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      Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity

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          Abstract

          Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.

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          Most cited references40

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          Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies.

          The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.
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            Molecular Structure of the Human CFTR Ion Channel.

            The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.
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              Structural identification of a hotspot on CFTR for potentiation

              Cystic fibrosis is a fatal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Two main categories of drugs are being developed: correctors that improve folding of CFTR and potentiators that recover the function of CFTR. Here, we report two cryo–electron microscopy structures of human CFTR in complex with potentiators: one with the U.S. Food and Drug Administration (FDA)–approved drug ivacaftor at 3.3-angstrom resolution and the other with an investigational drug, GLPG1837, at 3.2-angstrom resolution. These two drugs, although chemically dissimilar, bind to the same site within the transmembrane region. Mutagenesis suggests that in both cases, hydrogen bonds provided by the protein are important for drug recognition. The molecular details of how ivacaftor and GLPG1837 interact with CFTR may facilitate structure-based optimization of therapeutic compounds.
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                Author and article information

                Contributors
                Journal
                Front Physiol
                Front Physiol
                Front. Physiol.
                Frontiers in Physiology
                Frontiers Media S.A.
                1664-042X
                07 June 2021
                2021
                : 12
                : 652513
                Affiliations
                [1] 1Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory + Children’s Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children’s Healthcare of Atlanta , Atlanta, GA, United States
                [2] 2Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University , Atlanta, GA, United States
                Author notes

                Edited by: John Wilson, Cystic Fibrosis Service, Alfred Hospital, Australia

                Reviewed by: Naomi Niisato, Kyoto University of Advanced Science (KUAS), Japan; Robert Tarran, University of North Carolina at Chapel Hill, United States

                *Correspondence: Nael A. McCarty, namccar@ 123456emory.edu

                This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

                Article
                10.3389/fphys.2021.652513
                8215275
                34163370
                334e1c9b-4496-4fcf-a5fb-ddbc4513f41e
                Copyright © 2021 Cui, Cottrill, Strickland, Mashburn, Koval and McCarty.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 13 January 2021
                : 23 April 2021
                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 40, Pages: 11, Words: 0
                Funding
                Funded by: Cystic Fibrosis Foundation 10.13039/100000897
                Award ID: MCCART17G0
                Award ID: MCCART18G0
                Categories
                Physiology
                Original Research

                Anatomy & Physiology
                cystic fibrosis,cholesterol,modulator,potentiator,pharmacology,ivacaftor,kalydeco
                Anatomy & Physiology
                cystic fibrosis, cholesterol, modulator, potentiator, pharmacology, ivacaftor, kalydeco

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