Cholesterol Interaction Directly Enhances Intrinsic Activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Abstract

The recent cryo-electron microscopy structures of zebrafish and the human cystic fibrosis transmembrane conductance regulator (CFTR) provided unprecedented insights into putative mechanisms underlying gating of its anion channel activity. Interestingly, despite predictions based on channel activity measurements in biological membranes, the structure of the detergent purified, phosphorylated, and ATP-bound human CFTR protein did not reveal a stably open conduction pathway. This study tested the hypothesis that the functional properties of the detergent solubilized CFTR protein used for structural determinations are different from those exhibited by CFTR purified under conditions that retain associated lipids native to the membrane. It was found that CFTR purified together with phospholipids and cholesterol using amphipol: A8-35, exhibited higher rates of catalytic activity, phosphorylation dependent channel activation and potentiation by the therapeutic compound, ivacaftor, than did CFTR purified in detergent. The catalytic activity of phosphorylated CFTR detergent micelles was rescued by the addition of phospholipids plus cholesterol, but not by phospholipids alone, arguing for a specific role for cholesterol in modulating this function. In summary, these studies highlight the importance of lipid interactions in the intrinsic activities and pharmacological potentiation of CFTR.

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

“…The blots were developed with ECL (Amersham) using the Li-Cor Odyssey Fc (LI-COR Biosciences, Lincoln, NE, USA) in a linear range of exposure (2–45 min) [ 24 , 25 ]. Relative levels of CFTR proteins were quantitated by densitometry of immunoblots using ImageStudioLite (LI-COR Biosciences, Lincoln, Nebraska, USA) [ 26 , 27 ].…”

Distinctive lipid signatures of bronchial epithelial cells associated with cystic fibrosis drugs, including Trikafta

“…An important contribution to CFTR stability at the cell membrane also comes from the action of flippases (4), enzymes known to regulate the movement of phospholipids across the cell membrane. Very recently, Bear and colleagues demonstrated that membrane cholesterol plays a significant role in CFTR activity (5). Despite this work clearly demonstrating a crucial role for lipid composition in CFTR trafficking and activity, to our knowledge there has not yet been an untargeted lipidomic profiling of CF-relevant cell models directed toward the whole lipidome and not selected lipid species.…”

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis‐causing mutation

“…hCFTR was detected with hCFTR‐specific murine mAb 596 (1:5000) or rabbit Ab Anti‐GFP (1:5000). The blots were developed with ECL (Amersham) on the Li‐Cor Odyssey Fc (LI‐COR Biosciences) in a linear range of exposure …”

“…The blots were developed with ECL (Amersham) on the Li-Cor Odyssey Fc (LI-COR Biosciences) in a linear range of exposure. [44][45][46][47] To evaluate protein glycosylation status, HEK293 cells were transiently transfected with WT-zCftr-GFP or F507del-zCftr-GFP. The cells were grown at 37°C for 24 hours and subsequently lysed in modified RIPA buffer as described above.…”