The intestinal and renal proximal tubule brush border (BB) Na + -H + exchanger NHE3 binds to members of the NHERF (Na + -H + exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable series of proteins in NHE3-containing complexes in a dynamic manner that is altered by changes in signal transduction which affects NHE3 activity. The specific roles of these proteins in terms of NHE3 regulation as well as interactions with each other and with their many other substrates are only now being defined. Specificity for only one member of the NHERF family in one example of NHE3 regulation, inhibition by elevation in cGMP, is used to describe how NHERF family proteins are involved in NHE3 complex formation and its regulation. In this case, NHERF2 directly binds cGKII in the brush border to form an NHE3 complex, with cGKII also associating with the BB via its myristoylation.

Electroneutral NaCl absorption mediated by Na؉ /H ؉ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na ؉ homeostasis.cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. In vitro conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 in vitro, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.The rapid elevation of intestinal cAMP and cGMP levels by activation of adenylate cyclase and guanylate cyclase, respectively, inhibits intestinal NaCl absorption, either moderately as part of normal digestive physiology or excessively in diarrheal diseases. Some details of the mechanisms of acute regulation of intestinal NaCl absorption by cAMP are understood. Hormones such as vasoactive intestinal peptide or secretin and enterotoxins such as cholera toxin activate adenylate cyclase and increase cellular cAMP content. According to the current model, based on studies in PS120 fibroblasts and the polarized OK 1 renal proximal tubule cell line, acute elevation of cAMP inhibits NHE3 by stimulating its endocytosis plus decreasing its exocytosis and, additionally, by decreasing the NHE3 turnover number (1-4). NHE3 and cAMP-dependent protein kinase type II (PKAII) are part of the same signaling complex that is scaffolded by either of two brush border (BB)-associated PDZ domain containing proteins, NHERF1 (also called NHERF or EBP50) or NHERF2 (also called E3KARP) (5, 6). NHERF1/ NHERF2 each contain two homologous PDZ domains (PDZ1 and PDZ2) and an ERM (ezrin-radixin-moesin) binding domain, which anchors NHERF and its binding partners to the actin cytoskeleton via NHERF binding to ezrin. Ezrin binds both NHERF1/NHERF2 and PKAII and acts as a low affinity cAMP kinase-anchoring protein (AKAP), positioning PKAII so it can phosphorylate NHE3, which is required for cAMP inhibition of NHE3 (1, 6,7).In some cases, cGMP regulates intracellular events by mechanisms analogous to those demonstrated for cAMP. However, the effects of cGMP in the small intestine are not fully elucidated. The intrinsic ileal peptide guanylin and the Escherichia coli heat-stable enterotoxin (STa) both bind to the same BB receptor, guanylate cyclase-C, and within minutes increase intracellular cGMP content (8). STa, guanylin, and cGMP all rapidly i...

SummaryThe epithelial brush border Na/H exchanger NHE3 is active under basal conditions and functions as part of neutral NaCl absorption in the intestine and renal proximal tubule, where it accounts for the majority of total Na absorbed. NHE3 is highly regulated. Both stimulation and inhibition occur post-prandially. This digestion related regulation of NHE3 is mimicked by multiple extracellular agonists and intracellular second messengers. The regulation of NHE3 depends on its C-terminal cytoplasmic domain, which acts as a scaffold to bind multiple regulatory proteins and links NHE3 to the cytoskeleton. The cytoskeletal association occurs by both direct binding to ezrin and by indirect binding via ezrin binding to the C-terminus of the multi-PDZ domain containing proteins NHERF1 and NHERF2. This is a review of the domain structure of NHE3 and of the scaffolding function and role in the regulation of NHE3 of the NHE3 C-terminal domain.

The epithelial brush border (BB) Na+/H+ exchanger, NHE3, plays a major role in transcellular Na+ absorption in the renal proximal tubule. NHE3 activity is rapidly regulated by neurohumoral substances and growth factors via changes in its amount on the BB by a process partially involving vesicle trafficking. The PDZ domain-containing proteins, NHERF1/2, are scaffold proteins that link NHE3 to the actin cytoskeleton via their binding to both ezrin and NHE3. NHERF1/2 interact with both an internal C-terminal domain of NHE3 and the N-terminus of ezrin. We used fluorescence recovery after photobleaching (FRAP) to study the effect of NHERF1/2 on NHE3 mobility in the brush border of opossum kidney (OK) proximal tubule cells. A confocal microscope was used to allow the selective study of apical membrane versus intracellular NHE3. A chimera of NHE3-EGFP was transiently expressed in OK cells and its lateral diffusion in the apical membrane was measured with FRAP and confocal microscopy at 37°C. The contribution of intracellular NHE3-EGFP to recovery on the OK surface not directly over the juxtanuclear area (non-JN) was negligible as exposure to the water soluble crosslinker BS3 (10 mM) at 4°C resulted in no recovery of this component of surface NHE3-EGFP after photobleaching. The mobile fraction (Mf) of apical NHE3-EGFP was 47.5±2.2%; the effective diffusion coefficient (Deff) was (2.2±0.3) ×10–10 cm2/second. Overexpression of NHERF2 in OK cells decreased the Mf to 29.1±3.1% without changing Deff. In the truncation mutant, NHE3585-EGFP (aa 1-585), which lacks the NHERF1/2 binding domain, Mf increased to 66.4±2.2%, with no change in Deff, whereas NHE3660-EGFP, which binds NHERF1/2, had Mf (48.3±3.0%) and Deff both similar to full-length NHE3. These results are consistent with the PDZ domain proteins NHERF1 and NHERF2 scaffolding NHE3 in macromolecular complexes in the apical membrane of OK cells under basal conditions, which limits the lateral mobility of NHE3. It is probable that this is one of the mechanisms by which NHERF1/2 affects rapid regulation of NHE3 by growth factors and neurohumoral mediators. By contrast, disrupting the actin cytoskeleton by latrunculin B treatment (0.05 μM, 30 minutes) reduced the NHE3 Mf (21.9±4.5%) without altering the Deff. Therefore the actin cytoskeleton, independently of NHERF1/2 binding, is necessary for apical membrane mobility of NHE3.

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCalpha pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCalpha from cytosol to membrane. PKCalpha bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCalpha and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCalpha is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

Based on physiological studies, the epithelial brush-border (BB) Na+/H+ antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be alpha-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.

Background & Aims-Oral rehydration solutions (ORS) reduce diarrhea-associated mortality by unclear mechanisms. Sodium absorption is mediated by the Na + /H + hydrogen exchanger NHE3 and is increased by Na + -glucose co-transport in vitro, but the mechanisms of this process are only partially understood and its in vivo relevance has not been determined.

Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-NЈ-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (k off ), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower k off value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 ϩ/Ϫ hypertonic saline for CF lung disease.The pulmonary disease in patients with cystic fibrosis (CF) reflects genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that produce defective epithelial ion transport. The CF airway epithelial ion transport abnormalities lead to a well described pathophysiological cascade that adversely affects the innate defense Article, publication date, and citation information can be found at