Structural mechanisms of CFTR function and dysfunction

Hwang, Tzyh Chang; Yeh, Jiunn-Tyng; Yu, Ying; Yeh, Han I.; DeStefano, Samantha

doi:10.1085/jgp.201711946

Cited by 105 publications

(109 citation statements)

References 238 publications

(507 reference statements)

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“…That report also echoes the functional importance of CFTR's NBD2 (Hwang et al . ) since the Q1412X mutation with only the last two β‐strands in NBD2 deleted exhibits severe defects in gating. As the W1282 residue is located further from the C‐terminus of CFTR than Q1412, we expected that the truncated proteins of the W1412X mutation should present with gating defects.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the C‐terminus truncated protein out of W1282X is unlikely to respond to ATP (Hwang & Sheppard, ; Hwang et al . ). On the other hand, the tiny fraction of ATP‐sensitive currents observed in W1282X‐CFTR comes from the full‐length read‐through proteins.…”

Section: Discussionmentioning

confidence: 97%

“…, ; Hwang et al . ), CF or CFTR can serve as an ideal model system to investigate the consequences of PTCs at a fundamental level.…”

Section: Introductionmentioning

confidence: 99%

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Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR

Yeh

Hwang

2019

The Journal of Physiology

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Key points Biochemical and biophysical characterizations of three nonsense mutations of cystic fibrosis transmembrane conductance regulator (CFTR) associated with a severe form of cystic fibrosis (CF) reveal the importance and heterogenous effects of the position of the premature termination codon (PTC) on the CFTR protein function. Electrophysiological studies of W1282X‐CFTR, whose PTC is closer to the C‐terminus of CFTR, suggest the presence of both C‐terminus truncated CFTR proteins that are poorly functional and read‐through, full‐length products. For G542X‐ and E60X‐CFTR, the only mechanism capable of generating functional proteins is the read‐through, but the outcome of read‐through products is highly variable depending on the interplay between the missense mutation caused by the read‐through and the structural context of the protein. Pharmacological studies of these three PTCs with various CFTR modulators suggest position‐dependent therapeutic strategies for these disease‐inflicting mutations. Abstract About one‐third of genetic diseases and cancers are caused by the introduction of premature termination codons (PTCs). In theory, the location of the PTC in a gene determines the alternative mechanisms of translation, including premature cessation or reinitiation of translation, and read‐through, resulting in differential effects on protein integrity. In this study, we used CFTR as a model system to investigate the positional effect of the PTC because of its well‐understood structure‐function relationship and pathophysiology. The characterization of three PTC mutations, E60X‐, G542X‐ and W1282X‐CFTR revealed heterogenous effects of these PTCs on CFTR function. The W1282X mutation results in both C‐terminus truncated and read‐through proteins that are partially or fully functional. In contrast, only the read‐through protein is functional with E60X‐ and G542X‐CFTR, although abundant N‐terminus truncated proteins due to reinitiation of translation were detected in E60X‐CFTR. Single‐channel studies of the read‐through proteins of E60X‐ and G542X‐CFTR demonstrated that both mutations have a single‐channel amplitude similar to wild type (WT), and good responses to high‐affinity ATP analogues, suggesting intact ion permeation pathways and nucleotide binding domains (NBDs), albeit with reduced open probability (Po). The comparison of the Po of these mutations with the proposed missense mutations revealed potential identities of the read‐through products. Importantly, a majority of the functional protein studied responds to CFTR modulators like GLPG1837 and Lumacaftor. These results not only expand current understanding of the molecular (patho)physiology of CFTR, but also infer therapeutic strategies for different PTC mutations at large.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

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Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR

Yeh

Hwang

2019

The Journal of Physiology

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“…Either way, the molecular coupling between TMDs and NBDs is not obligatory (Hwang et al . ). In the former case, the pore architecture can change without separation of NBD dimer.…”

