The venom from predators such as snakes, toads and spiders have been an excellent source of molecules with diverse properties that scientists have put to use for studying properties of neurons. For years, neurobiologists have used alpha-bungarotoxin from the venom of cobras to inactivate an acetylcholine receptor in neurons, with numerous applications for experimental research. Dr. Michael Nitaback of the Yale School of Medicine and colleagues have developed an innovative method, dubbed “toxineering,” to screen approximately 100 different peptides (i.e., small proteins) found in the venom of a Peruvian green-velvet tarantula (Thrixopelma pruriens) to see if any of these peptides might be used as a painkiller.
Toxineering uses techniques of molecular biology to generate a small library of different toxins found in the spider venom. The library is then screened to identify a molecule which binds and blocks only a specific ion channel of interest. The research team was focused in particular on an ion channel called TRPA1, found in the membranes of neurons that transduce pain signals caused by neuropathy or inflammation. Their goal was to isolate a molecule from tarantula venom that blocks this ion channel alone for use as a painkiller.
Their initial screen identified a peptide 35 amino acids in length called ProTx-I that binds TRPA1 with high affinity. The protein has been found capable of blunting activity in the neurons that transmit pain. The trouble with ProTx-I, however, is that it had already been found to bind a voltage-gated sodium (NaV) channel – a completely different ion channel than the one they were specifically interested in. Naturally occurring toxins frequently suffer from this non-specificity, potentially reacting with several different ion channels, and muddying an understanding of their activity. The researchers wanted a molecule that specifically bound their TRPA1 channel, leaving all other channels unaffected.
The toxineering technique developed by these researchers is conducive to this. The method allows the researchers to look not just at naturally occurring toxins from spider (especially tarantula) venom, but also at subtle molecular variants. After they pulled ProTx-I out of the initial screen, they created another library, but this time made up entirely of ProTx-I molecules that had been mutated so that each molecule was slightly different from the others in the library. They then screened this library against both NaV1.2 and TRPA1, the ion channels that the original ProTx-I could not distinguish.
Notably, this process (generally known as mutagenesis) eventually yielded two novel ProTx-I variants that are active against either TRPA1 alone or NaV1.2 alone. By means of further experimentation, the investigators were even able to identify the sub-domains of the TRPA1 ion channel that were bound by the modified ProTx-I.
The current study on the potential use of elements of tarantula venom as a painkiller, is published in Current Biology with Dr. Nitabach as lead author. These experiments successfully established the technique of “toxineering,” for quickly screening libraries of peptides for molecules with desired properties. More work for the team is ahead, as they plan to use toxineering to screen tens of thousands of new toxins for specific peptides that bind specific ion channels to target specific neurons in specific pathways.
By Laura Prendergast