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A single amino acid substitution in CFTR converts ATP to an inhibitory ligand
Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Rockefeller University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178940/ https://www.ncbi.nlm.nih.gov/pubmed/25225552 http://dx.doi.org/10.1085/jgp.201411247 |
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author | Lin, Wen-Ying Jih, Kang-Yang Hwang, Tzyh-Chang |
author_facet | Lin, Wen-Ying Jih, Kang-Yang Hwang, Tzyh-Chang |
author_sort | Lin, Wen-Ying |
collection | PubMed |
description | Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (P(o)) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its P(o), has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the γ-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF. |
format | Online Article Text |
id | pubmed-4178940 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-41789402015-04-01 A single amino acid substitution in CFTR converts ATP to an inhibitory ligand Lin, Wen-Ying Jih, Kang-Yang Hwang, Tzyh-Chang J Gen Physiol Research Articles Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (P(o)) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its P(o), has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the γ-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF. The Rockefeller University Press 2014-10 /pmc/articles/PMC4178940/ /pubmed/25225552 http://dx.doi.org/10.1085/jgp.201411247 Text en © 2014 Lin et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). |
spellingShingle | Research Articles Lin, Wen-Ying Jih, Kang-Yang Hwang, Tzyh-Chang A single amino acid substitution in CFTR converts ATP to an inhibitory ligand |
title | A single amino acid substitution in CFTR converts ATP to an inhibitory ligand |
title_full | A single amino acid substitution in CFTR converts ATP to an inhibitory ligand |
title_fullStr | A single amino acid substitution in CFTR converts ATP to an inhibitory ligand |
title_full_unstemmed | A single amino acid substitution in CFTR converts ATP to an inhibitory ligand |
title_short | A single amino acid substitution in CFTR converts ATP to an inhibitory ligand |
title_sort | single amino acid substitution in cftr converts atp to an inhibitory ligand |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178940/ https://www.ncbi.nlm.nih.gov/pubmed/25225552 http://dx.doi.org/10.1085/jgp.201411247 |
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