Molecular mechanism of CFTR channel gating: transmission of conformational signals originating at the catalytic site

Lead Research Organisation: University College London
Department Name: Neuroscience Physiology and Pharmacology

Abstract

Cystic fibrosis is the most common life-threatening inherited disease in the UK. The disease is caused by absence or malfunction of a protein called CFTR. CFTR is a channel protein: a specialized machine that allows movement of ions into and out of cells. It consists of several parts, or domains. One part, the transmembrane domain, is embedded in the cells’ membrane and forms a pore, a pathway through which the ions can flow. A molecule called ATP remotely controls access to the pore by binding to CFTR at its nucleotide-binding domains (or NBDs). What shape changes occur in the NBDs when ATP binds or when it is cleaved? How are these shape changes transmitted to the transmembrane domain, where the pore opens or closes? Answering these basic questions could suggest new ways of treating some cystic fibrosis patients. Many other proteins, related to CFTR, have NBDs. We will use the evolutionary record of this family, as well as sensitive recordings of tiny electrical signals generated by CFTR molecules, to probe the general mechanism by which NBDs drive the changes in shape of adjacent domains -- in CFTR itself and in other, harder to investigate, members of the same protein family.

Technical Summary

CFTR, whose dysfunction causes the fatal disease cystic fibrosis (CF), belongs to the superfamily of ATP binding cassette (ABC) proteins, which includes many other members associated with human diseases. ABC proteins couple hydrolytic cycles at conserved nucleotide-binding domains (NBDs) to diverse cellular functions, such as active transport of molecules across membranes. CFTR is unique among ABC proteins in that its trans-membrane domains (TMDs) comprise an ion channel. Opening and closing (gating) of CFTR?s permeation pathway has been linked to binding and hydrolysis, respectively, of ATP at its two NBDs. The experiments proposed here are aimed at understanding how conformational signals originating at the catalytic site are propagated through the protein and ultimately to the TMDs, resulting in the coupling of hydrolytic and gating cycles.
By analyzing alignments of thousands of homologous NBD sequences from different ABC proteins one can measure whether amino acid distribution at any two positions varies in a correlated way. Such pair-wise coupling values can then be clustered to extract a core network of positions with strong mutual coupling. Does this statistical coupling in evolutionary space result from conservation of a network of residues - energetically coupled in real space - which mediate transmission of allosteric signals in individual ABC proteins? We will address this question experimentally by measuring energetic coupling between pairs of CFTR amino-acid side-chains, selecting targets for mutagenesis at statistically coupled positions and on the basis of available structural information. For each pair of targets, we will use single-channel kinetic analysis to measure several parameters (apparent affinity for ATP, opening rate, open probability). For each parameter we will apply thermodynamic mutant cycle analysis and determine energetic coupling values, allowing us to infer how coupling changes as the channel binds ATP, opens, or moves between open and closed states. Together, these studies will compose a molecular description - in both space (within the protein?s structure), and time (along the reaction coordinate of CFTR?s functional cycle) of the allosteric coupling mechanism by which conformational signals are transmitted from NBD active site towards the TMD.
The results will provide basic knowledge directly relevant to the understanding of some forms of cystic fibrosis. The experimental design, relying on the evolutionary record for the selection of targets, broadens the scope of such relevance to include other human diseases, linked to ABC proteins that, unlike CFTR, cannot be studied with high-resolution single-molecule techniques.

Publications

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Csanády L (2013) Conformational changes in the catalytically inactive nucleotide-binding site of CFTR. in The Journal of general physiology

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Csanády L (2019) STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL. in Physiological reviews

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Csanády L (2010) Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations. in Proceedings of the National Academy of Sciences of the United States of America

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Muallem D (2009) Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

 
Description 2012 Research Grants
Amount £69,055 (GBP)
Funding ID CFT Project No: RS31 
Organisation Cystic Fibrosis Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description Outgoing Short Visit Grant
Amount £4,332 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2007 
End 03/2007
 
Description Sparks Project Grant
Amount £150,000 (GBP)
Funding ID 15UCL04 
Organisation Sparks 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 11/2018
 
