Acute Regulation of Proximal Tubule Apical Membrane Na/H Exchanger NHE-3

The mammalian proximal tubule reabsorbs approximately 80% of the NaHCO3 and approximately 50% of the filtered NaCl from the glomerular filtrate (1,2,3). All of the transcellular NaCl and two-thirds of the transcellular NaHCO3 absorption are mediated by the proximal tubule apical membrane Na/H exchanger (4,5). In addition, transcellular NaHCO3 absorption also provides the driving force for paracellular NaCl absorption (6). Quantitatively, this translates to severalfold of the total extracellular NaCl and NaHCO3 content being turned over by a single transporter protein throughout the course of a day. Exquisitely tight regulation of the apical membrane Na/H exchanger is paramount to extracellular fluid volume and acidbase homeostasis. This article summarizes some of the recent developments in the understanding of mechanisms of acute regulation of the proximal tubule apical membrane Na/H exchanger. The review focuses on several aspects: (1) NHE-3 phosphorylation; (2) NHE-3 trafficking; (3) changes in intrinsic activity of NHE-3; and (4) accessory regulatory cofactors. NHE Gene Family Mammalian Na/H exchangers (NHE) are ubiquitous membrane ion transporters belonging to a gene family of related proteins that utilize a downhill transmembrane Na+ gradient to energize H+ extrusion up an electrochemical gradient in an electroneutral 1:1 stoichiometry. Although Na+ follows first order Michaelis-Menton kinetics (7,8), intracellular H+ serves as a substrate as well as an allosteric activator (9). The seminal paper by Sardet and coworkers describes the cloning of the first NHE isoform using an elegant genetic complementation approach (10). Based on the first cDNA, a total of six members of this gene family has been identified in mammals thus far (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). In addition, piscine, crustacean, and prokaryotic homologues exist with variable structural and functional similarities to the mammalian exchangers (25, 26, 27, 28, 29, 30). The different mammalian NHE isoforms have distinct pharmacokinetic characteristics, regulatory mechanisms, and tissue and cellular distributions. Several excellent recent general reviews are available (31, 32, 33, 34). The primary amino acid sequence of the NHE predicts a putative topology of the transporter protein as shown in Figure 1. An amino terminus transmembrane domain of approximately 400 amino acids performs Na/H exchange function, and a carboxy terminus cytoplasmic domain receives and interacts with incoming regulatory signals and exerts control over the transmembrane transporting domain. Although the current paradigm of the structural and functional domains are largely correct, the truth is likely more complex than the model of a strictly bipartite functional and structural division. Biemesderfer et al. propose that while the carboxy-terminal domain may be largely cytoplasmic, the carboxy-terminal tail may actually be extracellular (35). It is currently unknown how the cytoplasmic tail with virtually no hydrophobic regions can traverse the lipid bilayer. From the functional standpoint, in contrast to the NHE-1 isoform (36), the region of the molecule responsible for acute regulation of NHE-3 by hypertonicity likely resides in the transmembrane rather than the cytoplasmic domain (37,38).Figure 1: . Putative structure of mammalian Na/H exchangers. All members of the mammalian NHE gene family share a common similar predicted structure with approximately 400 amino acids in the N-terminal half of the protein spanning the plasma membrane 12 times. The transmembrane domain of the molecule is mainly responsible for the Na/H exchange transport function. Approximately 400 amino acids in the carboxy-terminal half of the protein constitute the cytoplasmic domain, which is mainly regulatory in function. The cytoplasmic domain contains phosphorylation sites that are targets for protein kinases and domains that bind to various regulatory cofactors.The distinct NHE isoforms likely evolved from the same ancestral genomic template to serve specialized needs of highly differentiated cells. Due to the diverse heterogeneity of specialized cellular phenotypes along the nephron, all known NHE isoforms are expressed in