Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

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 patchclamp 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.

Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression

“…Whole Cell Patch Clamp Assays-Individual dishes of transfected COS-7 cells were used in electrophysiological recordings as described previously (15). One modification is that PClamp 8.0 software was used in this study.…”

Cortical Actin Organization: Lessons from ERM (Ezrin/Radixin/Moesin) Proteins

“…Interestingly, because NHE-RF regulates the NHE3 function in a protein kinase A (PKA)-dependent manner and because ezrin specifically binds to the RII subunit of PKA (38), ezrin appears to play an important role in recruiting PKA to the NHE3⅐NHE-RF complex to regulate the function of NHE3. Furthermore, NHE-RF and E3KARP were found to also be associated with other integral membrane proteins such as the ␤ 2 -adrenergic receptor and the cystic fibrosis transmembrane conductance regulator (39,40), which are not always co-localized with ERM proteins (41). The physiological relevance of the existence of two mechanisms of binding of ERM proteins to integral membrane proteins, direct and indirect, is an interesting subject for future study.…”

A Golgi-associated PDZ Domain Protein Modulates Cystic Fibrosis Transmembrane Regulator Plasma Membrane Expression

“…Immunoprecipitation and Immunoblotting-Cells were harvested and processed as described previously (23). Briefly, cells were solubilized and lysates were spun at 14,000 ϫ g for 15 min to pellet insoluble material.…”

“…Surface Biotinylation-Biotinylated CFTR at the plasma membrane was precipitated as described in detail previously with some modifications (23). Lysates were incubated with immobilized NeutrAvidin beads (Pierce, catalog number 53151), and bound proteins were eluted with 2ϫ Laemmli sample buffer supplemented with 100 M DTT at 42°C for 30 min.…”

“…Subsequently, cells were washed extensively with DMEM containing 10 mM of non-labeled methionine and 10 mM of unlabeled cysteine and chased in this solution for 0, 30, 60, 90, or 120 min. Biotinylated CFTR at the plasma membrane was immunoprecipitated as described previously (23). Radiolabeled CFTR was analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).…”