Abstract
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The licensing of the CFTR potentiator ivacaftor (VX-770, Vertex Pharmaceuticals Inc.), provided proof of concept for the use of small molecules to treat CF. However, the mechanism of action of ivacaftor is not well understood. Studies exploiting species differences in CFTR pharmacology have previously been used to identify binding sites of CFTR modulators. We hypothesised that differences in the response of CFTR orthologues to ivacaftor may be used to determine regions of the protein involved in its mechanism of action.CFTR orthologues from human, pig, sheep, ferret, mouse and zebrafish were studied using single-channel patch-clamp recordings. These studies identified differences in the single-channel current amplitude (i) and open probability (Po) of CFTR orthologues. Single-channel recordings also demonstrated that the Po of mouse F508del-CFTR after rescue of its plasma membrane expression was not reduced compared to mouse wild-type (WT)-CFTR and channel activity remained stable at 37 °C in contrast to human F508del-CFTR. Furthermore, neither mouse WT-CFTR nor rescued mouse F508del-CFTR were potentiated by ivacaftor. We therefore developed a high-throughput assay utilising automated whole-cell patch-clamp recording to test a selection of human-mouse CFTR chimeras for their response to ivacaftor. From these studies, we identified five sequence alterations from the human to the mouse sequence, A326T, L327V, K329N, I332V and A349S, located in the third extracellular loop (ECL3) and on transmembrane helix 6 (TM6), that when expressed in human CFTR prevented potentiation by ivacaftor. These experiments were further supported by single-channel recordings of human-mouse CFTR chimeras.
In conclusion, this study has identified structural regions within the transmembrane helices of CFTR that may be targeted for the development of novel CFTR potentiators to be used in conjunction with CFTR correctors for the treatment of CF.
| Date of Award | 23 Jan 2019 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | David N Sheppard (Supervisor) |