the kidney in various levels. Immunohistochemistry with several well-characterized antisera have unequivocally localized NHE-3 to the apical membrane of the proximal convoluted tubule and the thick ascending limb and, to a lesser extent, in the upper portion of long descending thin limbs (39,40). Although both NHE-2 and NHE-4 possess inhibitor kinetics that somewhat resemble the proximal tubule apical membrane Na/H exchanger (16, 17, 18,41, 42, 43, 44), localization with antisera against NHE-2 and NHE-4 failed to label the proximal tubule apical membrane (45, 46, 47). Transport data in apical membrane vesicles also revealed no evidence of HOE694-inhibitable (characteristic of NHE-2) ΔpH-driven Na flux (48). Similarly, no HOE694-sensitive Na flux was detected in the in vivo perfused proximal tubules in NHE-3-deficient mice (49,50). In concert, based on the current body of data, few, if any, isoforms other than NHE-3 are major players in the proximal tubule apical membrane Na transport. However, preliminary reports have described NHE-2 activity and antigen in proximal tubule-enriched preparations (51). Baum has found abundant NHE-2 transcript using reverse transcription-PCR in microdissected rabbit proximal tubules, where there is no possible contamination from distal segments (Michel Baum, personal communication). At present, although there is still some controversy whether NHE-3 is the singular isoform, there is little doubt that NHE-3 is the predominant NHE isoform in the proximal tubule apical membrane. Role of NHE-3 Phosphorylation The apical membrane Na/H exchanger is acutely regulated by a number of agonists. Table 1 presents a summary. Almost all of the hormones that modulate NHE-3 are coupled to protein kinases as part of their intracellular signaling cascades, and the hormonal effects on apical NHE can be completely or partially blocked by pharmacologic kinase inhibitors. In addition to pharmacologic antagonists, the effect of cAMP on NHE-3 activity is completely abolished by either the pseudosubstrate peptide inhibitor of PKA (117) or a dominant negative mutant regulatory subunit of protein kinase A (PKA) (118), indicating that the inhibitory effect of cAMP on NHE-3 is completely through PKA rather than a direct effect of the cyclic nucleotide. The carboxy-terminal domain of NHE-3 harbors numerous putative phosphorylation target motifs for various kinases (13). Deletion of the cytoplasmic domain of NHE-3 renders it largely constitutive, although it retains intact albeit reduced transport activity (117,119,120). Domain swapping experiments have shown that regulatory characteristics of one NHE isoform can be conferred upon another by the cytoplasmic domain of the first (120, 121, 122). For example, NHE-1 is largely cAMP-insensitive, whereas NHE-3 is inhibited by cAMP addition (33). A chimera consisting of the transmembrane domain of NHE-1 and the cytoplasmic domain of NHE-3 is inhibited by cAMP, whereas the reverse chimera is not regulated by cAMP (120). All of the above findings suggest that NHE-3 phosphorylation may play a role in its functional regulation. Three questions emerge concerning the role of NHE-3 phosphorylation in its functional regulation: (1) Does kinase activation lead to NHE-3 phosphorylation in vivo? (2) Is phosphorylation necessary to regulate NHE-3 activity? and (3) Is phosphorylation sufficient to regulate NHE-3 activity?Table 1: Acute regulation of proximal tubule apical membrane Na/H exchangeaNHE-3 was shown to exist as a phosphoprotein in intact cells with mainly phosphoserines (37,86,118,123,124). Although the cytoplasmic domain of NHE-3 is a direct substrate for purified PKA and protein kinase C (PKC) in vitro (37,117), direct phosphorylation of NHE-3 by a particular protein kinase in vivo is more difficult to prove. Two lines of indirect evidence support the notion that PKA and PKC directly phosphorylate NHE-3 in the intact cell. First, Wiederkehr and coworkers compared target phosphorylation sites on NHE-3 from in vitro phosphorylation by purified PKC to the in vivo phosphorylation by phorbol ester addition in intact cells and observed common phosphopeptides (37) Second, Zhao and coworkers used a back-phosphorylation assay and showed that activation of PKA in vivo can occupy PKA target sites with