Biophysical analysis of interactions between peptide toxins and human sodium channel voltage-sensor domains

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Voltage-gated sodium channels regulate the rapid and specific flow of sodium ions through the cell membrane. They are of great importance for functions in the human body such as the regulation of the heartbeat and electrical signaling in nerve cells. Examples of diseases caused by mutations in sodium channels include fatal cardiac arrhythmias, epilepsy, neuromuscular disorders and severe migraines. Furthermore, sodium channels are also promising targets in the treatment of pain and potentially in the prevention of cancer metastasis. Sodium channels are targeted by a vast array of natural toxins, many of them highly selective peptide toxins that animals use for defense or to subdue their prey. These toxins represent a treasure trove of bioactive compounds with potential applications as tools for basic research as well as in the development of drugs for the treatment of sodium channel-related diseases. Some spider, scorpion and sea anemone toxins that target sodium channels change the voltage of activation of the channels by binding to the voltage sensor domains (VSDs) of the channel. These kinds of toxins are known as gating-modifier toxins and are of special interest because of their potential to control channel activity in a subtle and controlled way with high specificity. Current knowledge of the mode of action of gating-modifier toxins is mostly based on functional and mutational studies. A few direct structural studies of toxin-channel complexes have also been reported, but the resolution in the regions where the toxin binds is generally poor due to the dynamic nature of the VSDs and the large size of the sodium channels (~2000 amino acid residues). In this project, isolated sodium channel VSDs from two human sodium channel isoforms will be used as targets for toxin isolation, and the toxins will then be functionally and structurally characterized. Additionally, structural details of the interactions between new and/or known toxins and these VSDs will be elucidated through different biophysical techniques. For conducting these experiments, VSDs from two human sodium channels (the cardiac sodium channel NaV1.5 and NaV1.7 of the peripheral nervous system that is involved in pain transmission) will be recombinantly expressed in bacteria and reconstituted in a membrane mimetic system suitable for toxin pull-down experiments and biophysical interaction studies. The recombinant VSDs will be used to fish out interacting toxins from the crude venoms of several animal species that are known to contain gating-modifier toxins. Such toxins will be subjected to mass spectrometry analysis for determining their peptide sequence. They will then be chemically or biosynthetically produced for further NMR structural analysis. The details of interactions between VSDs and known or new interacting toxins will be elucidated by measuring how the mutation of different residues on the toxin and VSD affect the binding affinities and channel modulation as measured by electrophysiology and direct binding assays. The results of these experiments will provide useful structural information that can be exploited in the development of drugs targeting ion channels to treat disorders including cardiac arrhythmias and pain.