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

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 CFTRdependent 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 ϳ30% 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 ϳ35% 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.

Regulation of G Protein-Coupled Receptor Function by Na+/H+Exchange Regulatory Factors

“…The mice developed normally, lacked gross phenotypic abnormalities, and exhibited normal mineral-ion homeostasis (Kocher et al, 2003). Like NHERF2-null mice, the absence of NHERF3 was associated with modest reduction of forskolin-induced Na and HCO 3 Ϫ transport (Hillesheim et al, 2007). These results contrast with others showing reduced basal but normal forskolin-induced HCO 3 Ϫ secretion ).…”

“…No evident morphological, behavioral, mineral-ion, or uric acid wasting phenotype was evident. (Broere et al, 2007;Cunningham et al, 2008). Recent results indicate morphological changes of the intestine of NHERF2-null mice with shorter villi, deeper crypts, and decreased epithelial cell number Murtazina et al, 2011).…”

“…NHERF2-null mice were generated by a retroviral genetrapping procedure that resulted in protein abolition from kidney, lung, stomach, heart, brain, and colon (Broere et al, 2007). No evident morphological, behavioral, mineral-ion, or uric acid wasting phenotype was evident.…”

Altered cGMP Dynamics at the Plasma Membrane Contribute to Diarrhea in Ulcerative Colitis

“…In additional studies that used NHERF1-and NHERF2-deficient mice, NHERF1 was shown to be critical for apical membrane placement of CFTR in native intestinal epithelium and optimizing cAMP-and cGMP-dependent regulation of CFTR, whereas placing NHERF2 not being of importance for these physiologic roles. 43 NHERF2 confers inhibition of CFTR activity by coupling the lysophosphatidic acid receptor to CFTR on lysophosphatidic acid stimulus. 11,42 Therefore, NHERF proteins follow specific physiologic and pathophysiologic contexts.…”

Characterization of CFTR High Expresser cells in the intestine

“…The observation that the CHE cells express NHERF-1, but not NHE3, was somewhat unexpected because NHERF-1 was originally thought to be functionally related to NHE3, hence its name. However, NHERF-1 (EBP50) also seems important for the stabilization of apical CFTR (12). NHERF-1 is also expressed in nonepithelial cells like T cells.…”