Targeting PI3Kγ anchoring enhances CFTR membrane localization and modulator efficacy via PKD1
Alessandra Murabito, Marco Mergiotti, Valeria Capurro, Alessia Loffreda, Mingchuan Li, Paola Peretto, Kai Ren, Andrea Raimondi, Carlo Tacchetti, Dario Diviani, Nicoletta Pedemonte, Emilio Hirsch, Alessandra Ghigo
Alessandra Murabito, Marco Mergiotti, Valeria Capurro, Alessia Loffreda, Mingchuan Li, Paola Peretto, Kai Ren, Andrea Raimondi, Carlo Tacchetti, Dario Diviani, Nicoletta Pedemonte, Emilio Hirsch, Alessandra Ghigo
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Research Article Cell biology Pulmonology

Targeting PI3Kγ anchoring enhances CFTR membrane localization and modulator efficacy via PKD1

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride channel, cause cystic fibrosis (CF), the most common life-threatening inherited disorder among White individuals. Current CFTR correctors and potentiators, such as elexacaftor-tezacaftor-ivacaftor (ETI), only partially restore the function of the most prevalent mutant, F508del-CFTR, resulting in residual disease in people with CF. Here, we demonstrate that a mimetic peptide targeting the A-kinase–anchoring protein (AKAP) function of PI3Kγ (PI3Kγ MP), and driving localized cAMP elevation, enhances F508del-CFTR membrane localization, maximizing ETI efficacy in restoring chloride secretion. Mechanistically, PI3Kγ MP activates an AKAP-Lbc–anchored pool of PKD1, a known regulator of membrane trafficking. Consistently, PKD1 inhibition prevents PI3Kγ MP from enhancing the membrane expression of ETI-corrected F508del-CFTR. Overall, our findings reveal a regulatory pathway controlling CFTR membrane abundance via the AKAP function of PI3Kγ, which can be targeted to overcome the limitations of current CFTR modulator therapies.

Authors

Alessandra Murabito, Marco Mergiotti, Valeria Capurro, Alessia Loffreda, Mingchuan Li, Paola Peretto, Kai Ren, Andrea Raimondi, Carlo Tacchetti, Dario Diviani, Nicoletta Pedemonte, Emilio Hirsch, Alessandra Ghigo

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Figure 1

PI3Kγ MP increases the plasma membrane density of F508del-CFTR in combination with ETI.

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PI3Kγ MP increases the plasma membrane density of F508del-CFTR in combin...
(A) Representative immunogold electron microscopy images showing the distribution of overexpressed F508del-CFTR-GFP in HEK293T cells. Cells were treated for 24 hours with DMSO (Veh), ETI (3 μM VX-445, 10 μM VX-661, and 1 μM VX-770) plus control peptide (ETI + 25 μM CP), or ETI plus PI3Kγ MP (ETI + 25 μM PI3Kγ MP). HEK293T cells expressing WT CFTR-GFP served as positive controls. Arrowheads: CFTR channels located at the plasma membrane. Insets: higher magnification of selected regions. Scale bar: 1 μm. (B) Quantification of plasma membrane gold density as shown in A; 99–118 cells from 19–20 images were quantified. ****P < 0.0001 by Kruskal-Wallis test with Dunn’s multiple-comparison test. (C) Representative Western blot of CFTR and GAPDH in plasma membrane fractions and total lysates (TLs) of F508del-CFBE41o- cells treated for 24 hours with ETI alone or ETI + PI3Kγ MP (25 μM). GAPDH was absent in plasma membrane fractions and used as a loading control in TLs. (D) Quantification of plasma membrane CFTR band C intensity as shown in C, expressed as fold-change relative to ETI alone. n = 4–6. *P < 0.05, ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple-comparison test. (E) Representative Western blot of CFTR and GAPDH levels in F508del-CFBE41o- cells treated for 24 hours with ETI alone or ETI + PI3Kγ MP (25 μM) and cycloheximide (CHX, 1 μg/mL) exposure. Cells were lysed at the indicated times after CHX treatment. (F) Quantification of CFTR band C as shown in E, expressed as fold-change relative to t = 0. n = 3. ##P < 0.01, ###P < 0.001 versus t = 0 by 1-way ANOVA with Bonferroni’s post hoc test; *P < 0.05 for ETI versus ETI + PI3Kγ MP by Welch’s t test. Data shown as mean ± SEM. n: number of independent experiments. Data points represent independent biological replicates.