Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel by genetic mutations causes the inherited disease cystic fibrosis (CF). CF lung disease that involves ...multiple disorders of epithelial function likely results from loss of CFTR function as an anion channel conducting chloride and bicarbonate ions and its function as a cellular regulator modulating the activity of membrane and cytosol proteins. In the absence of CFTR activity, abundant mucus accumulation, bacterial infection and inflammation characterize CF airways, in which inflammation-associated tissue remodeling and damage gradually destroys the lung. Deciphering the link between CFTR dysfunction and bacterial infection in CF airways may reveal the pathogenesis of CF lung disease and guide the development of new treatments. Research efforts towards this goal, including high salt, low volume, airway surface liquid acidosis and abnormal mucus hypotheses are critically reviewed.
Key points
The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by ...intracellular ATP.
To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH.
We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains.
Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects.
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP‐binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single‐channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open‐time constant (τo) of wild‐type CFTR. By contrast, the fast closed‐time constant (τcf), which describes the short‐lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three‐state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP‐binding site 1, including K464A, D572N and the CF‐associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP‐dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP‐binding site 1 modulates intraburst gating.
Key points
The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP.
To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH.
We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains.
Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects.
Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters ...electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR −/− and CFTR ΔF508/ΔF508 airway epithelia lacked anion conductance, and they did not hyperabsorb Na + . Therefore, we asked whether Cl − and Na + conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na + channel inhibitor amiloride produced similar reductions in Gt and Na + absorption, indicating that Na + conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl − conductance. These results indicate that Na + conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.
ABSTRACTPeople with the genetic disease cystic fibrosis (CF) often carry a deletion mutation ∆F508 on the gene encoding the CF transmembrane conductance regulator (CFTR) Cl− channel. This mutation ...greatly reduces the CFTR maturation process and slows the channel opening rate. Here, we investigate whether residues near F508 contribute to these defects in ∆F508‐CFTR. Most deletion mutations, but not alanine substitutions, of individual residues from positions 503 to 513 impaired CFTR maturation. Interestingly, only protein processing of ∆Y512‐CFTR, like that of ∆F508‐CFTR, was greatly improved by low‐temperature culture at 27°C or small‐molecule corrector C18. The 2 mutant Cl− channels were equally slow to open, suggesting that they may share common structural flaws. Studies on the H3‐H4 loop that links residues F508 and Y512 demonstrate that G509A/V510G mutations, moving G5091 position backward in the loop, markedly enhanced ∆F508‐CFTR maturation and opening rate while promoting protein stability and persistence of the H3 helix in ∆F508 nucleotide‐binding domain 1. Moreover, V510A/S511A mutations noticeably increased ∆Y512‐CFTR maturation at 27°C and its opening rate. Thus, loop abnormalities may contribute to ∆F508‐ and ∆Y512‐CFTR defects. Importantly, correcting defects from G509 displacement in ∆F508‐CFTR may offer a new avenue for drug discovery and CF treatments.—Chen, X., Zhu, S., Zhenin, M., Xu, W., Bose, S. J., Wong, M. P.‐F., Leung, G. P. H., Senderowitz, H., Chen, J.‐H. A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis‐causing mutation. FASEB J. 33, 5126–5142 (2019). www.fasebj.org
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by protein kinase A (PKA) phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain ...residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing one to three protein segments of the R domain including residues 708–759 (ΔR708–759), R760–783, and R784–835, each of which contains one or two PKA phosphorylation sites. Deletion of R708–759 or R760–783 had little effect on CFTR gating, whereas all mutations lacking R784–835 reduced CFTR activity by decreasing the mean burst duration and increasing the interburst interval (IBI). The data suggest that R784–835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ΔR784–835 had minor impact on gating potentiation by 2′dATP, CaATP, and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R809–835 shortened the IBI of ΔR708–835-CFTR. Consistently, ΔR815–835, but not ΔR784–814, enhanced IBI, whereas both reduced mean burst duration. These data suggest that the entirety of R784–835 is required for stabilizing the open state of CFTR; however, R815–835, through interactions with the channel, is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR708–783-CFTR. Conversely, the IBI of ΔR708–814–CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R784–814–mediated autoinhibition that prevents IBI shortening by R815–835. This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.
