Abstract
The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl-) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl- secretion by stimulating CFTR Cl- channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl- secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl- secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.
Highlights
The cystic fibrosis transmembrane conductance regulator b Supported by a pre-doctoral fellowship from the Dolores Zohrab Liebmann Foundation
By using quantitative confocal fluorescence microscopy, cellsurface biotinylation, and short circuit current (Isc) analyses, we demonstrate that the predominant mechanism by which cAMP stimulates green fluorescent protein (GFP)-CFTR-mediated ClϪ secretion is by activating channels resident in the apical membrane and not by stimulating insertion of channels into the apical membrane or inhibiting retrieval of channels from the apical membrane
GFP-CFTR Functions as a cAMP-activated ClϪ Channel— Because CFTR ClϪ channel function is necessary for normal CFTR trafficking [8], we performed experiments to examine whether fusion of GFP to the N terminus of CFTR affected function as a cAMP-activated ClϪ channel
Summary
B Supported by a pre-doctoral fellowship from the Dolores Zohrab Liebmann Foundation. (CFTR), a cAMP-activated chloride (ClϪ) channel, is targeted to the apical plasma membrane region in many epithelial cells, including those in the kidney [1,2,3], and is defective in the genetic disease cystic fibrosis [4]. The second mechanism is more controversial; in intestinal epithelial cells, some investigators have found positive effects of cAMP on CFTR trafficking to the apical membrane [12], whereas other investigators have not [2, 13]. To begin to understand the trafficking of CFTR, we constructed a jellyfish green fluorescent protein (GFP)-CFTR expression vector in which GFP was ligated to the N terminus of wild-type CFTR, and we used GFP fluorescence to localize CFTR in living and fixed cells. GFP generates a bright green fluorescence, is resistant to photobleaching, does not require any exogenous cofactors or substrates to fluoresce, and, when ligated to other proteins, generally does not alter fusion protein function or localization [18, 20]
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