phosphates and block the subsequent in vitro phosphorylation of NHE-3 by purified PKA (118). These findings in concert strongly suggest that NHE-3 is phosphorylated in vivo by PKA and PKC. Interestingly, the phosphorylation pattern of total cellular and plasma membrane NHE-3 from intact cells appeared not to differ significantly (118,124). However, it is conceivable that there may be a pool of intracellular NHE-3 that is sequestered from kinases and phosphatases and thus have little phosphate turnover. This pool of transporters will be oblivious to the phosphopeptide mapping technique (118,124) that relies on phosphate exchange between the phosphoprotein and γ32P-ATP. NHE-3 is phosphorylated bu PKA on multiple sites in the intact cell. Using site-directed mutagenesis, Kurashima and coworkers showed that Ser605 and Ser634 of rat NHE-3 are crucial for regulation of NHE-3 by PKA (Table 2). Mutation of either site greatly diminished and mutation of both sites completely abrogated the effect of PKA activation on NHE-3 activity (123). Interestingly, only Ser605 is phosphorylated in vivo and Ser634 is not a phosphoserine. The study of Zhao and coworkers showed that Ser552 and Ser605 are both phosphorylated in vivo in a dose profile similar to functional inhibition and mutations of either Ser552 or Ser606 abolished cAMP responsiveness (118). In the study of Kurashima and coworkers, Ser552 is neither phosphorylated nor functionally important. The disparity between the two studies is unclear. It is possible that there is some redundancy in the system and that PKA can modify NHE-3 activity via phosphorylation on different sites. Different cellular context may result in the use of different regulatory phosphoserines. Regardless of the reasons for the discrepancy, current data do support that NHE-3 phosphorylation is necessary for PKA to exert its inhibition. An NHE-3 mutant harboring six serine mutations (Ser513, Ser552, Ser575, Ser661, Ser690, Ser804) displayed intact Na/H transport function but no regulation of activity by phorbol ester in vivo (37). In addition to data based on mutant exchangers in transfected cells, parallel changes in phosphorylation and activity in native NHE-3 in opossum kidney (OK) cells in terms of a dose-and time-response have been described with activation by endothelin (86) and inhibition by dopamine (99). In the intact animal, Fan and coworkers have shown that acute infusion of parathyroid hormone (PTH) leads to inhibition of renal cortical apical membrane NHE-3 activity and an increase in NHE-3 phosphorylation after 30 and 60 min (125). A parallel decrease in NHE-3 activity with increased NHE-3 phosphorylation was reported by Collazo and coworkers in OK cells (126). The current cumulative data strongly suggest that NHE-3 phosphorylation is important for its functional regulation. The mechanism by which phosphorylation of NHE-3 leads to changes in NHE-3 activity is currently unknown.Table 2: Point mutations of key serines in rat NHE-3aIf NHE-3 phosphorylation is necessary, is NHE-3 phosphorylation sufficient to regulate its activity? This is a more difficult question to address. Thus far, NHE function has only been studied in perfused tubules, native membrane vesicles, or cultured cells either as a native or heterologously expressed protein. The presence of other proteins in the system renders definitive conclusions regarding sufficiency rather difficult. Yip and coworkers found that rabbit NHE-3 when expressed in fibroblasts can be regulated by phorbol esters without change in phosphorylation pattern of NHE-3 (124). Although it is somewhat unusual that the exchanger in this study is highly phosphorylated even at the baseline state, the key finding in this study is that transporter function was inhibited without changes in NHE-3 phosphorylation (124). This indicates that factors other than NHE-3 phosphorylation can regulate NHE-3 activity. The effect of PKC activation by phorbol ester on proximal tubule Na absorption (127, 128, 129), apical membrane NHE activity (130,131), native NHE-3 activity in culture cells (132), and fibroblast transfected with NHE-3 (37,119,121,133) has been studied. Depending on the system used, PKC activation can lead to either stimulation (37,128,130,131) or inhibition (37,119,127, 128, 