Regulated recycling of mutant CFTR partially restored by pharmacological treatment

Abstract

SummaryEfficient trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) to and from the cell surface is essential for maintaining channel density at the plasma membrane (PM) and ensuring proper physiological activity. The most common mutation, F508del, exhibits reduced surface expression and impaired function despite treatment with currently available pharmacological small molecules, called correctors. To gain more detailed insight into whether CFTR enters compartments that allow corrector stabilization in the cell periphery, we investigated the peripheral trafficking itineraries and kinetics of wild type (WT) and F508del in living cells using high-speed fluorescence microscopy together with fluorogen activating protein detection. We directly visualized internalization and accumulation of CFTR WT from the PM to a perinuclear compartment that colocalized with the endosomal recycling compartment (ERC) markers Rab11 and EHD1, reaching steady-state distribution by 25 minutes. Stimulation by protein kinase A (PKA) depleted this intracellular pool and redistributed CFTR channels to the cell surface, elicited by reduced endocytosis and active translocation to the PM. Corrector or temperature rescue of F508del also resulted in targeting to the ERC and exhibited subsequent PKA-stimulated trafficking to the PM. Corrector treatment (24 hours) led to persistent residence of F508del in the ERC, while thermally destabilized F508del was targeted to lysosomal compartments by 3 hours. Acute addition of individual correctors, C4 or C18, acted on peripheral trafficking steps to partially block lysosomal targeting of thermally destabilized F508del. Taken together, corrector treatment redirects F508del trafficking from a degradative pathway to a regulated recycling route, and proteins that mediate this process become potential targets for improving the efficacy of current and future correctors.

Rescuing Trafficking Mutants of the ATP-binding Cassette Protein, ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease

“…Note that VX-809 increased the steady-state protein levels of both the wt and mutant ABCA4. However, even though C18 and VX-809 are similar classes of compounds (16), the effect of C18 on the mutants was greater than that of VX-809.…”

Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

“…Interestingly, ENaC and CFTR that are both regulated by Nedd4-2 also seem to be regulated by such a PKA-mediated dual response. PKA activation leads to increased surface expression of both ENaC and CFTR (12,31). In addition, the open probability of ENaC and the activity of CFTR are increased in response to activation of PKA (9,34).…”

Rab GTPases regulate the trafficking of channels and transporters – a focus on cystic fibrosis

“…21 Recent data have also reported that the Rab11 machinery is involved in the recycling of corrector-rescued F508del-CFTR, confirming that chemical correction redirects F508del trafficking from the degradation pathway to regulated recycling -with the proteins that are involved in the process being obvious targets for the improvement of the available correctors. 22 Interestingly, this study reports an improvement in mutant CFTR stability due to decreased lysosomal accumulation/targeting when correctors are added acutely, suggesting that i) the compounds may also act on peripheral quality control and ii) that temperature/corrector-promoted F508del-CFTR escape from the ER does not correspond to its full correction in terms of misfolding. 22 Rab4 was found initially to have only a minor role in CFTR direct recycling to the PM from early endosomes in BHK cells.…”

“…22 Interestingly, this study reports an improvement in mutant CFTR stability due to decreased lysosomal accumulation/targeting when correctors are added acutely, suggesting that i) the compounds may also act on peripheral quality control and ii) that temperature/corrector-promoted F508del-CFTR escape from the ER does not correspond to its full correction in terms of misfolding. 22 Rab4 was found initially to have only a minor role in CFTR direct recycling to the PM from early endosomes in BHK cells. 13 Later studies reported that overexpression of Rab4 inhibits CFTR-mediated current in the colonic epithelial cell line HT29 and that, reversely, Rab4 inhibition (by overexpression of an anti-Rab4 antibody) reverses the observed inhibition of CFTR.…”

“…48 The pharmacological or temperature rescue of F508del-CFTR has been shown to result in its targeting to the endosomal recycling compartment (ERC), colocalizing with Rab11 and EHD1 ERC markers. 22 Moreover, corrector-rescued F508del-CFTR in the ERC exhibited subsequent PKA-stimulated trafficking to the PM, indicating that corrector treatment redirects F508del trafficking from a degradative pathway to a regulated recycling route. This suggested that the proteins that mediate this process, namely Rab11, could become potential targets for improving the efficacy of current and future CFTR modulators.…”