Select Amino Acids Increase Anion Secretion in Human Bronchial Epithelial Cells (HBEC) with F508del and Nonsense Mutations: Alternative Approach to Restoring Airway Anion Secretion in Cystic Fibrosis

Abstract

ObjectiveChloride and bicarbonate transport across the apical membrane via CFTR is critical for homeostasis of airway surface liquid and mucociliary clearance. Cystic fibrosis (CF) is caused by mutations in the CFTR gene resulting in mucus buildup, bacterial colonization, inflammation and respiratory insufficiency. F508del and nonsense mutations are the most common mutations characterized by defective protein processing and trafficking (F508del) or early termination of CFTR synthesis (G542X, R1162X). A combination of CFTR modulators (VX445/661/770; VX) has largely improved CFTR function in patients with F508del but is ineffective in nonsense mutations. We have shown that select amino acids (SAA) can modulate trafficking and activity of CFTR and alternative anion‐secretory pathways, and thus could improve clinical outcomes in CF patients.HypothesisSAA can stimulate anion secretion in primary HBEC with F508del/F508del, F508del/R1162X and G542X/G542X mutation.MethodsTransepithelial short‐circuit current (Isc) was measured in differentiated HBEC treated with DMSO or VX for 24h and bathed in vehicle or SAA in Ussing chambers. After blocking ENaC with benzamil, benzamil‐insensitive (anion) Isc was either continuously recorded for 90min, or sequentially blocked with CFTRinh172, CaCCinhA01 and bumetanide in 15min intervals. Isotope (36Cl) flux studies and whole‐cell patch clamp technique were used to confirm chloride movements, and CFTR and alternative anion‐secretory channel expression was analyzed by immunofluorescence.ResultsAnion Isc increased in all mutations and was more sustained when exposed to SAA compared to vehicle (F508del: 15.4±0.5 vs 1.4±0.2; R1162X: 19.4±0.3 vs 2.3±0.3; G542X: 17.2±0.5 vs 1.6±0.2 µA.cm‐2; P<0.001). SAA recovered 48.7, 57.3 and 40.9% of normal Isc (21.9±0.5 µA.cm‐2). Treatment with VX increased anion Isc in F508del and R1162X (11.4±0.7 and 5.7±0.4 µA.cm‐2; P<0.001) but not in G542X (1.7±0.2 µA.cm‐2). In F508del, the CFTR response increased from 20.7% (non‐treated cells) to 32.9 and 85.5% of the anion Isc when treated with SAA or VX, and in R542X from 3.7% to 46.3 and 16.0%, respectively. Both, SAA and VX did not affect CaCC, but SAA increased the bumetanide‐sensitive Isc from 31.0 to 50.5% in F508del, and from 33.3 to 56.3% in R1162X, while VX reduced the bumetanide response to 5.4 and 4.5%. Isotope flux studies showed that SAA (‐0.21±0.08 μEq.h‐1.cm‐2) and VX (‐0.19±0.02 μEq.h‐1.cm‐2) increased net Cl‐ secretion in F508del when compared to vehicle (‐0.03±0.01 μEq.h‐1.cm‐2; P<0.05). SAA, but not VX increased Cl‐ secretion in G542X (‐0.21 0.07 vs ‐0.08±0.02 μEq.h‐1.cm‐2). In patch clamp recordings, SAA and VX resulted in a significant reduction in CFTRinh172 whole cell current in F508del. Immunofluorescence imaging demonstrated increased CFTR and alternative anion‐secretory channel expression along the apical membrane in SAA cells while VX showed increased expression of only CFTR.ConclusionsThese studies demonstrate that SAA stimulates anion current and increases Cl‐ secretion via CFTR and alternative secretory pathways in different mutations, and could be beneficial in nonsense mutations but needs further investigation.