The most prevalent mutation ( ∆ F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a ⌬ F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from ⌬ F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous ⌬ F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse ⌬ F508-CFTR is a temperature sensitive processing mutant. The ⌬ F508 mouse is therefore a valid in vivo model of human ⌬ F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis. ( J. Clin. Invest. 1996. 98:1304-1312.)

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na+bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na+/H+exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl−-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl−secretion by goblet cells and Cl−and HCO3−secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.

No abstract

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, DeltaF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

The striking similarities between microvillus inclusions (MIs) in enterocytes in microvillus inclusion disease (MID) and vacuolar apical compartment in tissue culture epithelial cells, led us to analyze endoscopic biopsies of duodenal mucosa of a patient after the samples were used for diagnostic procedures. Samples from another patient with an unrelated disease were used as controls. The MID enterocytes showed a decrease in the thickness of the apical F-actin layer, and normal microtubules. The immunofluorescence analysis of the distribution of five apical membrane markers (sucrase isomaltase, alkaline phosphatase, NHE-3 Na + /H + exchanger, cGMP-dependent protein kinase, and cystic fibrosis trans-membrane conductance regulator), showed low levels of these proteins in their standard localization at the apical membrane as compared with normal duodenal epithelium processed in parallel. Instead, four of these markers were found in a diffuse distribution in the apical cytoplasm, below the terminal web (as indicated by co-localization with F-actin and cytokeratin 19), and in MIs as well. The basolateral protein Na + -K + ATPase, in contrast, was normally localized. These results support the hypothesis that MID may represent the first genetic defect affecting apical membrane traffic, possibly in a late step of apical exocytosis.