The role of intracellular Ca ² ⁺ in cigarette smoke-induced CFTR internalisation

Abstract

Chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide, is characterised by airflow obstruction and is primarily caused by smoking. In contrast, another obstructive pulmonary disease, cystic fibrosis (CF), has orphan disease status. However, patients with either COPD or CF present with similar clinical lung problems. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activity is reduced in both diseases. Recent work from our lab showed that cigarette smoke-induced increases in cytosolic Ca2+ were consequential in reducing plasma membrane expression of CFTR by an unknown mechanism. Therefore, the major aim of my project was to identify the molecular mechanism underlying the loss of CFTR activity brought about by an increase in cytosolic Ca2+. Whole cell patch clamp recordings in HEK 293T cells transiently transfected with CFTR showed that an increase in cytosolic Ca2+ significantly reduced CFTR-mediated conductance. Characterisation of the dynamic changes in cytosolic Ca2+, induced by a range of agonists, showed that a sustained increase in Ca2+ was not essential for the loss of CFTR-mediated conductance, but it did involve a dynamin-dependent internalisation of the channel. Confocal imaging further confirmed that an increase in cytosolic Ca2+ caused a reduction in plasma membrane CFTR expression, and a reciprocal increase in intracellular CFTR. Activation of the MEK/ERK pathway has previously been linked to smoke-induced internalisation of CFTR. Similarly, inhibition of the pathway prevented a Ca2+-induced internalisation of CFTR, indicating this pathway also plays a role in Ca2+-induced CFTR internalisation. Importantly, inhibition of the Ca2+ dependent phosphatase calcineurin with cyclosporin A prevented both Ca2+ as well as smoke-induced loss of CFTR, suggesting that the mechanism of internalisation is linked to dephosphorylation, possibly of CFTR itself. Furthermore, either an increase in Ca2+, or exposure to cigarette smoke, increased calcineurin activity, further implicating this phosphatase as a key effector. Functionally, inhibition of calcineurin prevented against a smoke-induced reduction in ASL height whilst having no effect on physiological changes in height induced by G protein-coupled receptor agonists; signifying calcineurin only gets activated under conditions of stress. These findings highlight a role for cytosolic Ca2+ in modulating CFTR activity. Additionally, these data may lead to novel therapeutic strategies aimed at correcting ASL hydration in smokers as well as in people with CF.