Chimeric Human-NavMs Sodium Channel Constructs for Structure/Function Studies of Selective Toxin Binding

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Voltage-gated sodium channels are responsible for a wide range of neurological and cardiovascular channelopathies, and hence are important current and future targets for the development of novel and highly specific pharmaceutical drugs.This studentship will combine training and research in molecular biology (cloning, expression, purification and characterization of membrane proteins), structural biology (crystallography, cryoEM), binding studies (circular dichroism spectroscopy, surface plasmon resonance, microscale thermophoresis(MST)), and bioinformatics/computational biology as part of an interdisciplinary discovery/design project on sodium channels aimed at identifying different sites within the voltage sensor regions of sodium channels that confer selectivity and sensitivity by the different channel isoforms for different naturally-occurring toxins. The nine sodium channel isoforms are found in different tissues throughout the human body have different physiological roles, but have high {~90-95%} sequence identities and are targets for development of anti-epileptic, anti-arrhythmic, and analgesic drugs. Such drugs must take into account the significant homologies to prevent unwanted side effects (ie. analgesic effects on cardiac suppression). Highly specific toxsins, both natural and synthetic, which target different isoforms are excellent tools used in the pharmaceutical industry as the basis for rational drug design and development of new targeted compounds (either synthetic toxins or small molecules).The access to both novel methods for characterization and novel compounds provided by the industrial partner will greatly enhance the potential of this project for development of novel and potent isoform-specific drugs, as well as enhancing our understanding of sodium channel structure and function. In order to identify the regions within the different sodium channel domains that are responsible for specific binding, we will construct chimeras/mutants based on the prokaryotic NavMs full length crystal structure recently determined in the Wallace lab (1), in which specific regions/residues are replaced with isoform-specific regions of human sodium channels. NavMs has been shown to be an excellent and accessible functional orthologue for drug binding characteristics of human sodium channels (6) and chimeric/mutant structures created based on human isoforms can provide lead information on disease-related regions of the molecule that can be targeted for drug/isoform-specific toxin binding (1,2). Naturally-occurring (and mutant) toxins which bind differentially in the voltage-sensor domains and sub-domains will be used as guides to target regions/sequences associated with selectivity between, for example Nav1.7, 1.8, and 1.9 human channels. The binding will be initially assessed using biophysical methods such as SPR and thermal melt circular dichroism spectroscopy (3,5), available at either the industrial or academic site, and then extended using MST and other in house methods that Vertex uses for screening. Once suitable stable toxin-channel complexes are prepared, they can be examined in detail using structural biology techniques available via either/both sites (crystallography, cryoEM). This project will thus give the student the opportunity to undertake and develop studies in both an academic and company environment, giving them exposure to a range of techniques, compounds and interactions with colleagues otherwise not available at one of the sites alone; this project will also develop further ties between the partner groups.