Increases in cytosolic Ca2+ induce dynamin- and calcineurin-dependent internalisation of CFTR

Waseema Patel,Waseema Patel,Waseema Patel,Michael A Gray,Robert Tarran,Andrei A Aleksandrov,Margarida D Amaral,Robert Tarran,Patrick J Moore,Miquéias Lopes-Pacheco,M Flori Sassano

doi:10.1007/s00018-018-2989-3

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated, apical anion channel that regulates ion and fluid transport in many epithelia including the airways. We have previously shown that cigarette smoke (CS) exposure to airway epithelia causes a reduction in plasma membrane CFTR expression which correlated with a decrease in airway surface hydration. The effect of CS on CFTR was dependent on an increase in cytosolic Ca2+. However, the underlying mechanism for this Ca2+-dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca2+ on CFTR function. We show that an increase in cytosolic Ca2+ induced a time-dependent reduction in whole cell CFTR conductance, which was paralleled by a loss of cell surface CFTR expression, as measured by confocal and widefield fluorescence microscopy. The decrease in CFTR conductance and cell surface expression were both dynamin-dependent. Single channel reconstitution studies showed that raising cytosolic Ca2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca2+-dependent phosphatase, suggesting that dephosphorylation of CFTR was linked to the loss of surface expression. In support of this, the calcineurin inhibitor, cyclosporin A, prevented the Ca2+-induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism.

Highlights

CFTR is a cAMP- and ATP-regulated anion channel whose function is critical for transepithelial anion and fluid secretion in multiple organs, including the conducting airways, intestines, reproductive tracts, and kidneys [1, 2]
We have previously demonstrated that acute exposure of fully differentiated primary airway epithelial cultures to cigarette smoke (CS) caused a time-dependent loss of CFTR from the plasma membrane, which correlated with a reduction in airway surface liquid (ASL) volume [4, 22]
To investigate the effect of raising cytosolic C a2+ on CFTR channel activity, we first studied the effect of altering cytosolic C a2+ on whole cell CFTR C l− currents in CFTR-transfected HEK293T cells

Summary

Introduction

CFTR is a cAMP- and ATP-regulated anion channel whose function is critical for transepithelial anion and fluid secretion in multiple organs, including the conducting airways, intestines, reproductive tracts, and kidneys [1, 2]. The importance of the channel is underscored by the hereditary disease cystic fibrosis (CF), where loss of anion permeability through CFTR has major detrimental effects in the lungs, gastrointestinal and reproductive tracts. The deterioration of lung function is a major determinant of morbidity in CF [1]. Others, have recently linked the loss of CFTR function to the development and progression of the chronic bronchitis (CB) form of chronic obstructive pulmonary disease (COPD) [3, 4]. CB bears many phenotypic similarities to CF, such as increased mucus stasis and airway inflammation [5]

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Results

Conclusion