1. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
- Author
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Anna Seelig, Manuel Hellstern, Cinzia Esposito, and Matthias Zwick
- Subjects
0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,biology ,ATP-binding cassette transporter ,Cell Biology ,respiratory system ,Anion channel activity ,Biochemistry ,Cystic fibrosis transmembrane conductance regulator ,respiratory tract diseases ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,ATP hydrolysis ,Membrane Biology ,Chloride channel ,biology.protein ,Biophysics ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Adenosine triphosphate - 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.
- Published
- 2016