We recently found that electroneutral intestinal salt absorption is dysregulated in PDZK1-deficient mice. We now studied the role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes.

Methods: NHE3 transport rates were assessed fluorometrically in BCECF-loaded colonic crypts in the NHE3- expressing cryptal openings by measuring acid-activated, Na+- dependent, Hoe621-insensitive proton efflux rates. NHE3 mRNA and protein expression levels were determined by quantitative PCR and Western analysis and immunohistochemistry.

Results: In pdzk1 -/- colonic surface cells, acid-activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin, 8-Br-cGMP, as well as ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3-selective inhibitor S1611 inhibited acid- activated NHE3 activity in pdzk1 -/- and +/+ mice, suggesting that functional NHE3 is present in pdzk1-deficient colonocytes. No difference in brush border membrane NHE3 abundance or distribution was found in the absence of PDZK1.

Conclusions: Lack of the PDZ-adapter protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca2+-dependent pathways.