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J. Biol. Chem., Vol. 282, Issue 52, 37575-37584, December 28, 2007

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Cystic Fibrosis Transmembrane Conductance Regulator Activation Is Reduced in the Small Intestine of Na⁺/H⁺ Exchanger 3 Regulatory Factor 1 (NHERF-1)- but Not NHERF-2-deficient Mice^*

Nellie Broere, Jutta Hillesheim, Biguang Tuo, Huub Jorna, Adriaan B. Houtsmuller^¶, Shirish Shenolikar^||1, Edward J. Weinman^**, Mark Donowitz, Ursula Seidler, Hugo R. de Jonge, and Boris M. Hogema²

From the Department of Biochemistry, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands, the Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, 30625 Hannover, Germany, the ^¶Optical Image Center, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands, the ^||Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, the ^**Departments of Medicine and Physiology, University of Maryland School of Medicine and the Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland 21201, and the Departments of Medicine and Physiology, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195

Binding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to the Na⁺/H⁺ exchanger 3 regulatory factor 1 (NHERF-1) and NHERF-2 scaffolding proteins has been shown to affect its localization and activation. We have for the first time studied the physiological role of these proteins in CFTR regulation in native tissue by determining CFTR-dependent chloride current in NHERF-1- and NHERF-2-deficient mice. The cAMP- and cGMP-activated chloride current and the basal chloride current in basolaterally permeabilized jejunum were reduced by 30% in NHERF-1-deficient mice but not in NHERF-2-deficient mice. The duodenal bicarbonate secretion was affected in a similar way, whereas no significant differences in CFTR activity were observed in ileum. CFTR abundance as determined by Western blotting was unaltered in jejunal epithelial cells and brush border membranes of NHERF-1 and NHERF-2 mutant mice. However, semi-quantitative detection of CFTR by confocal microscopy showed that the level of apically localized CFTR in jejunal crypts was reduced by 35% in NHERF-1-deficient and NHERF-1/2 double deficient mice but not in NHERF-2 null mice. Together our results indicate that NHERF-1 is required for full activation of CFTR in murine duodenal and jejunal mucosa and that NHERF-1 affects the local distribution of CFTR in or near the plasma membrane. These studies provide the first evidence in native intestinal epithelium that NHERF-1 but not NHERF-2 is involved in the formation of CFTR-containing functional complexes that serve to position CFTR in the crypt apical membrane and/or to optimize its function as a cAMP- and cGMP-regulated anion channel.

Received for publication, June 13, 2007 , and in revised form, October 15, 2007.

^* This work was supported by Sophia Foundation Grant 506-2006 (to H. R. D. J.); Deutsche Forschungsgemeinschaft Grants Se 460/13-1/2 and Se 460/17-1; Sonderforschungsbereich 621/project C9 (to U. S.); Dutch Stomach-Liver-Intestine Foundation (MLDS) Grant MWO 03-15 (to H. R. D. J.); National Institutes of Health Grant DK55881 (to E. J. W. and S. S.); funds from the Research Service of the Department of Veteran Affairs (to E. J. W.); NIDDK, National Institutes of Health Grants RO1DK26523, RO1 DK61765, PO1 DK072084, and R24 DK64388 (to M. D.); and the Hopkins Basic Research Digestive Diseases Development Core Center (to M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Pfizer Global Research and Development, Ann Arbor, Michigan 48105.

² To whom correspondence should be addressed: Erasmus University Medical Center, Dept. of Biochemistry, Dr. Molewaterplein 50, Ee634b, 3015 GE Rotterdam, The Netherlands. Tel.: 31-10-7043323; Fax: 31-10-7044747; E-mail: B.Hogema{at}ErasmusMC.nl.

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