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 Ca 2+ . However, the underlying mechanism for this Ca 2+ -dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca 2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca 2+ on CFTR function. We show that an increase in cytosolic Ca 2+ 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 Ca 2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca 2+ -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 Ca 2+ -induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca 2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism. Electronic supplementary material The online version of this article (10.1007/s00018-018-2989-3) contains supplementary material, which is available to authorized users.
Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis. To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen. Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet. The association of Hrs and SNAP-25 is mediated via coiled-coil interactions. Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions. Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools. Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells. Moreover, overexpression of Hrs in PC12 cells inhibits Ca(2+)-dependent exocytosis. These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.
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