Biophysical analysis of interactions between peptide toxins and human sodium channel voltage-sensor domains - 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. The gating-modifier toxins are a subgroup of these toxins that change the
voltage of activation of the channels by binding to the voltage sensor domains (VSDs) of the channel. 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. 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 channel NaV1.5 and
NaV1.7 of the peripheral nervous system that is involved in pain transmission) will be 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 isolate interacting toxins from the crude venoms of several animal
species that are known to contain gating-modifier toxins. Initial identification of such toxins will be achieved by
MALDI-TOF mass spectrometry. The instrument currently used for this purpose no longer works reliably and
cannot be fixed since it is out of support from the vendor. Funding for the purchase of a replacement mass
spectrometer is therefore requested, since this instrument is essential in the first step of toxin characterization.
Following this toxin identification, toxins of interest will be further characterized by amino acid sequencing,
chemical or recombinant synthesis and 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.
