Abstract

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 approximately 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 approximately 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.

Highlights

  • 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

  • The exploitation of Na؉/H؉ exchanger regulatory factor 1 (NHERF-1)- and NHERF-2-deficient and NHERF-1/2 double-mutant mice in this study has enabled us to investigate, for the first time, the role of these PDZ domain proteins in CFTR expression and regulation in native small intestinal epithelium

  • In addition to NHERF-1 and NHERF-2, PDZK1 and the single PDZ domain proteins CAL (CFTR-associated ligand) and Shank2 are known binding partners of CFTR and candidate PDZ proteins binding to CFTR in vivo in enterocytes [37]

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Summary

EXPERIMENTAL PROCEDURES

Animals—Using a previously described retroviral trapping method [23], Lexicon Genetics generated NHERF-2-deficient mice using Omnibank clone OST2298. The mice were genotyped by two-allele three-primer PCR, using genomic DNA isolated from tail clips; primers 1 (5Ј-TTCTATAAGCCTCCATTTCCTCT-3Ј) and 3 (5Ј-CCCACCCCCATCGCTGCTC-3Ј) were used for detection of the wild type (WT) allele. Epithelial tissue and BBMVs were resuspended in SDS-PAGE loading buffer containing protease inhibitors (Complete; Roche Applied Science) and homogenized by brief sonication on ice. The protein concentrations were determined using the RC DC protein assay kit (Bio-Rad). Sections were incubated with anti-CFTR (R3195, 1:100) or anti-NHERF-1 (1:500) antibodies in phosphate-buffered saline with 2% bovine albumin for 1.5 h at room temperature. For (semi)quantification of signal from jejunal crypts, images from longitudinal sections at Z-steps of 0.5 ␮m were analyzed by determining total background-subtracted fluorescent signal using KS400 Zeiss software. Statistical significance was established using Student’s unpaired two-tailed t test

RESULTS
B-3 Glucose
Total fluores cence intens ity
DISCUSSION
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