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Updated: Jun 10, 2026
Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae
Published on: May 10, 2014
CFTR function, pathology and pharmacology at single-molecule resolution
Jesper Levring1, Daniel S Terry2, Zeliha Kilic2
1Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY, USA.
Abstract:
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4-6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.
Insights
Cystic fibrosis transmembrane conductance regulator (CFTR) NBDs dimerize before opening, controlling chloride flow. This mechanism explains how CFTR potentiators work and informs new cystic fibrosis therapies.
Area of Science:
- Biophysics
- Molecular Biology
- Ion Channel Physiology
Background:
- The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial anion channel regulating epithelial salt and fluid balance.
- Dysfunction of CFTR leads to cystic fibrosis, a severe, incurable genetic disorder.
- Previous studies analyzed CFTR's electrophysiology and determined its structure in distinct conformations, but lacked direct functional correlations.
Purpose of the Study:
- To elucidate the gating mechanism of the human CFTR channel.
- To correlate CFTR's structure with its function in regulating ion transport.
- To understand how disease-causing mutations and potentiator drugs affect CFTR function.
Main Methods:
- Ensemble functional measurements
- Single-molecule fluorescence resonance energy transfer (smFRET)
- Electrophysiology
- Kinetic simulations
Main Results:
- The two nucleotide-binding domains (NBDs) of CFTR dimerize prior to channel opening.
- CFTR operates via an allosteric gating mechanism involving NBD dimerization and ATP hydrolysis.
- Potentiators like ivacaftor increase channel activity by enhancing pore opening during NBD dimerization.
- Disease-associated mutations (G551D, L927P) impair NBD dimerization efficiency.
Conclusions:
- A detailed gating mechanism for CFTR has been proposed, involving NBD dimerization as a key step.
- Understanding this mechanism provides insights into the action of potentiator drugs.
- These findings pave the way for developing more effective therapeutic strategies for cystic fibrosis.
