Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB113

Poster Communications

Cystic fibrosis mutations at position S549 modulate CFTR Cl- channel processing, gating and responsiveness to mutation-specific therapies

M. K. Al Salmani1, A. Rab2, J. S. Hong2, H. li1, Z. Cai1, U. W. Fass3, E. J. Sorscher2, D. N. Sheppard1

1. School of Physiology, Pharmacology and Neuroscience, The University of Bristol, Bristol, United Kingdom. 2. School of Medicine and Children's Hospital of Atlanta, Emory University, Atlanta, Georgia, United States. 3. Department of Biochemistry, Oman Medical College, Sohar, Oman.


Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- ion channel evolved from an ATP binding cassette (ABC) transporter, widely expressed in epithelial tissues. It is activated by protein kinase A (PKA) and gated by the binding and hydrolysis of ATP at two nucleotide-binding domains (NBDs). ATP binding opens the channel by initiating NBD dimerization, while ATP hydrolysis partially separates the NBD dimer to close the channel. Position S549 is highly conserved in ABC transporters and is believed to play a role in facilitating ATP binding and hydrolysis. Three CF mutations at this position have been reported in patients, S549N, S549R and S549I. To better understand the molecular dysfunction caused by these mutations, we studied wild-type and mutant CFTR protein processing, channel gating and responsiveness to CFTR modulators. We also investigated two additional mutations, S549C- and S549F-CFTR, to probe the role of S549 in channel gating. For our studies, we utilised recombinant Fischer Rat Thyroid (FRT) and CHO cells and Western-blotting, Ussing chambers and patch-clamp recording. The amount of fully-glycosylated S549N-CFTR expressed in FRT cells was similar to wild-type CFTR when cellular mRNA levels were held constant. By contrast, the expression of fully-glycosylated S549R- and S549I-CFTR were markedly diminished. Lumacaftor (3 µM) increased the amount of fully-glycosylated S549N- and S549R-CFTR by approximately 2-fold and enhanced the magnitude of CFTR-mediated Cl- current. By contrast, lumacaftor (3 µM) failed to rescue S549I-CFTR protein and had little effect on the magnitude of CFTR-mediated Cl- currents. Ivacaftor (5 µM) doubled the activity of S549N- and S549R-CFTR, but did not potentiate the activity of S549I-CFTR. To further understand the molecular defects introduced by these mutations, we studied single channels using excised inside-out membrane patches. To enhance the plasma membrane expression of mutant Cl- channels, we incubated transfected CHO cells at 27 °C for > 48 hours. The single channel activities of S549N- and S549R-CFTR were disrupted severely when compared to wild-type CFTR with bursts of channel openings separated by prolonged channel closures. By contrast, S549I-CFTR modulated, but did not disrupt, CFTR channel gating. Moreover, the apparent ATP affinities of S549N- and S549R-, but not S549I-CFTR, were profoundly decreased compared to wild-type CFTR. To probe further the role of S549, we examined the gating properties of S549C- and S549F-CFTR. S549C-CFTR differs from wild-type CFTR by only one atom, whereas S549F-CFTR introduces a bulky hydrophobic amino acid. Like S549I-CFTR, both S549C- and S549F-CFTR modulated, but did not disrupt CFTR channel gating. Altogether, the above investigations suggest that S549 plays a key role in CFTR folding, gating and responsiveness to CFTR modulators.

Where applicable, experiments conform with Society ethical requirements