129,133) of NHE-3 activity. Wiederkehr and coworkers expressed NHE-3 in AP-1 fibroblasts and found that individual clones of transfected cells express heterogenous phenotypes (37). They observed inhibition, stimulation, or no regulation of NHE-3 by phorbol ester depending on the individual clone examined (37). These authors did not provide data for the basis of the heterogeneous response. However, the critical finding is that while phorbol ester induces NHE-3 phosphorylation on identical serines on the cytoplasmic tail on all the clones as evident by identical phosphopeptide maps, the functional sequelae can be suppression, augmentation, or no effect on NHE-3 activity (Figure 2). This study provides definitive evidence that phosphorylation of NHE-3 per se is insufficient to alter its activity, and some factors extrinsic to NHE-3 have to be involved in its functional regulation. This notion is further supported by the effect of dopamine on NHE-3 activity and phosphorylation pattern in OK cells (99). Dopamine receptor-1 (DR-1) agonist inhibits NHE-3 activity involving phosphorylation of NHE-3 by PKA. While dopamine receptor-2 (DR-2) agonist per se had no effect on NHE-3 activity, DR-2 agonist plays a synergistic role by enhancing the inhibitory action of dopamine receptor-1 (DR-1) agonist (99). When NHE-3 phosphorylation was examined, DR-2 agonist was sufficient to phosphorylate NHE-3 on sites that are identical to combined DR-1/DR-2 stimulation (99). Again, the conclusion from this observation is that factors other than NHE-3 phosphorylation are required to effect its inhibition by dopamine.Figure 2: . Experimental evidence that phosphorylation per se is insufficient to regulate NHE-3 activity. Rat NHE-3 was expressed in NHE-null AP-1 cells, and data from three clonal lines M1, M2, and M4 are shown. Phosphorylation pattern on NHE-3 was assessed by phosphopeptide mapping (32P pulse, immunoprecipitation of NHE-3, trypsin digestion, two-dimensional resolution, autoradiography). In general, one spot represents one specific phosphorylation site. NHE-3 activity was assayed fluorimetrically as Na-dependent cell pH recovery after an acid load using the pH-sensitive dye BCECF. Activation of protein kinase C with phorbol ester (100 nM × 20 min) led to identical phosphorylation patterns of NHE-3. However, despite the same phosphorylation patterns, NHE-3 function varies diversely depending on the particular clone of host cell. Factors extrinsic to the NHE-3 molecule must be involved in determining the activity of the transporter.In addition to protein kinases and NHE-3 phosphorylation, protein phosphatases and NHE-3 dephosphorylation may play a role in the regulation of NHE-3. It is unclear at present whether protein phosphatases are merely constitutively counterbalancing the kinase or whether both phosphorylation and dephosphorylation reactions are regulated by agonists. Phosphopeptide mapping of the dopamine-induced changes in NHE-3 phosphorylation reveals dephosphorylation of one of the serines, although the exact location and functional significance have not be identified (99). Dopamine is coupled to a regulated phosphatase in renal epithelia via the dopamine-regulated phosphoprotein, which has been shown to acutely regulate Na-K-ATPase activity via dephosphorylation (134,135). In summary, one may reasonably conclude that phosphorylation of NHE-3 by kinases is an important integral part of acute regulation of NHE-3. Future questions will be directed at how phosphorylation of certain residues leads to changes in NHE-3 activity. A most critical question is what accessory mechanisms are operational in regulating NHE-3 activity after NHE-3 is phosphorylated. In addition to kinases, the role of protein phosphatases in regulating NHE-3 activity will have to determined. Another question fundamental to signal transduction between an external stimuli and a cellular response is that the number of incoming signals greatly exceeds the available second messengers. The relevance of this concept to NHE-3 phosphorylation can be appreciated from the fact that in the thick ascending limb, PTH, calcitonin, vasopressin, glucagon, and β-adrenergic agonists all stimulate adenylyl cyclase, yet the cellular effects of these hormones are diverse (136). If two agonists