxications annually and several deaths, it has shown efficacy for the treatment of cancer-related pain in phase II and III clinical trials. Tetrodotoxin is a sodium channel blocker. It inhibits the firing of action potentials in neurons by binding to the voltage-gated sodium channels in nerve cell membranes and blocking the passage of sodium ions (responsible for the rising phase of an action potential) into the neuron. This prevents the nervous system from carrying messages and thus muscles from contracting in response to nervous stimulation. Its mechanism of action – selective blocking of the sodium channel – was shown definitively in 1964 by Toshio Narahashi and John W. Moore at Duke University, using the sucrose gap voltage clamp technique.
Sources in nature Apart from their bacterial species of most likely ultimate biosynthetic origin (see below), tetrodotoxin has been isolated from widely differing animal species, including:
Tarichatoxin was shown to be identical to TTX in 1964 by Mosher et al., and the identity of maculotoxin and TTX was reported in Science in 1978, and the synonymity of these two toxins is supported in modern reports (e.g., at Pubchem and in modern toxicology textbooks) though historic monographs questioning this continue in reprint. The toxin is variously used by animals as a defensive biotoxin to ward off predation, or as both a defensive and predatory venom (e.g., in octopuses, chaetognaths, and ribbon worms). Even though the toxin acts as a defense mechanism, some predators such as the common garter snake have developed insensitivity to TTX, which allows them to prey upon toxic newts. The association of TTX with consumed, infecting, or symbiotic bacterial populations within the animal species from which it is isolated is relatively clear; presence of TTX-producing bacteria within an animal's microbiome is determined by culture methods, the presence of the toxin by chemical analysis, and the association of the bacteria with TTX production by toxicity assay of media in which suspected bacteria are grown. As Lago et al. note, "there is good evidence that uptake of bacteria producing TTX is an important element of TTX toxicity in marine animals that present this toxin." TTX-producing bacteria include Actinomyces, Aeromonas, Alteromonas, Bacillus, Pseudomonas, and Vibrio species; in the following animals, specific bacterial species have been implicated:
The association of bacterial species with the production of the toxin is unequivocal – Lago and coworkers state, "[e]ndocellular symbiotic bacteria have been proposed as a possible source of eukaryotic TTX by means of an exogenous pathway", and Chau and coworkers note that the "widespread occurrence of TTX in phylogenetically distinct organisms... strongly suggests that symbiotic bacteria play a role in TTX biosynthesis" – although the correlation has been extended to most but not all animals in which the toxin has been identified. To the contrary, there has been a failure in a single case, that of newts (Taricha granulosa), to detect TTX-producing bacteria in the tissues with highest toxin levels (skin, ovaries, muscle), using PCR methods, although technical concerns about the approach have been raised. Critically for the general argument, Takifugu rubripes puffers captured and raised in laboratory on controlled, TTX-free diets "lose toxicity over time", while cultured, TTX-free Takifugu niphobles puffers fed on TTX-containing diets saw TTX in the livers of the fishes increase to toxic levels. Hence, as bacterial species that produce TTX are broadly present in aquatic sediments, a strong case is made for ingestion of TTX and/or TTX-producing bacteria, with accumulation and possible subsequent colonization and production. Nevertheless, without clear biosynthetic pathways (not yet found in animals, but shown for bacteria), it remains uncertain whether it is simply via bacteria that each animal accumulates TTX; the question remains as to whether the quantities can be sufficiently explained by ingestion, ingestion plus colonization, or some other mechanism.
Biochemistry Tetrodotoxin binds to what is known as site 1 of the fast voltage-gated sodium channel. Site 1 is located at the extracellular pore opening of the ion channel. Any molecule bound to this site will block sodium ions from going into the nerve cell through this channel (which is ultimately necessary for nerve conduction). Saxitoxin, neosaxitoxin, and several of the conotoxins also bind the same site. The use of this toxin as a biochemical probe has elucidated two distinct types of voltage-gated sodium channels (VGSCs) present in mammals: tetrodotoxin-sensitive voltage-gated sodium channels (TTX-s Na+ channels) and tetrodotoxin-resistant voltage-gated sodium channels (TTX-r Na+ channels). Tetrodotoxin inhibits TTX-s Na+ channels at concentrations of around 1–10 nM, whereas micromolar concentrations of tetrodotoxin are required to inhibit TTX-r Na+ channels. Nerve cells containing TTX-r Na+ channels are located primarily in cardiac tissue, while nerve cells containing TTX-s Na+ channels dominate the rest of the body. TTX and its analogs have historically been important agents for use as chemical tool compounds, for use in channel characterization and in fundamental studies of channel function. The prevalence of TTX-s Na+ channels in the central nervous system makes tetrodotoxin a valuable agent for the silencing of neural activity within a cell culture.
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