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Molecular & Cellular Proteomics 4:1762-1775, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Blue Native/SDS-PAGE Analysis Reveals Reduced Expression of the mClCA3 Protein in Cystic Fibrosis Knock-out Mice^*

Franck Brouillard^,, Noura Bensalem^,¶, Alexandre Hinzpeter^,¶, Danielle Tondelier, Stéphanie Trudel, Achim D. Gruber^||,**, Mario Ollero and Aleksander Edelman^,,

From the INSERM U467, 156 rue de Vaugirard, Paris F-75015 and Université Paris-Descartes, Faculté de Médecine, 15 rue Ecole de Médecine, Paris F-75005, France, Proteomic Core Facilities of Institut Féderatif de Recherche 94, Faculté de Médecine, Université Paris-Descartes, 156 rue de Vaugirard Paris, F-75015 France, and ^|| Department of Pathology, School of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr^–/–). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr^–/– mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr^–/– mice. In an ex vivo assay, we found that the Ca²⁺-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 µM) was impaired in the distal colon of cftr^–/– mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

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