stimulate adenylyl cyclase but different proteins are targeted for phosphorylation, the freely diffusable second-messenger cAMP must be translated to active PKA only in a specific well-defined microenvironment. The role of kinase anchoring proteins will be discussed further below. Role of NHE-3 Trafficking A number of experiments have suggested the role of protein trafficking in the acute regulation of NHE-3 even before isoform-specific reagents were available. Hensley and coworkers developed a density gradient centrifugation technique to examine Na/H exchange activity in different membrane fractions (137). PTH administered to cortical slices resulted in the acute decrease in Na/H exchange activity in a membrane fraction enriched in alkaline phosphatase and a commensurate increase in Na/H exchange activity in another fraction enriched in acid phosphatase and galactosyltransferase (138). The findings in this study are compatible with changes in NHE-3 trafficking as a mechanism for acute regulation, although no inhibitor pharmacokinetic or antigenic data were available to secure that the exchange activity was indeed NHE-3. Intracellular staining of NHE-3 was not evident in two immunohistochemical studies (39,40). However, Biemesderfer and coworkers have presented electron micrographs showing that in addition to the apical membrane, NHE-3 antigen exists in subapical vesicles (139). Intracellular location of NHE-3 is also noted in nonepithelial cells transfected with NHE-3 (140, 141, 142), and in renal epithelial cells expressing native NHE-3 (Moe, unpublished observations). Zhang, McDonough, and coworkers were the first to correlate acute changes in apical membrane Na/H exchange activity with NHE-3 antigen by fractionation technique of Hensley as well as immunohistochemistry using a model of hypertension-induced natriuresis (143, 144, 145, 146). Pressure-induced acute proximal tubule natriuresis was associated with rapid and reversible decreases in apical membrane Na/H exchange activity and redistribution of NHE-3 antigen to membranes of higher density enriched in microvilli cleft and endosomal markers (145). Interestingly, the distribution was observed with both acute and chronic hypertension (146). The rapid reversibility of the internalization presents a strong impetus to explore whether the endocytosed NHE-3 is recycled back to the apical membrane or whether a nascent pool destined for future insertion is rapidly recruited. D'Souza, Kurashima, and coworkers have shown in NHE-3-transfected nonepithelial cells that NHE-3 is present as a functionally active exchanger in recycling endosomes and that the steady-state surface population of NHE-3 is controlled by dynamic endocytosis and recycling via phosphatidylinositol 3′ kinase-dependent pathways (141,142). Janecki and coworkers examined the parallel changes in NHE-3 activity and surface NHE-3 antigen in response to phorbol esters in Caco-2 cells and reported changes in NHE-3 antigen that accounted for 50% of the change in NHE-3 activity (147). In a preliminary report, Biemesderfer and coworkers described the existence of apical membrane NHE-3 in rat kidney cortex in two distinct states with differential detergent solubility and sedimentation coefficients (148). The multimeric complexed NHE-3 was found to be associated with megalin. Megalin is a >600-kD membrane protein belonging to the LDL receptor family expressed in various polarized epithelia performing vectorial transport across two fluid compartments (149, 150, 151). In the mammalian proximal tubule, megalin likely performs endocytotic scavenger function for an extremely broad spectrum of macromolecular substrates filtered at the glomerulus, including B12-transcobalamin complex, B12-intrinsic factor complex, albumin, proteinases, proteinase-inhibitor complex, and drugs such as gentamicin (149,150). The exact functional role of the megalin-NHE-3 interaction is not clear at present. Given the wide scope of extracellular substrates for megalin, it is conceivable that megalin may serve in a generic capacity in overall endocytotic function in the proximal tubule of membrane proteins as well as extracellular ligands. Several recent preliminary reports have further strengthened the role of NHE-3 trafficking in regulating NHE-3 activity in