Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na(+)/H(+) exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.

Direct phosphorylation of sodium hydrogen exchanger type 3 (NHE3) is a well-established physiological phenomenon; however, the exact role of NHE3 phosphorylation in its regulation remains unclear. The objective of this study was to evaluate whether NHE3 phosphorylation at serines 552 and 605 is physiologically regulated in vivo and, if so, whether changes in phosphorylation at these sites are tightly coupled to changes in transport activity. To this end, we directly compared PKA-induced NHE3 inhibition with site-specific changes in NHE3 phosphorylation in vivo and in vitro. In vivo, PKA was activated using an intravenous infusion of parathyroid hormone in Sprague-Dawley rats. In vitro, PKA was activated directly in opossum kidney (OKP) cells using forskolin and IBMX. NHE3 activity was assayed in microvillar membrane vesicles in the rat model and by (22)Na uptake in the OKP cell model. In both cases, NHE3 phosphorylation at serines 552 and 605 was determined using previously characterized monoclonal phosphospecific antibodies directed to these sites. In vivo, we found dramatic changes in NHE3 phosphorylation at serines 552 and 605 with PKA activation but no corresponding alteration in NHE3 activity. This dissociation between NHE3 phosphorylation and activity was further verified in OKP cells in which phosphorylation clearly preceded transport inhibition. We conclude that although phosphorylation of NHE3 at serines 552 and 605 is regulated by PKA both in vivo and in vitro, phosphorylation of these sites does not directly alter Na(+)/H(+) exchange activity.

Background: cGKII acutely inhibits brush-border NHE3, but the mechanism is unknown. Results: cGMP/cGKII phosphorylates NHE3 at three sites. All are necessary for NHE3 inhibition. One of these is also phosphorylated by SGK1 to stimulate NHE3. Conclusion: cGKII inhibits NHE3 by phosphorylating and reducing NHE3 surface amount. Significance: Phosphorylation of the same site in a protein can alter function differently based on phosphorylation of additional sites.

Dietary potassium (K(+)) restriction and hypokalemia have been reported to change the abundance of most renal Na(+) and K(+) transporters and aquaporin-2 isoform, but results have not been consistent. The aim of this study was to reexamine Na(+), K(+) and H(2)O transporters' pool size regulation in response to removing K(+) from a diet containing 0.74% NaCl, as well as from a diet containing 2% NaCl (as found in American diets) to blunt reducing total diet electrolytes. Sprague-Dawley rats (n = 5-6) were fed for 6 days with one of these diets: 2% KCl, 0.74% NaCl (2K1Na, control chow) compared with 0.03% KCl, 0.74% NaCl (0K1Na); or 2% KCl, 2%NaCl (2K2Na) compared with 0.03% KCl, 2% NaCl (0K2Na, Na(+) replete). In both 0K1Na and 0K2Na there were significant decreases in: 1) plasma [K(+)] (<2.5 mM); 2) urinary K(+) excretion (<5% of control); 3) urine osmolality and plasma [aldosterone], as well as 4) an increase in urine volume and medullary hypertrophy. The 0K2Na group had the lowest [aldosterone] (172.0 ± 17.4 pg/ml) and lower blood pressure (93.2 ± 4.9 vs. 112.0 ± 3.1 mmHg in 2K2Na). Transporter pool size regulation was determined by quantitative immunoblotting of renal cortex and medulla homogenates. The only differences measured in both 0K1Na and 0K2Na groups were a 20-30% decrease in cortical β-ENaC, 30-40% increases in kidney-specific Ste20/SPS1-related proline/alanine-rich kinase, and a 40% increase in medullary sodium pump abundance. The following proteins were not significantly changed in both the 0 K groups: Na(+)/H(+) exchanger isoform 3; Na(+)-K(+)-Cl(-) cotransporter; Na(+)-Cl(-) cotransporter, oxidative stress response kinase-1; renal outer medullary K(+) channel; autosomal recessive hypercholesterolemia; c-Src, aquaporin 2 isoform; or renin. Thus, despite profound hypokalemia and renal K(+) conservation, we did not confirm many of the changes that were previously reported. We predict that changes in transporter distribution and activity are likely more important for conserving K(+) than changes in total abundance.

ϩ exchanger (NHE) regulatory factor 2 (NHERF2) is necessary for multiple aspects of acute regulation of NHE3 in intact mouse small intestine, distal ileal NHE3 activity was determined using two-photon microscopy/SNARF-4F in a NHERF2-null mouse model. The NHERF2-null mouse ileum had shorter villi, deeper crypts, and decreased epithelial cell number. Basal rates of NHE3 activity were reduced in NHERF2-null mice, which was associated with a reduced percentage of NHE3 in the apical domain and an increase in intracellular NHE3 amount but no change in total level of NHE3 protein. cAMP, cGMP, and elevated Ca 2ϩ due to apical exposure to UTP all inhibited NHE3 activity in wild-type mouse ileum but not in NHERF2-null mice, while inhibition by hyperosmolarity occurred normally. The cAMP-increased phosphorylation of NHE3 at aa 552; levels of PKAII␣ and cGMP-dependent protein kinase II (cGKII); and elevation of Ca 2ϩ were similar in wild-type and NHERF2-null mouse ileum. Luminal lysophosphatidic acid (LPA) stimulated NHE3 in wild-type but not in NHERF2-null ileum. In conclusion, 1) there are subtle structural abnormalities in the small intestine of NHERF2-null mouse which include fewer villus epithelial cells; 2) the decreased basal NHE3 activity and reduced brush border NHE3 amount in NHERF2-null mice show that NHERF2 is necessary for normal basal trafficking or retention of NHE3 in the apical domain; 3) hyperosmolar inhibition of NHE3 occurs similarly in wild-type and NHERF2-null ileum, demonstrating that some inhibitory mechanisms of NHE3 are not NHERF2 dependent; 4) cAMP inhibition of NHE3 is NHERF2 dependent at a step downstream of cAMP/PKAII phosphorylation of NHE3 at aa 552; 5) cGMP-and UTP-induced inhibition of NHE3 are NHERF2 dependent at steps beyond cGKII and the UTP-induced increase of intracellular Ca 2ϩ ; and 6) LPA stimulation of NHE3 is also NHERF2 dependent. Ca 2ϩ ; adenosine 3=,5=-cyclic monophosphate; guanosine 3=,5=-cyclic monophosphate; trafficking; Na ϩ /H ϩ exchanger regulation; Na ϩ /H ϩ exchanger regulatory factor; lysophosphatidic acid THE BRUSH BORDER sodium/hydrogen exchanger, NHE3, belongs to the SLC9A gene family of proteins and is predominantly expressed in the apical surface of small and large intestine and proximal tubules of the kidney where it accounts for the majority of intestinal and renal Na ϩ absorption (39). NHE3 activity is highly regulated. Much of this regulation involves large signaling complexes which NHE3 forms with multiple binding partners, some of which bind to the intracellular COOH-terminal ϳ377 aa of NHE3 (12, 14, 15). The Na ϩ /H ϩ exchanger regulatory factor (NHERF) family of multi-PDZ domain containing proteins (NHERFs 1-4) are scaffolds that bind to the middle of the intracellular COOH terminus of NHE3 and form some of the signaling complexes that regulate NHE3 (12-14, 36, 38). The NHERF proteins are present in the apical domain of intestinal and renal proximal tubule Na-absorptive cells and bind multiple proteins in addition to NHE3. These scaffold proteins contain t...