Final Report Summary - ß-TOXIN INSECTICIDE (The ß-scorpion toxin binding site as a potential target for new insecticides) The IEF training project β-TOXIN INSECTICIDE investigated the interaction of compounds such as toxins, local anesthetics and small-molecule channel blockers with the voltage-gated sodium channel of mammals and insects. A multidisciplinary approach was applied including patch-clamp electrophysiology of heterologously expressed channels, which allowed the training fellow to master this technique. We first studied the interaction of the environmental pollutant bisphenol A (BPA) with the heart sodium channel. BPA is an additive of plastics and is well characterized as an endocrine disruptor. The many reports on its adverse health effects have led to its nation-wide ban in Denmark, while other states are discussing this issue. As BPA exposure is linked with cardiovascular disorders, we hypothesized that it may be interacting with the cardiac sodium channel to interfere with cardiac electrophysiology. We discovered that BPA blocks the current flowing through the channel, although the IC50 needed for block is well above the highest blood concentration measured so far (O’Reilly 2012, PlosOne). We used mutagenesis studies to locate the BPA binding site in the channel and determined that it overlaps with the local anesthetic receptor site. Using 3D molecular modeling studies with steered molecular dynamics simulations, we showed that BPA may enter the sodium channel via side fenestrations in the pore, which is a new access pathway that may also be relevant for the entry of local anesthetics. This may help the design of new drugs targeting sodium channel subtypes relevant for pain, heart diseases such as arrhythimas or muscle diseases (e.g. myotonia). Our study providing the first detailed report on the effects of BPA on human heart sodium channels may aid policy makers in their decisions on safe exposure limits to this ubiquitous environmental compound. Our initial difficulty expressing a scorpion toxin for our studies was resolved very successfully through the development of a novel expression system. Scorpion toxins have multiple disulphide bridges and misfolding and aggregation are the typical results when these proteins are produced using the E.coli bacterial expression system. We co-expressed chaperones called protein disulphide isomerases to act on misfolded toxin, thereby producing disulphide shuffling to obtain the correct optimally-stable native fold. Bj-xtrIT from Hottentotta judaica was successfully purified and crystallized using this method (O’Reilly et al. 2013, BBA), which is the first reported instance of a scorpion toxin correctly folding inside a bacterium. This expression strategy may also work with other difficult-to-produce toxins and may therefore become a valuable method to overcome an intractable problem that has so far hindered toxin research.We used in silico methods of molecular modeling and ligand binding studies to investigate why some members of the pyrethroid family of insecticides are poor insecticides yet are very effective at targeting other arthropods including ticks and mites. Our studies suggest that a single amino acid difference in the pyrethroid binding site of sodium channels is important; large pyrethroids can fit into the spacious binding site of tick/mite channels whereas the more physically-constricted binding site of insects prevents through steric hindrance the accommodation of these same pyrethroids (O’Reilly et al. 2013, Pest Man Sci). These results and a related study on the neonicotinoid class of insecticides interacting with tick acetylcholine receptors (Erdmanis et al., 2012) may provide useful structural insight to aid in the design of new compounds that specifically target ticks and mites in the presence of agriculturally important insect species. A important and topical example of this scenario is the targeting of Varroa destructor mites that parasitize honeybees and which are an important contributing factor to the phenomenon of colony collapse disorder.In conclusion, our development of a novel expression method enables the routine production and mutagenesis of animal toxins for application as probes for structure-function studies of sodium channels. Many scorpion and spider toxins are insect-specific and therefore represent valuable bioactive compounds that may be exploited directly or as pharmacophore templates for rational insecticide design. The results of our studies may therefore aid in the development of novel insecticides that could supercede in terms of specificity and environmental friendliness the widely-used pyrethroid and neonicotinoid classes of insecticides, thereby safe-guarding global food security.
