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Updated: Mar 20, 2026
Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Published on: March 10, 2012
How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance
Matthias Zwick1, Cinzia Esposito1, Manuel Hellstern1
1From the Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
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.
Insights
Cystic fibrosis transmembrane conductance regulator (CFTR) phosphorylation by PKA drives most ATP hydrolysis, not CFTR-ATPase activity. Phosphorylation requires a functional nucleotide binding domain and is linked to CFTR channel function.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Physiology
Background:
- Cystic fibrosis transmembrane conductance regulator (CFTR) is an ABC transporter crucial for anion transport.
- CFTR function depends on phosphorylation and ATP hydrolysis, but these processes are poorly understood.
- Existing research has focused on CFTR anion channel activity, neglecting phosphorylation and ATPase mechanisms.
Purpose of the Study:
- To develop a label-free, real-time method for measuring CFTR phosphorylation and ATPase activity in live cells.
- To elucidate the relative contributions of PKA-mediated phosphorylation and CFTR-ATPase activity to ATP hydrolysis.
- To investigate the relationship between CFTR phosphorylation, ATP hydrolysis, and anion channel function.
Main Methods:
- Utilized live cells stably transfected with CFTR for non-invasive, real-time measurements.
- Employed label-free techniques to monitor phosphorylation and CFTR-ATPase activity simultaneously.
- Investigated a specific CFTR mutant (E1371S) defective in ATP hydrolysis.
Main Results:
- PKA-mediated phosphorylation accounts for the majority (≥90%) of CFTR-related ATP hydrolysis.
- CFTR-ATPase activity contributes minimally (≤10%) to the overall ATP hydrolysis rate.
- The CFTR-E1371S mutant, despite being conductive, showed no phosphorylation, indicating a requirement for a functional nucleotide binding domain and post-hydrolysis state for phosphorylation.
- CFTR-ATPase activity was found to be inversely proportional to CFTR anion flux.
Conclusions:
- CFTR phosphorylation by PKA is the primary driver of its ATP hydrolysis.
- Phosphorylation occurs in the post-hydrolysis transition state, dependent on a functional nucleotide binding domain.
- CFTR-ATPase activity is inversely correlated with anion transport, suggesting a regulatory role in channel gating.
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