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