Peripheral nervous system abnormalities, including neuropathy, have been reported in people with cystic fibrosis. These abnormalities have largely been attributed to secondary manifestations of the ...disease. We tested the hypothesis that disruption of the cystic fibrosis transmembrane conductance regulator (CFTR) gene directly influences nervous system function by studying newborn CFTR ⁻/⁻ pigs. We discovered CFTR expression and activity in Schwann cells, and loss of CFTR caused ultrastructural myelin sheath abnormalities similar to those in known neuropathies. Consistent with neuropathic changes, we found increased transcripts for myelin protein zero , a gene that, when mutated, can cause axonal and/or demyelinating neuropathy. In addition, axon density was reduced and conduction velocities of the trigeminal and sciatic nerves were decreased. Moreover, in vivo auditory brainstem evoked potentials revealed delayed conduction of the vestibulocochlear nerve. Our data suggest that loss of CFTR directly alters Schwann cell function and that some nervous system defects in people with cystic fibrosis are likely primary.
Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain.
CFTR
−/−
pigs ...spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo.
CFTR
−/−
epithelia showed markedly reduced Cl
- and HCO
3
- transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na
+ or liquid absorption or reduce periciliary liquid depth. Like human CF,
CFTR
−/−
pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl
- conductance caused the change, not increased Na
+ transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl
- and HCO
3
- in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.
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► Airway epithelia in a porcine model of cystic fibrosis lack Cl
- and HCO
3
- transport ► In contrast to a widely held hypothesis, the CF epithelia do not hyperabsorb Na
+ ► Missing Cl
- conductance causes voltage and current alterations seen in CF epithelia
Background
Cystic fibrosis (CF) is a rare condition in Asians. Since 1985, only about 30 Chinese patients have been reported with molecular confirmation.
Method
Using our in‐house next‐generation ...sequencing (NGS) pipeline for childhood bronchiectasis, we identified disease‐causing CFTR mutations in CF patients in Hong Kong. After identifying p.I1023R in multiple patients, haplotype analysis was performed with genome‐wide microarray to ascertain the likelihood of this being a founder mutation. We also assessed the processing and gating activity of the mutant protein by Western hybridization and patch‐clamp test.
Results
Molecular diagnoses were confirmed in four patients, three of whom shared a missense mutation: CFTR:c.3068T>G:p.I1023R. The results suggested that p.I1023R is a founder mutation in southern Han Chinese. In addition, the processing and gating activity of the mutant protein was assessed by gel electrophoresis and a patch‐clamp test. The mutant protein exhibited trafficking defects, suggesting that the dysfunction is caused by reduced cell surface expression of the fully glycosylated proteins.
Conclusion
Together with other previously reported mutations, the specific founder mutation presented herein suggests a unique CFTR mutation spectrum in the southern Chinese populations, and this finding has vital implications for improving molecular testing and mutation‐specific treatments for Chinese patients with CF.
We identified a shared haplotype of CFTR:p.I1023R encompassing 381 SNPs. The estimated physical length of the haplotype was 1,958,052 bp, which was equivalent to a genetic distance of 1.28 cM. We did not identify the specific haplotype in any of the control samples. The haplotype analysis suggested that the mutation is a founder mutation among our population.
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common ...CF-associated mutation is ΔF508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-ΔF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ΔF508 mutation in vivo, we produced CFTR(ΔF508/ΔF508) pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTR(ΔF508/ΔF508) airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3',5'-monophosphate (cAMP) agonists were less potent at stimulating current in CFTR(Δ)(F508/)(Δ)(F508) epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTR(ΔF508/ΔF508) pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.
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