various physiologic models. Amemiya and coworkers showed in OK cells that increased expression of plasma membrane NHE-3 preceded increases in total cellular NHE-3 in response to potassium depletion (152). Biemesderfer and coworkers showed that while chronic metabolic acidosis did not change NHE-3 antigen in total renal cortex, NHE-3 antigen is redistributed from a nonapical to an apical fraction on a density centrifugation gradient (153). Peng and coworkers showed that the acute stimulation of NHE-3 activity by endothelin in OK cells is associated with increased surface but not total cellular NHE-3 (154). Fan and coworkers studied the effect of PTH on renal cortical apical membrane NHE-3 activity and antigen in parathyroidectomized rats. After 4 h of PTH infusion, decreased apical membrane NHE-3 activity was accompanied by a clear decrease in apical membrane but not cortical membrane NHE-3 antigen (125). When the animals were pretreated with the microtubule-disrupting agent colchicine, the decrease in NHE-3 antigen was blocked. The PTH-induced decrease in apical membrane NHE-3 is distinct from the PTH-induced decrease in the type IIa Na-PO4 cotransporter (NaPi-2) in three ways. First, the kinetics of apical membrane NaPi-2 antigen show a peak decrease in less than 1 h (125,155), while the decrease in NHE-3 is not evident until 4 h (125). Second, it appears that the insertion of NaPi-2 into the apical membrane is microtubule-dependent, whereas the retrieval of NaPi-2 from the apical membrane is not (156,157). In contrast, the PTH-induced decrease in apical NHE-3 is completely blocked by colchicine suggesting microtubule-dependent retrieval (125). Third, the endocytosed NaPi-2 is destined for lysosomal degradation (158,159), while the endocytosed NHE-3 appears not to be degraded (125). These results suggest that PTH-induced retrieval of NaPi-2 and NHE-3 are regulated by and proceed via distinct vesicular transport pathways with differential fates. Decreased surface NHE-3 in response to PTH was also seen in OK cells in a preliminary study by Collazo and coworkers (126). After 30 min of PTH, the decrease in NHE-3 activity was accompanied by decreased surface NHE-3 antigen (126). The decreased NHE-3 antigen was shown to be due predominantly to increased NHE-3 endocytosis rather than decreased NHE-3 insertion (126). Dynamin is a GTP-binding protein/GTPase that binds to the interior of the invaginated coated pit concomitant with and subsequent to the assembly of the clathrin tri-skeleton in its apo- or GDP-bound form (160,161). Upon GTP-GDP exchange, dynamin redistributes itself to the constricted neck of the budding vesicles, and GTP hydrolysis initiates the severing of the neck to form a clathrin-coated vesicle. Using a dominant-negative GTP-binding defective mutant of dynamin, Collazo and coworkers could block the late inhibition and endocytosis of NHE-3 induced by PTH (126). In summary, NHE-3 exist in multiple compartments in addition to the apical membrane. Even within the apical membrane, there may be NHE-3 transporters that are sequestered from the functional pool. Additional studies are needed to define each of these functional and anatomic compartments. It is most likely that modulation of both exocytosis and endocytosis is used to acutely regulate NHE-3 antigen on the cell surface. A host of questions abound. What are the specific exocytotic and endocytotic pathways? How are these pathways regulated? Within each specific pathway, what are the adaptor proteins that confer the specificity to selectively recruit NHE-3 for membrane insertion or retrieval? Is it possible that endocytosed NHE-3 may be temporarily detained in a "holding population" awaiting further instructions to decide its fate of either recycling or degradation? Changes in the Intrinsic Transport Activity of NHE-3 How else can one modulate the activity of NHE-3? One way is to modify the substrate kinetic parameters of the transporter itself without changing the number of transporters. There are several lines of evidence suggesting that such mechanisms may be functional in regulating NHE-3. A number of studies have described regulation of Na/H exchange activity in response to direct addition of cAMP analogues (162), phorbol ester (130,131), or dopamine (96,98) to apical