Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.During digestion, the distal intestine needs to reabsorb large quantities of salt and water that have been secreted into the gastrointestinal lumen by more proximal organs or are ingested through the mouth. We here show that the major salt absorptive transporter, sodium/hydrogen exchanger isoform 3 (NHE3), requires the PDZ-adaptor protein NHERF2 to determine its localization along the terminal web-microvillar axis in the apical pole of the enterocyte. The NHERF-2 deficient murine intestine has also lost the ability of certain secretagogues to inhibit NHE3 absorptive function and thereby to cause diarrhoea. NHERF-2 is therefore an important regulator of intestinal fluid transport, and is an interesting drug target, encoded by a potential disease-causing modifier gene.
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