Impact of the F508del Mutation on Pig Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a Cl- Channel with Enhanced ATP-Dependent Channel Gating

Physiology 2021 (2021) Proc Physiol Soc 48, OC34

Oral Communications: Impact of the F508del Mutation on Pig Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a Cl- Channel with Enhanced ATP-Dependent Channel Gating

Demi R S Ng1, Samuel J Bose1, David N Sheppard1

1 School of Physiology, Pharmacology & Neuroscience, Biomedical Sciences Building, University of Bristol, BS8 1TD. , Bristol, United Kingdom

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Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel, causes the genetic disorder cystic fibrosis (CF) (1).  F508del, the most common CF mutation, disrupts CFTR processing and intracellular trafficking, reduces CFTR stability at the plasma membrane and alters channel gating (2).  To understand CF pathogenesis and test new therapeutics, CF pigs with the F508del mutation were generated (3).  Here, we investigate the single-channel behaviour of pig CFTR and the impact on it of the F508del mutation.  Using the patch-clamp technique, we studied CFTR Cl- channels in excised inside-out membrane patches from CHO cells transiently transfected with CFTR constructs voltage-clamped at -50 mV using a large Cl- concentration gradient ([Cl-]i, 147 mM; [Cl-]o, 10 mM); temperature was 37 °C (4).  Like human CFTR, pig CFTR formed low conductance Cl–selective channels regulated by protein kinase A-dependent phosphorylation and intracellular ATP.  However, distinct differences were observed in the frequency and duration of channel openings.  At 1 mM ATP, channel openings of pig CFTR were 4-fold longer than those of human CFTR, whereas the long closures separating channel openings of pig CFTR were similar in duration to those of human CFTR.  Consequently, the open probability (Po) of pig CFTR (0.68 ± 0.12, n = 13; means ± SD) was 2-fold higher than that of human CFTR (0.39 ± 0.07, n = 15) (Student’s unpaired t-test, P < 0.0001).  To explore these gating differences, we examined the ATP dependence of Po between 0.03 mM and 3 mM ATP.  By fitting mean Po data with Michaelis-Menten functions, we found that ATP regulated pig CFTR with increased apparent affinity and efficacy (human: KD = 180 µM, Po(max) = 0.61, r2 = 0.96, n = 4 – 16; pig: KD = 25 µM, Po(max) = 0.76, r2 = 0.78, n = 4 – 7).  Consistent with previous studies (4, 5), the severity of the F508del mutation varied across species.  Human F508del-CFTR exhibited a severe gating defect characterised by infrequent channel openings and marked thermoinstability demonstrated by Po diminishing from ~0.15 to 0 within 8 minutes at 37 °C (4).  By contrast, the F508del mutation had reduced impact on pig CFTR.  First, the F508del mutation decreased the Po of pig CFTR by only 0.5-fold (0.38 ± 0.18, n = 9) (Student’s unpaired t-test, P < 0.001 vs. pig wild-type CFTR), but that of human CFTR by 5-fold (0.08 ± 0.03, n = 7) (Student’s unpaired t-test, P < 0.0001 vs. human wild-type CFTR).  Second, pig F508del-CFTR showed greater thermostability at 37 °C, with Po only gradually declining from ~0.26 to ~0.10 over a 20-minute period (n = 5).  We conclude that i) pig CFTR forms a regulated Cl- channel with enhanced ATP-dependent channel gating, and ii) the F508del mutation has distinct consequences in human and pig CFTR.  Thus, these data provide insights into species-specific differences that illuminate analyses of CFTR structure and function with the potential to inform the development of new CF therapeutics.



Where applicable, experiments conform with Society ethical requirements.

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