cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of <200 amino acids). Thus it is not surprising that CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca 2+ -mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through G αq can stimulate CFTR channels by activating Ca 2+ -dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations.

BACKGROUND AND PURPOSEThe most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a 'potentiator' to increase channel open probability and a 'corrector' that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood. EXPERIMENTAL APPROACHCF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (I sc ) across airway epithelial monolayers expressing F508del CFTR. KEY RESULTSThe potentiated I sc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached 11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTR inh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40 mg·mL À1 ). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated I sc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator. CONCLUSIONS AND IMPLICATIONSChronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770. Abbreviations

Bicarbonate facilitates mucin unpacking and bacterial killing however its transport mechanisms in the airways are not well understood. cAMP stimulates anion efflux through the CFTR (ABCC7) anion channel and this is defective in CF. The anion exchanger pendrin (SLC26A4) also mediates HCO3− efflux and is upregulated by proinflammatory cytokines. Here we examined pendrin and CFTR expression and their contributions to HCO3− secretion by human nasal and bronchial epithelia. In native tissue, both proteins were most abundant at the apical pole of ciliated surface cells with little expression in submucosal glands. In well‐differentiated primary nasal and bronchial cell cultures, IL‐4 dramatically increased pendrin mRNA levels and apical immunostaining. Exposure to low‐Cl− apical solution caused intracellular alkalinization (ΔpHi) that was enhanced 4‐fold by IL‐4 pretreatment. ΔpHi was unaffected by DIDS or CFTRinh‐172 but was reduced by adenoviral shRNA targeting pendrin. Forskolin increased ΔpHi and this stimulation was prevented by CFTRinh‐172 implicating CFTR, yet forskolin only increased ΔpHi after pendrin expression had been induced by IL‐4. The dependence of ΔpHi on pendrin suggests there is minimal electrical coupling between Cl− and HCO3− fluxes and that CFTR activation increases anion exchange‐mediated HCO3− influx. Conversely, inducing pendrin expression increased forskolin‐stimulated, CFTRinh‐172‐sensitive current by ~2‐fold in epithelial and non‐epithelial cells. We conclude that pendrin mediates most HCO3− secretion across airway surface epithelium during inflammation and enhances electrogenic Cl− secretion via CFTR as described for other SLC26A transporters. Support or Funding Information Support: Cystic Fibrosis Canada, Canadian Institutes of Health Research, Canada Foundation for Innovation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background:The canonical regulation of CFTR is mediated by serine/threonine phosphorylation. Results: GPCR stimulation of CFTR mutants lacking PKA/PKC sites revealed alternate pathways which involve tyrosine kinases. Conclusion: Pyk2 and Src family tyrosine kinases mediate part of CFTR stimulation by the M3 muscarinic receptor. Significance: This provides the first evidence that physiological secretagogues can activate CFTR through tyrosine phosphorylation.

Trikafta, currently the leading therapeutic in Cystic Fibrosis (CF), has demonstrated a real clinical benefit. This treatment is the triple combination therapy of two folding correctors elexacaftor/tezacaftor (VX445/VX661) plus the gating potentiator ivacaftor (VX770). In this study, our aim was to compare the properties of F508del-CFTR in cells treated with either lumacaftor (VX809), tezacaftor, elexacaftor, elexacaftor/tezacaftor with or without ivacaftor. We studied F508del-CFTR function, maturation and membrane localisation by Ussing chamber and whole-cell patch clamp recordings, Western blot and immunolocalization experiments. With human primary airway epithelial cells and the cell lines CFBE and BHK expressing F508del, we found that, whereas the combination elexacaftor/tezacaftor/ivacaftor was efficient in rescuing F508del-CFTR abnormal maturation, apical membrane location and function, the presence of ivacaftor limits these effects. The basal F508del-CFTR short-circuit current was significantly increased by elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor compared to other correctors and non-treated cells, an effect dependent on ivacaftor and cAMP. These results suggest that the level of the basal F508del-CFTR current might be a marker for correction efficacy in CF cells. When cells were treated with ivacaftor combined to any correctors, the F508del-CFTR current was unresponsive to the subsequently acute addition of ivacaftor unlike the CFTR potentiators genistein and Cact-A1 which increased elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor-corrected F508del-CFTR currents. These findings show that ivacaftor reduces the correction efficacy of Trikafta. Thus, combining elexacaftor/tezacaftor with a different potentiator might improve the therapeutic efficacy for treating CF patients.

Cystic fibrosis (CF), a genetic disease caused by mutations in the CFTR gene, is a life-limiting disease characterized by chronic bacterial airway infection and severe inflammation. Some CFTR mutants have reduced responsiveness to cAMP/PKA signaling; hence, pharmacological agents that elevate intracellular cAMP are potentially useful for the treatment of CF. By inhibiting cAMP breakdown, phosphodiesterase (PDE) inhibitors stimulate CFTR in vitro and in vivo. Here, we demonstrate that PDE inhibition by RPL554, a drug that has been shown to cause bronchodilation in asthma and chronic obstructive pulmonary disease (COPD) patients, stimulates CFTR-dependent ion secretion across bronchial epithelial cells isolated from patients carrying the R117H/F508del CF genotype. RPL554-induced CFTR activity was further increased by the potentiator VX-770, suggesting an additional benefit by the drug combination. RPL554 also increased cilia beat frequency in primary human bronchial epithelial cells. The results indicate RPL554 may increase mucociliary clearance through stimulation of CFTR and increasing ciliary beat frequency and thus could provide a novel therapeutic option for CF.

These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl(-) permeation pathway of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl؊ channel physiologically important in fluidtransporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest ␤ strands (␤ c 5 and ␤ c 6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the ␤ c 5-␤ c 6 hairpin, within the ␤ c 5 strand (H620Q), within the ␤-turn linking the two ␤ strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the ␤ c 6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.Mapping ligand binding sites within ionic channel structures is important to understand the mechanism of action of channel modulators (i.e. activators and inhibitors), to identify regions required for selective interaction with modulators, and to design more selective and more potent agents. Such mapping, called pharmaco-topology, has been performed for the sulfonylurea receptor (SUR) regulatory subunits of K ATP channels (reviewed in Ref.