Section: Discussionmentioning

confidence: 97%

“…Taking the P‐loop (phosphate‐binding loop, also known as Walker A motif) for example, this is a crucial motif for ATP binding and hydrolysis in CFTR and is well‐conserved across ABC protein superfamily (Wilkens, ; Hwang et al . ). The P‐loop is usually flanked by a beta‐strand and an alpha‐helix, which are formed by amino acids R1239–L1243 and G1249–R1259, respectively (Liu et al .…”

Section: Discussionmentioning

confidence: 97%

Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe Class VI pathogenic mutation in cystic fibrosis

Yeh

Hwang

2018

The Journal of Physiology

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Key points Electrophysiological characterization of Q1412X‐CFTR, a C‐terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of cystic fibrosis (CF), reveals a gating defect that has not been reported previously. Mechanistic investigations of the gating deficit in Q1412X‐CFTR suggest that the reduced open probability in Q1412X‐CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs). Detailed comparisons of several mutations with different degrees of truncation in the C‐terminal region of NBD2 reveal the importance of the last two beta‐strands in NBD2 for maintaining proper gating functions. The results of the present study also show that the application of clinically‐approved drugs (VX‐770 and VX‐809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations. Abstract Cystic fibrosis (CF) is caused by loss‐of‐function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation‐activated but ATP‐gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease‐associated mutations are categorized into six classes. Among them, Class VI, whose members include some of the C‐terminal truncation mutations such as Q1412X, is defined as decreased membrane expression because of a faster turnover rate. In the present study, we characterized the functional properties of Q1412X‐CFTR, a severe‐form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (Po). Detailed kinetic studies support the idea that the gating defect is the result of a dysfunctional ATP‐binding site 2 in the nucleotide binding domains (NBDs). Because the Q1412X mutation results in a deletion of the last two beta‐strands in NBD2 and the whole C‐terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C‐terminus of CFTR at the same time as keeping an intact NBD2 (i.e. D1425X and S1455X) assume gating function almost identical to that of wild‐type channels. However, the deletion of the last beta‐strand in the NBD2 (i.e. N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that clinically‐approved VX‐809 (Lumacaftor, Vertex Pharmaceuticals, Boston, MA, USA) and VX‐770 (Ivacaftor, Vertex Pharmaceuticals, Boston, MA, USA) significantly enhance the overall function of Q1412X‐CFTR provides the conceptual basis for the treatment of patients carrying this mutation.

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Strategies for cystic fibrosis transmembrane conductance regulator inhibition: from molecular mechanisms to treatment for secretory diarrhoeas

et al. 2020

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Cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter. It acts as an anion‐selective channel that drives osmotic fluid transport across many epithelia. In the gut, CFTR is crucial for maintaining fluid and acid‐base homeostasis, and its activity is tightly controlled by multiple neuro‐endocrine factors. However, microbial toxins can disrupt this intricate control mechanism and trigger protracted activation of CFTR. This results in the massive faecal water loss, metabolic acidosis and dehydration that characterize secretory diarrhoeas, a major cause of malnutrition and death of children under 5 years of age. Compounds that inhibit CFTR could improve emergency treatment of diarrhoeal disease. Drawing on recent structural and functional insight, we discuss how existing CFTR inhibitors function at the molecular and cellular level. We compare their mechanisms of action to those of inhibitors of related ABC transporters, revealing some unexpected features of drug action on CFTR. Although challenges remain, especially relating to the practical effectiveness of currently available CFTR inhibitors, we discuss how recent technological advances might help develop therapies to better address this important global health need.

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Structural mechanisms of CFTR function and dysfunction

Abstract: Hwang et al. integrate new structural insights with prior functional studies to reveal the functional anatomy of CFTR chloride channels.

Cited by 105 publications

References 238 publications

Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR

Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR

Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe Class VI pathogenic mutation in cystic fibrosis

Strategies for cystic fibrosis transmembrane conductance regulator inhibition: from molecular mechanisms to treatment for secretory diarrhoeas

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