Chin, S. and Yang, D. and Miles, Andrew J. and Eckford, P.D.W. and Molinski, S. and Wallace, Bonnie A. and Bear, C.E. (2016) Attenuation of Phosphorylation-dependent activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by disease-causing mutations at the transmission interface. Journal of Biological Chemistry 292 (5), pp. 1988-1999. ISSN 0021-9258.
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Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.
Metadata
| Item Type: | Article |
|---|---|
| Keyword(s) / Subject(s): | cysteine-mediated cross-linking, cystic fibrosis transmembrane conductance regulator (CFTR), ion channel, phosphorylation, spectroscopy, synchrotron radiation circular dichroism |
| School: | Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences |
| Depositing User: | Administrator |
| Date Deposited: | 10 Mar 2017 14:21 |
| Last Modified: | 02 Aug 2023 17:32 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/18322 |
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