membrane vesicles, which is unlikely to retain competence for protein trafficking. Weinman and Shenolikar performed a series of experiments in which native NHE-3 is solubilized from apical membrane vesicles, phosphorylated by purified PKA in vitro, and reconstituted into proteoliposomes (163, 164, 165, 166, 167). In this system, in which there is absolutely no possibility of a trafficking event, PKA clearly inhibits Na/H exchange, indicating that the intrinsic transport activity of NHE-3 can be modified. Further evidence stems from kinetic analysis of the acute inhibition of NHE-3. The activation of the NHE-1 isoform by serum and growth factors is mediated by a shift in the sensitivity of NHE-1 to intracellular pH (pHi), whereas the regulation of NHE-3 have been largely attributed to changes in Vmax in both native and transfected proteins (31, 32, 33). Miller and Pollock noticed that the acute inhibition of native NHE-3 in OK cells by PTH involves a decrease in Vmax and a shift in KH without a change in KNa (168). This finding of a combined KH and Vmax effect was recently reproduced by Lamprecht and coworkers studying the effect of cAMP analogues on NHE-3 expressed in fibroblasts (169). Although the change in Vmax is compatible with a reduction of surface NHE-3 antigen, there is no tangible way that changes in number of surface NHE-3 transporters can explain the shift in KH. Addition mechanisms must be operational. In addition to circumstantial evidence, there are direct antigenic data of changes in NHE-3 activity without changes in NHE-3 antigen. Soleimani and coworkers described increases in Na/H exchange activity but not in NHE-3 antigen in apical membrane vesicles prepared from renal tubules acutely incubated with acid medium (170). Janecki and coworkers could account for only 50% of the changes in NHE-3 activity by changes in NHE-3 antigen in Caco-2 cells treated with phorbol esters (147). The response of NHE-3 to PTH in rats (125) and OK cells (126) proceeds in a biphasic pattern involving dual mechanisms. Immediately after the addition of PTH, NHE-3 activity is inhibited clearly without changes in NHE-3 antigen. Associated with the functional inhibition is increased NHE-3 phosphorylation by PTH in both rats and OK cells. The kinetic properties of NHE-3 were not specifically examined in these studies, but the ion gradients of the assays used were such that a change in KH alone cannot account for the change in NHE-3 activity. This suggests that the acute change in Vmax of NHE-3 can be mediated by changes other than the number of transporter proteins. Further data to support this notion are presented here in Figure 3. In the first experiment (Figure 3A), OK cells were treated with different doses of 8-Br-cAMP for 10 min, surface native NHE-3 antigen were measured as the biotinylatable streptavidin-precipitated NHE-3 antigen, and NHE-3 activity was assayed as Na-dependent cell alkalinization under Vmax conditions (extracellular [Na] = 140, pHi = 6.8). The decrease in NHE-3 activity was not accompanied by any detectable decrease in surface NHE-3 antigen. In the second experiment (Figure 3B), rat NHE-3 was expressed in sf9 insect cells, 0.1 mM 8-Br-cAMP was added for 20 min, and NHE-3 activity and antigen were determined in variable amounts of membrane vesicles prepared from the cells as pH-driven (pHi = 5.5; pHo = 7.5) 22Na flux. When NHE-3 activity is normalized to actual NHE-3 antigen in the same vesicles where transport was studied, one can see that activation of PKA clearly inhibited NHE-3 activity.Figure 3: . Changes in NHE-3 activity without change in NHE-3 antigen. (A) Opossum kidney cells were treated with varying concentrations of 8-Br-cAMP (× 10 min) to activate protein kinase A. NHE-3 activity was measured fluorimetrically using the pH-sensitive dye BCECF as Na-dependent cell pH recovery after an acid load, and surface NHE-3 antigen was measured as biotin-accessible immunoreactive NHE-3. (B) Rat NHE-3 was heterologously expressed in sf9 insect cells. Endogenous protein kinase A was activated by 8-Br-cAMP (0.1 mM × 20 min), membrane vesicles were prepared, and NHE-3 activity was assayed as ΔpH-driven 22Na flux in varying amounts of membrane, hence NHE-3 protein. Transport activity was normalized to NHE-3 antigen as