Both NHERF3 and NHERF2 are necessary for multiple aspects of acute regulation of NHE3 by elevated Ca2+, cGMP, and lysophosphatidic acid.

The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its COOH terminus that include the four multi-PDZ domain-containing NHERF family proteins. All bind to NHE3 and take part in different aspects of NHE3 regulation. Because the roles of each NHERF appear to vary on the basis of the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in C57BL/6 mouse jejunum using homozygous NHERF2 and NHERF3 knockout mice. NHE3 activity was determined with two-photon microscopy and the dual-emission pH-sensitive dye SNARF-4F. The jejunal apical membrane of NHERF3-null mice appeared similar to wild-type (WT) mice in surface area, microvillus number, and height, which is similar to results previously reported for jejunum of NHERF2-null mice. NHE3 basal activity was not different from WT in either NHERF2- or NHERF3-null jejunum, while d-glucose-stimulated NHE3 activity was reduced in NHERF2, but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca2+) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2- and NHERF3-null jejunum. Forskolin inhibited NHE3 in NHERF3-null jejunum, but the extent of inhibition was reduced compared with WT. The forskolin inhibition of NHE3 in NHERF2-null mice was too inconsistent to determine whether there was an effect and whether it was altered compared with the WT response. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca2+, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush-border NHERF3-NHERF2 heterodimer or sequential NHERF-dependent effects in these aspects of NHE3 regulation. NEW & NOTEWORTHY NHERF2 and NHERF3 are apical membrane multi-PDZ domain-containing proteins that are involved in regulation of intestinal NHE3. This study demonstrates that NHERF2 and NHERF3 have overlapping roles in NHE3 stimulation by LPA and inhibition by elevated Ca2+ and cGMP. These results are consistent with their role being as a NHERF3-NHERF2 heterodimer or via sequential NHERF-dependent signaling steps, and they begin to clarify a role for multiple NHERF proteins in NHE3 regulation.