Abstract
Large animal models are valuable to investigate the pathophysiology of the genetic disease cystic fibrosis (CF) and test new therapies. Due to species-specific differences in the function of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, which is defective in CF, this study aims to characterise the single-channel properties of two CFTR orthologues, pig and ferret CFTR, and their responses to CFTR modulators using cells heterologously expressing CFTR constructs.Pig and ferret CFTR formed Cl- channels regulated by phosphorylation with protein kinase A and intracellular ATP. Both CFTR orthologues had larger single-channel conductances than human CFTR. ATP regulated pig CFTR with stronger binding affinity and efficacy relative to human CFTR, whereas channel activity of ferret and human CFTR were similar. When compared with its functional consequences on human CFTR, the major CF-causing CFTR variant, F508del, had reduced impact on pig CFTR, evidenced by its modest effects on channel gating and the greater thermostability of pig F508del-CFTR at 37 °C. To restore function to pig F508del-CFTR, the clinically-approved triple combination therapy elexacaftor-tezacaftor-ivacaftor was applied to heterologous cells. When compared with rescue by low temperature incubation, elexacaftor-tezacaftor-ivacaftor increased current flow through the channel and improved the thermostability of a sub-population of channels without enhancing channel gating.
In other studies, the rare CF-causing CFTR variant F312del, which affects a residue in the binding site for ivacaftor and GLPG1837, was investigated. F312del modestly impacted CFTR gating, but abolished potentiation by ivacaftor and GLPG1837.
In conclusion, this study characterised the behaviour of individual pig and ferret CFTR Cl- channels, examined the effects of the F508del variant on pig CFTR and its rescue by elexacaftor-tezacaftor-ivacaftor, and analysed the F312del variant. Species-specific differences in CFTR function inform structural interpretation of CFTR and the use of CF animal models, and hence, the discovery of novel CF therapeutics.
| Date of Award | 6 Dec 2022 |
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| Original language | English |
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| Supervisor | David N Sheppard (Supervisor) & Chrissy L Hammond (Supervisor) |