Delta F508 in cystic fibrosis: willing but not able (original) (raw)

Molecular mechanisms of cystic fibrosis – how mutations lead to misfunction and guide therapy

Bioscience Reports

Cystic fibrosis, the most common autosomal recessive disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride and bicarbonate channel that regulates ion and water transport in secretory epithelia. Although all mutations lead to the lack or reduction in channel function, the mechanisms through which this occurs are diverse – ranging from lack of full-length mRNA, reduced mRNA levels, impaired folding and trafficking, targeting to degradation, decreased gating or conductance, and reduced protein levels to decreased half-life at the plasma membrane. Here, we review the different molecular mechanisms that cause cystic fibrosis and detail how these differences identify theratypes that can inform the use of directed therapies aiming at correcting the basic defect. In summary, we travel through CFTR life cycle from the gene to function, identifying what can go wrong and what can be targete...

Functional characterization of a novel CFTR mutation P67S identified in a patient with atypical cystic fibrosis

CELLULAR PHYSIOLOGY AND BIOCHEMISTRY, 2007

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR serves as a cAMP-stimulated chloride channel in a wide range of epithelial tissues and its dysfunction is a hallmark of CF. Over 1400 mutations in the CFTR gene are known, but functional data exist only for a minority of the mutant channels. The aim of the present study was to functionally characterize a novel CFTR mutation identified in a patient with atypical CF. Full length sequencing of the patient's CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Mutant and wild-type CFTR were heterologously expressed in Xenopus laevis oocytes. CFTR whole-cell currents were studied using the two-electrode voltage-clamp technique. Channel surface expression was assessed by a chemiluminescence assay. Expression of P67S-CFTR resulted in functional CFTR chloride channels. However, the CFTR chloride conductance observed in oocytes expressing the mutant channel averaged only 24% of that in oocytes expressing wild-type CFTR. Similarly, surface expression of the mutant channel was reduced. In contrast, the mutation did not alter the anion selectivity of the channel, and Western blot analysis indicated a similar protein expression level of mutant and wild-type CFTR. Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. The mild disease phenotype of the patient indicates that the residual function of the mutant channel is sufficient to prevent the development of severe CF symptoms.

Mutations in the Nucleotide Binding Domain 1 Signature Motif Region Rescue Processing and Functional Defects of Cystic Fibrosis Transmembrane Conductance Regulator Delta F508

Journal of Biological Chemistry, 2002

The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylationand nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (⌬F508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTR⌬F508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTR⌬F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTR⌬F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. The data show that the ⌬F508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.

Cystic fibrosis and CFTR

Pfl�gers Archiv European Journal of Physiology, 2001

Cystic fibrosis (CF) is a complex disease affecting epithelial ion transport. There are not many diseases like CF that have triggered such intense research activities. The complexity of the disease is due to mutations in the CFTR protein, now known to be a Clchannel and a regulator of other transport proteins. The various interactions and the large number of diseasecausing CFTR mutations is the reason for a variable genotype-phenotype correlation and sometimes unpredictable clinical manifestation. Nevertheless, the research of the past 10 years has resulted in a tremendous increase in knowledge, not only in regard to CFTR but also in regard to molecular interactions and completely new means of ion channel and gene therapy.

The Biogenesis, Traffic, and Function of the Cystic Fibrosis Transmembrane Conductance Regulator

International Review of Cytology, 1997

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMPactivated chloride channel that is encoded by the gene that is defective in cystic fibrosis. This ion channel resides at the luminal surfaces and in endosomes of epithelial cells that line the airways, intestine, and a variety of exocrine glands. In this article we discuss current hypotheses regarding how CFTR functions as a regulated ion channel and how CF mutations lead to disease. We also evaluate the emerging notion that CFTR is a multifunctional protein that is capable of regulating epithelial physiology at several levels, including the modulation of other ion channels and the regulation of intracellular membrane traffic. Elucidating the various functions of CFTR should contribute to our understanding of the pathology in cystic fibrosis, the most common lethal genetic disorder among Caucasians. Protein processing.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents

Proceedings of the National Academy of Sciences, 1995

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as ...

Mutations in the Nucleotide Binding Domain 1 Signature Motif Region Rescue Processing and Functional Defects of Cystic Fibrosis Transmembrane Conductance Regulator ΔF508

Journal of Biological Chemistry, 2002

The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylationand nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (⌬F508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTR⌬F508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTR⌬F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTR⌬F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. The data show that the ⌬F508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.