Impact of the F508del mutation on ovine CFTR, a Cl⁻ channel with enhanced conductance and ATP‐dependent gating

Abstract

Key pointsr Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF).r Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR.r We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function.r Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del.Abstract Cross-species comparative studies are a powerful approach to understanding the epithelial Cl − channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl − channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl − currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl − channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.

Potentiation of the cystic fibrosis transmembrane conductance regulator by VX‐770 involves stabilization of the pre‐hydrolytic, O1 state

“…However, gating of F508del‐CFTR is likely to be largely hydrolytic, as demonstrated by the strong potentiation by 5‐nitro‐2‐(3‐phenylpropylamino)benzoate (NPPB) (Csanády and Töröcsik, but also see Lin et al ., ) and by almost 100‐fold slowing of F508del‐CFTR closing rate by the E1371S mutation (Kopeikin et al ., ). The apparent discrepancy between the effect of VX‐770 on WT‐CFTR and the mutants investigated in this paper and its effects on F508del‐CFTR might be related to the very severe defect in opening measured in the latter mutant (Miki et al ., ; Cai et al ., ). While the opening transition of WT, K1250R, D1370N and K464A mutants (for which measured opening rates at saturating ATP concentrations vary less than threefold: Vergani et al ., ; Vergani et al ., ; Csanády et al ., ) is likely to occur via similar structural conformational changes, the energetic landscape visited during opening by F508del‐CFTR channels might be quite different, with bound VX‐770 providing a substantially facilitated pathway.…”

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis‐causing mutation

“…However, the misprocessing defect in delta F507del‐zCftr is not as severe as the defect induced by delta F508 in hCFTR with the C/C + B ratios for the zebrafish and human proteins being 43% and 16% of WT, respectively (see Figure A). Previous studies have highlighted differences in CFTR function, pharmacology, and the consequences of the F508del mutation across species. Some amelioration of the misprocessing defect, as shown in this study, has also been seen for other species including mouse, ferret, and pig .…”

“…CFTR, cystic fibrosis transmembrane conductance regulator; FLIPR, fluorometric imaging plate reader; FSK, forskolin; GFP, green fluorescent protein; HEK293, human embryonic kidney 293; WT, wild-type | 667 LASELVA Et AL C/C + B ratios for the zebrafish and human proteins being 43% and 16% of WT, respectively (see Figure 1A). Previous studies [53][54][55][56] have highlighted differences in CFTR function, pharmacology, and the consequences of the F508del mutation across species. Some amelioration of the misprocessing defect, as shown in this study, has also been seen for other species including mouse, ferret, and pig.…”

Lessons learned from the cystic fibrosis pig

“…Some of these include a short life span (~two years), small body size, lack of submucosal glands in lung airways, differences in modifier gene expression, etc. Since then, several other species have been targeted for study of CF including the pig [12], ferret [13], zebrafish [14], the rat [15] and others such as the sheep are being developed [16]. …”