The nociceptive system is a neural pathway that underlies our capacity to feel pain when we are threatened by potential tissue injury. Congenital Insensitivity to Pain (CIP) arises when genetic mutations disrupt molecular components that are essential to the development or postnatal function of this system. Understanding how these mutations alter the encoded protein, and subsequently impact sensory neuron signalling, can reveal new strategies to modulate their activity in maladaptive pain disorders.

The primary aim of this thesis was to confirm pathogenicity of several novel missense mutations from SCN9A-CIP patients, representing localised structural changes to the voltage-gated sodium channel (VGSC) Nav1.7. Studies revealed that all mutants were non-conducting, independent of protein folding or membrane trafficking defects, implying critical intramolecular roles for the affected residues. Through this project I developed a workflow for rapid stable cell line generation and functional assessment that could be applied to the interpretation of other VGSC variants.

We extended this project to examine the interactomes of wild-type human Nav1.7 channels and a null N-terminal mutation in the HEK293 expression system. While this approach did not conclusively identify any protein-protein interactions disturbed by the mutation, our results support some of the mass spectrometry hits detected in a similar study of the in vivo mouse Nav1.7 interactome.

Lastly, we uncovered a surprising genotype-phenotype correlation in another CIP gene, PRDM12. Unlike other loss-of-function mutations in this gene that cause global, lifelong painlessness, homozygous polyalanine expansions within a discrete range lead to facially-localised, enhanced itch in childhood (Midface Toddler Excoriation Syndrome – MiTES). Observations of the CIP- and MiTES-associated polyalanine expansion mutants in a non-neuronal cell line hint at a subtle difference in PRDM12 availability that may influence the maturation of polymodal pain- and itch-sensing neurons.