TY - JOUR
T1 - Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating
AU - Cai, Zhiwei
AU - Palmai-Pallag, Timea
AU - Khuituan, Pissared
AU - Mutolo, Michael J.
AU - Boinot, Clément
AU - Liu, Beihui
AU - Scott-Ward, Toby S.
AU - Callebaut, Isabelle
AU - Harris, Ann
AU - Sheppard, David N.
N1 - Funding Information:
This work was supported by the Cystic Fibrosis Foundation, Cystic Fibrosis Trust, the French Cystic Fibrosis Association Vaincre la Mucoviscidose and the National Institutes of Health (R01 HL094585); C.B. was supported by the Université de Poitiers and P.K. by the Strategic Scholarships Fellowships Frontier Research Networks, Office of the Higher Education Commission of Thailand.
Publisher Copyright:
© 2015 The Physiological Society.
PY - 2015/6/1
Y1 - 2015/6/1
N2 - 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.
AB - 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.
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U2 - 10.1113/JP270227
DO - 10.1113/JP270227
M3 - Article
C2 - 25763566
AN - SCOPUS:84929956909
VL - 593
SP - 2427
EP - 2446
JO - Journal of Physiology
JF - Journal of Physiology
SN - 0022-3751
IS - 11
ER -