title: Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface.
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.
authors.affiliation: From the Programme of Molecular Structure and Function, Hospital for Sick Children, Toronto M5G 0A4, Canada; the Department of Biochemistry, University of Toronto, Toronto, Canada.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Chin
authors.affiliation: From the Programme of Molecular Structure and Function, Hospital for Sick Children, Toronto M5G 0A4, Canada.
authors.forename: Donghe
authors.initials: D
authors.lastname: Yang
authors.affiliation: the Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Miles
authors.affiliation: From the Programme of Molecular Structure and Function, Hospital for Sick Children, Toronto M5G 0A4, Canada.
authors.forename: Paul D W
authors.initials: PDW
authors.lastname: Eckford
authors.affiliation: From the Programme of Molecular Structure and Function, Hospital for Sick Children, Toronto M5G 0A4, Canada; the Department of Biochemistry, University of Toronto, Toronto, Canada.
authors.forename: Steven
authors.initials: S
authors.lastname: Molinski
authors.affiliation: the Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom.
authors.forename: B A
authors.initials: BA
authors.lastname: Wallace
authors.affiliation: From the Programme of Molecular Structure and Function, Hospital for Sick Children, Toronto M5G 0A4, Canada; the Department of Biochemistry, University of Toronto, Toronto, Canada; the Department of Physiology, University of Toronto, Toronto, Canada. Electronic address: bear@sickkids.ca.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
mesh_terms: D019943:Amino Acid Substitution; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000235:genetics; D000072417:Protein Domains
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: cysteine-mediated cross-linking; cystic fibrosis transmembrane conductance regulator (CFTR); ion channel; phosphorylation; spectroscopy; synchrotron radiation circular dichroism