Am J Physiol Cell Physiol AJP: Lung Cellular and Molecular Physiology
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Am J Physiol Cell Physiol 274: C310-C318, 1998;
0363-6143/98 $5.00
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Vol. 274, Issue 2, C310-C318, February 1998

Hyperexpression of recombinant CFTR in heterologous cells alters its physiological properties

Raha Mohammad-Panah, Sophie Demolombe, David Riochet, Veronique Leblais, Gildas Loussouarn, Helene Pollard, Isabelle Baró, and Denis Escande

Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, Institut National de la Santé et de la Recherche Médicale CJF96-01, Hôpital Hotel-Dieu, 44093 Nantes, France

We investigated whether high levels of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) would alter the functional properties of newly synthesized recombinant proteins. COS-7, CFPAC-1, and A549 cells were intranuclearly injected with a Simian virus 40-driven pECE-CFTR plasmid and assayed for halide permeability using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe. With increasing numbers of microinjected pECE-CFTR copies, the baseline permeability to halide dose dependently increased, and the response to adenosine 3',5'-cyclic monophosphate (cAMP) stimulation decreased. In cells hyperexpressing CFTR, the high level of halide permeability was reduced when a cell metabolism poisoning cocktail was applied to decrease intracellular ATP and, inversely, was increased by orthovanadate. In CFPAC-1 cells investigated with the patch-clamp technique, CFTR hyperexpression led to a time-independent nonrectifying chloride current that was not sensitive to cAMP stimulation. CFPAC-1 cells hyperexpressing CFTR exhibited no outward rectifying chloride current nor inward rectifying potassium current either spontaneously or under cAMP stimulation. We conclude that hyperexpression of recombinant CFTR proteins modifies their properties inasmuch as 1) CFTR channels are permanently activated and not susceptible to cAMP regulation and 2) they lose their capacity to regulate heterologous ionic channels.

cystic fibrosis transmembrane conductance regulator; cystic fibrosis; expression; patch clamp; CFPAC-1 cells; A549 cells; epithelial cells


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