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- CFTR inhibitors (1)
- CFTR mutations (1)
- ENaC (1)
- In silico modelling (1)
- Off-target effects (1)
- Orai1 (1)
- R751L (1)
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The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.
Cysticfibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressiveand life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterized. Our aims were to describe theclinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individu-als harboring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made inprimary human bronchial epithelial cultures (HBEs) andXenopusoocytes. Molecular properties of R751L-CFTR were investigatedin the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotypeassociated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared with non-CF HBEs and asso-ciated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function inXenopusoocytes, together with folding and cell surface transport of R751L-CFTR, was not different from wild-type CFTR. Overall,R751L-CFTR was associated with reduced sodium chloride absorption but had functional properties similar to wild-type CFTR.This is thefirst report of R751L-CFTR that combines clinical phenotype with characterization of functional and biological proper-ties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and, importantly,inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to al-ternative non-CFTR factors, which require further investigation.
