Biochemical and structural studies of the voltage-gated sodium channels hNav1.8 and NavMs

Haste, Callum Alexander Franklin (2024) Biochemical and structural studies of the voltage-gated sodium channels hNav1.8 and NavMs. PhD thesis, Birkbeck, University of London.

Abstract

Voltage-gated sodium channels (VGSCs or Navs), responsible for the action potential in excitable tissues, are drug targets for a variety of serious diseases linked to Navs, termed channelopathies; however, many of these drugs cause significant side effects due to their non-specificity between the nine distinct human isoforms (hNavs). The treatment of these diseases is complicated by the multiple states cycled through during the action potential, with open, closed, and inactivated channels adopting different conformations, and the fact that drugs can bind differentially to each state. Recent structural studies of eukaryotic and prokaryotic sodium channels (which share high sequence identities at key residues, plus similar structural features and drug binding affinities to eukaryotic Navs) have revealed a wealth of information about their function and interaction with various animal toxins and small molecule drugs, but hNavs remain challenging targets for structural study. The aim of this research was to use a novel expression system, Tetrahymena thermophila, to aid in expressing and purifying the peripheral nervous system sodium channel hNav1.8, associated with pain disorders, in order to study it by cryo-transmission electron microscopy (cryo-TEM) and gain insights into the hNav1.8 mechanism. This research marked the first time our lab was able to visualise hNav1.8 particles on a cryo-TEM grid without graphene oxide support, however, it was deemed unsuitable for obtaining high-resolution structural data due to the high level of degradation of the protein. The next aim of the research was to develop a method for the cryo-TEM analysis of the prokaryotic VGSC NavMs so that this useful model for hNavs could be studied closer to physiological conditions and ideally in unstudied conformations, which with the use of membrane scaffold protein (MSP) nanodiscs was ultimately a success, leading to a 3.07 Å reconstruction and a model with features distinct from previously solved NavMs structures.

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