Description Strategic Research Centre
Amount £113,771 (GBP)
Funding ID SRC005 
Organisation Cystic Fibrosis Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description Wellcome Biomedical Vacation Scholarships
Amount £58,474 (GBP)
Funding ID 218411/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2019 
End 08/2019
 
Description Wellcome Trust Project Grant (Molecular dissection of the CFTR gating cycle, combining mathematical tools with experimental biophysics)
Amount £288,320 (GBP)
Funding ID 081298/Z/06/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2007 
End 04/2011
 
Title YFP-CFTR/mCherry assay 
Description A cell-based assay that uses fluorescent proteins to obtain information on membrane density and ion channel function of CFTR 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2019 
Provided To Others? Yes  
Impact The assay allows rapid screening of drug responses for a large panel of mutant CFTR versions 
URL https://www.biorxiv.org/content/10.1101/631614v8
 
Description Genentech: Studies on CFTR Inhibition 
Organisation Genentech, Inc
Country United States 
Sector Private 
PI Contribution Using our new CFTR monitoring assay (YFP-CFTR/mCherry) we tested the effect of 4 compounds identified by Genentech as active on a CFTR relative.
Collaborator Contribution Genentech provided compounds
Impact No outcomes yet. Interdisciplinary: Chemistry and physiology/Pharmacology
Start Year 2019
 
Description MD studies on CFTR 
Organisation University of Calgary
Country Canada 
Sector Academic/University 
PI Contribution knowledge on CFTR function and familiarity with literature describing it
Collaborator Contribution In depth understanding of tools used in homology modelling and molecular dynamics simulations
Impact Multidisciplinary: electrophysiology and Molecular dynamics
Start Year 2011
 
Description Mathematical Modelling of CF epithelia 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution background knowledge on epithelial physiology and cystic fibrosis
Collaborator Contribution Mathematical modelling
Impact Publication: Donal O'Donoghue et al., 2013 multidisciplinary (physiology/mathematical modelling)
Start Year 2010
 
Description Semmelweis University 
Organisation Semmelweiss University
Department Department of Medical Biochemistry
Country Hungary 
Sector Academic/University 
PI Contribution Initially, molecular biology (mainly preparation of new mutants and in vitro RNA transcription) was carried out only in our Lab, due to difficulties setting up the laboratory and the higher expense of getting materials in Budapest.
Collaborator Contribution My PostDoc spent some months in Dr Csanady's lab, during my maternity leave. Training in ion channel kinetic analysis was provided.
Impact Outputs: several Publications, see author lists; grant funding Collaborative Wellcome Trust Project Grant. Interdisciplinary: Budapest site provides expertise in mathematical analysis of data - London site provides Bioinformatic analysis of multiple sequence alignments - both sites carry out electrophysiological recordings
Start Year 2006
 
Description Be Inspired: Women Leaders 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Paola Vergani took part in a Careers talk for 16-18 year-old students at Westminster Kingsway College. The aim was to disband stereotypes on careers that are traditionally followed by white men, to increase diversity in STEM professions. Paola Vergani presented her work as a quantitative scientist, working on CFTR pharmacology.
Year(s) Of Engagement Activity 2019
URL https://www.westking.ac.uk/2019/04/women-leaders-inspire-westking-students/
 
Description School Visit (Highgate) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Paola Vergani visited a secondary school in her local area. She was asked to present basic research relevant to Biology A-level students, with potential applications to disease. Paola Vergani presented the CFTR protein's biogenesis, function, role in epithelial physiology and in the pathophysiology of cystic fibrosis. One of the students asked to spend three days in Paola Vergani's laboratory over the summer for work experience. She followed experiments and discussed research extensively with Paola Vergani and laboratory members, greatly increasing her awareness and understanding of cystic fibrosis.
Year(s) Of Engagement Activity 2018
 
Description School visits (Islington, Haringey) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I visited two primary schools in my local area, as part of a "STEM" week. The Education Professionals who contacted me were particularly interested in providing inspiring female "role models" for young girls. I talked about cystic fibrosis and developing drugs targeting the CFTR protein, especially for patients with rare mutations.
Year(s) Of Engagement Activity 2017