Abstract
Chronic obstructive pulmonary disease (COPD), which is most commonly caused by cigarette smoke (CS) exposure, is the third leading cause of death worldwide. The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane anion channel that is widely expressed in epithelia throughout the body. In the airways, CFTR plays an important role in fluid homeostasis and helps flush mucus and inhaled pathogens/toxicants out of the lung. Inhibition of CFTR leads to mucus stasis and severe airway disease. CS exposure also inhibits CFTR, leading to the decreased anion secretion/hydration seen in COPD patients. However, the underlying mechanism is poorly understood. Here, we report that CS causes CFTR to be internalized in a clathrin/dynamin-dependent fashion. This internalization is followed by retrograde trafficking of CFTR to the endoplasmic reticulum. Although this internalization pathway has been described for bacterial toxins and cargo machinery, it has never been reported for mammalian ion channels. Furthermore, the rapid internalization of CFTR is dependent on CFTR dephosphorylation by calcineurin, a protein phosphatase that is upregulated by CS. These results provide new insights into the mechanism of CFTR internalization, and may help in the development of new therapies for CFTR correction and lung rehydration in patients with debilitating airway diseases such as COPD.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated anion channel, which resides primarily in the apical membrane of glandular and surface airway epithelia
In HEK293T cells, air exposure did not change the subcellular localization of GFP-CFTR, whereas exposure to 13 puffs of freshly generated cigarette smoke (CS) decreased CFTR membrane fluorescence intensity, with a half-life of 10.2 min and a τ of 14.7 and intracellular CFTR appeared with similar kinetics after CS exposure (Fig. 1A,B)
We observed that CFTR was cleared from the plasma membrane following CS exposure with a half-life of ~10 min (Fig. 1A,B)
Summary
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated anion channel, which resides primarily in the apical membrane of glandular and surface airway epithelia. Deletion of F508, the most common CF mutation, causes CFTR misfolding and impaired trafficking to the plasma membrane This in turn results in diminished anion secretion, reduced mucociliary clearance and chronic airways infection and inflammation[6]. After formation in the ER, plasma membrane proteins usually pass through the Golgi apparatus, are trafficked to the plasma membrane and are internalized via endosomes They are either degraded in lysosomes or proteasomes or recycled back to the plasma membrane[7]. We have previously observed that CS causes plasma membrane CFTR channels to be rapidly internalized in multiple cell types[16,20] In airway epithelia, this leads to a CF-like decrease in anion secretion that contributes to dehydration of airway surface liquid. We have sought to determine how CS clears CFTR from the plasma membrane and to identify CFTR’s terminal intracellular location
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