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

doi:10.1074/jbc.273.34.21759

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

From the Departments of ^tOaPhysiology and ^tOhBiology, Dartmouth Medical School, Hanover, New Hampshire 03755, the ^tOgDepartment of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, and the ^tOdDepartment of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294

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.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants DK-45881 and DK-51067. Flow cytometry and confocal microscopy performed at the Dartmouth Medical School, in the Herbert C. Englert Cell Analysis Laboratory, were supported in part by Core Grant CA 23108 of the Norris Cotton Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵FNb Supported by a pre-doctoral fellowship from the Dolores Zohrab Liebmann Foundation.
↵FNc Supported by a post-doctoral fellowship from the Swiss National Science Foundation.
↵FNe Supported by a New Investigator grant from the Cystic Fibrosis Foundation.
↵FNf Supported by Grant CFF Ismail9710 from the Cystic Fibrosis Foundation.
↵i To whom correspondence should be addressed: Dept. of Physiology, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650-1775; Fax: 603-650-1130; E-mail:Bruce.A.Stanton{at}Dartmouth.edu.
Abbreviations:

CFTR

cystic fibrosis transmembrane conductance regulator

GFP

green fluorescent protein

MDCK

Madin-Darby canine kidney

I_sc

short circuit current

Endo H

endoglycosidase H

PNGase F

peptideN-glycosidase F

CPT-cAMP

8-(4-chlorophenylthio)-cAMP

DIDS

4,4′-diisothiocyanostilbene-2,2′-disulfonic acid

DPC

diphenylamine carboxylic acid

ER

endoplasmic reticulum

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

BSA

bovine serum albumin

MOPS

4-morpholinepropanesulfonic acid.
- Received April 21, 1998.
- Revision received May 27, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.