Integral voltage-gated sodium channels: production, characterisation and investigation into oligomeric state detergent dependence

Voltage-gated sodium channels underlie membrane potentials and electrical excitability in eukaryotes. Malfunctioning sodium channels give rise to conditions such as epilepsy, sensitivity to pain and cardiac arrhythmia. Structural and functional studies of voltage-gated sodium channels are therefore required in order to describe their mechanisms of action and subsequently develop specific drugs for treatments. Research into bacterial voltage-gated sodium channels started in 2001 when Ren et al. (2001) discovered their presence in bacteria and has escalated since the publication of the first structure in July 2011 with subsequent elucidation of two further bacterial voltage-gated sodium channel structures. In order to fully understand the mechanism of their eukaryotic counterparts there is a demand for further bacterial structures from a wide variety of species. One of the bottlenecks in amassing a database of bacterial structures is the protein behaviour during purification process leading to tetramer formation required for crystal formation. This study started in 2010 with the production and crystallisation of voltage-gated sodium channels from Pseudoalteromonas haloplanktis (NavPh) and Roseobacter litoralis (NavRl) and addressed the lack of procedures for preparation of tetramers. An investigation into the dependence of oligomeric state on detergents was performed. Patterns of behaviour were identified and preparation procedures were suggested. Both monomeric and tetrameric species of the selected targets were produced in milligram quantities, characterised and subjected to crystallisation trials. These investigations were supplemented with a study of the eukaryotic Nav1.1 voltage-gated sodium channel. A novel production in E. coli of the eukaryotic Nav1.1 dissected into its four domains was performed and the potential for reconstitution of the homotetramer and pseudotetramer is discussed. The viability of reconstitution of both prokaryotic and eukaryotic tetramers, however, remains irresolute.