Down regulation of small intestinal ion transport in PDZK1- (CAP70/NHERF3) deficient mice

Abstract

The PDZ-binding protein PDZK1 (CAP70/PDZ-dc-1/NHERF3) in vitro binds to cystic fibrosis transmembrane conductance regulator (CFTR), the anion exchangers SLC26A3 and SLC26A6 and the Na(+)/H(+) exchanger NHE3, all of which are major transport proteins for intestinal anion secretion and salt absorption. This study was undertaken to search for a role of PDZK1 in regulating electrolyte transport in native murine small intestine. Short circuit current (I (SC)) and HCO-(3) secretory rate (J(HCO-)(3)) were measured to assess electrogenic anion secretion; (22)Na(+) fluxes to assess sodium absorption in isolated small intestine. NHE3, CFTR, as well as NHERF1, NHERF2, and PDZK1 messenger RNA (mRNA) expression levels, and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative polymerase chain reaction (PCR) and Western analysis. NHE3 localization was performed by immunohistochemistry. In pdzk1 -/- jejunal mucosa, basal net Na(+) absorption as well as the inhibition of Na(+) absorption by forskolin was significantly reduced. In pdzk1 -/- duodenal mucosa, identical basal I (SC) and (J(HCO-)(3)) but a significant, yet mild, reduction of forskolin-stimulated Delta(J(HCO-)(3)) and DeltaI (SC) was observed compared to +/+ tissue. Tissue conductance, morphological features, and the DeltaI (SC) and increase in (22)Na(+) absorption in response to luminal glucose was identical in pdzk1 +/+ and -/- small intestine, ruling out a general absorptive defect. While CFTR mRNA expression levels were unchanged, NHE3 mRNA expression levels were significantly increased in small intestinal mucosa of pdzk1 -/- mice. Total enterocyte and BBM abundance was not significantly different, suggesting an increased NHE3 turnover, possibly due to reduced NHE3 membrane retention time. Lack of the PDZ-adapter protein PDZK1 in murine small intestine causes a mild reduction in maximal CFTR activation, but a severe defect in electroneutral Na(+) absorption.

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

“…1A). NHERF3 has been suggested to play a significant role in basal NHE3 activity in mouse intestine (21,22). We have previously demonstrated that NHERF1 and NHERF2 directly bind NHE3 between aa 589 and 667 (28).…”

“…Although NHERF3 has been shown to regulate NHE3 activity in the mouse intestine, the mechanism responsible for this regulation remains unclear (21,22). Therefore, the current study examined the role of NHERF3 in the PS120 fibroblast cell model, which lacks NHERF2 and NHERF4 as well as the polarized intestinal epithelial cell model, Caco-2BBe, which expresses all four members of the NHERF family.…”

“…NHERF1 was necessary for cAMP inhibition of NHE3 activity in renal proximal tubule but not in distal ileum) (10,39). Moreover, studies of the NHERF3 KO mouse demonstrated that whereas these mice appeared healthy, intestinal NHE3 activity was significantly reduced and these mice failed to respond to both cAMP and elevated [Ca 2ϩ ] i with NHE3 inhibition (21,22). Although these studies strongly suggest a role for NHERF proteins in NHE3 regulation, the cellular and molecular mechanisms responsible for NHERF-dependent regulation remain unclear.…”

“…In addition, studies of NHERF1 (6,40) and NHERF2 KO (40) mice have shown changes in expression of other NHERF protein family members, making it challenging to decide which NHERF protein accounts for changes in function. Intestinal studies of NHERF3 KO mice have not yet addressed whether changes in NHE3 regulation could also be due to compensatory changes of the other NHERF proteins (21,22).…”

“…Recent studies have begun evaluating the effect of NHERF3 on mouse intestinal Na ϩ and Cl Ϫ transport. Basal electroneutral sodium absorption was decreased by Ͼ40% in the NHERF3 null mouse jejunum (21) and by Ͼ80% in the colon (22). In addition, Cinar et al (22) demonstrated that cAMP and [Ca 2ϩ ] i inhibition of NHE3 activity was abolished in the NHERF3 null mouse colon.…”