title: How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.
authors.affiliation: From the Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Zwick
authors.affiliation: From the Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Esposito
authors.affiliation: From the Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
authors.forename: Manuel
authors.initials: M
authors.lastname: Hellstern
authors.affiliation: From the Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland anna.seelig@unibas.ch.
authors.forename: Anna
authors.initials: A
authors.lastname: Seelig
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006019:Glycolysis; D006868:Hydrolysis; D009154:Mutation; D010085:Oxidative Phosphorylation
chemical_list: D000838:Anions; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: ABC transporter; biosensor; cyclic AMP (cAMP); cystic fibrosis transmembrane conductance regulator (CFTR); protein kinase A (PKA)