determined by a standard curve using known quantities of purified recombinant NHE-3 protein. Immunoblot of NHE-3 antigen is shown.In summary, there is evidence in renal cortex, renal cell lines, and transfected cells that NHE-3 activity can be modified without changing NHE-3 antigenic abundance (Figure 3). Although this change in intrinsic transport activity is associated with changes in NHE-3 phosphorylation, the mechanism by which phosphorylation alters NHE-3 activity is still unknown. Experiments with functional purified NHE-3 in proteoliposomes will determine whether phosphorylation per se alters the kinetic parameters of NHE-3 or whether accessory proteins are required. What then are the biochemical and biophysical bases for alteration in KH and Vmax of the transporter protein? Regulatory Cofactors Although it is likely that NHE-3 phosphorylation is important to its functional regulation, it is fairly evident that NHE-3 phosphorylation per se is not the sole determinant of NHE-3 activity. As discussed in the previous section, mechanisms exist whereby NHE-3 activity can be modified without changes in NHE-3 antigen. It is conceivable that both of these findings are explained by binding of NHE-3 to regulatory cofactors. The question is what are they? If one uses NHE-3 phosphorylation as a reference point, one can theoretically partition these factors into: (1) ones that facilitate the phosphorylation of NHE-3, hence "pre-phosphorylation;" and (2) ones that cooperate with the phosphorylated NHE-3 to effect functional regulation either by changing its intrinsic transport property or its distribution, hence, "post-phosphorylation." Weinman and Shenolikar were the first to propose and isolate a regulatory factor for NHE-3 (163, 164, 165, 166, 167). Apical membrane proteins were solubilized and reconstituted into proteoliposomes, and Na/H exchange activity was measured. These investigators found that either limited trypsin digestion or size fractionation of the solubilized apical membrane proteins increased baseline Na/H exchange function and eliminated the inhibitory action of PKA on Na/H exchange activity. Reconstitution of a certain size/charge chromatographic fraction containing a 45- to 55-kD PKA substrate to the trypsinized or size-fractionated proteins restored PKA regulation of Na/H exchange. They went on to purify the cofactor, generated antiserum, and showed that they can confer PKA responsiveness to Na/H exchange activity in otherwise PKA-unresponsive size-fractionated apical membranes using the immunoprecipitated cofactor (166,167). The cloned protein was termed NHERF (Na/H exchanger regulatory factor) and was found to have a ubiquitous distribution (167). Yun and coworkers used the cytoplasmic domain of NHE-3 as a bait in a yeast-two-hybrid system and cloned E3KARP (exchanger-3 kinase A regulatory protein) as an NHE-3 binding partner (171), which is closely related to NHERF (52% identity). Both NHERF and E3KARP bind NHE-3 in vitro and in vivo (171). Using PS120 fibroblasts and Caco-2 cells (which do not express native NHERF or E3KARP) as hosts, Yun, Weinman, Donowitz, and coworkers showed that NHE-3 is cAMP-unresponsive, but cAMP responsiveness can be conferred by transfection of either NHERF or E3KARP (171). This was the first demonstration in an intact cell system in which proteins outside the protein kinase-NHE-3 axis are required for functional regulation of NHE-3. NHERF (synonym: EBP50) and E3KARP (synonyms: TKA-1, NHERF-2) are members of the same protein family containing two tandem PDZ domains that are conserved modules that mediate protein-protein interaction (172). The human homologue of NHERF, EBP50 binds the epithelial microvillus cytoskeletal protein ezrin (173, 174, 175, 176). Ezrin is a member of the ERM (ezrin-radixin-moesin) family that performs a regulatory and structural role in the assembly of microvilli by virtue of its ability to link cytoskeletal to plasma membrane proteins (174,176). Yun and coworkers showed that the carboxy-terminal 200 amino acids of E3KARP, which contains the second PDZ domain, interacts with an internal region of the cytoplasmic domain of NHE-3 (amino acids 585 to 660) (140). In addition, the same region on E3KARP also interacts with the protein ezrin. Unlike its interaction with NHE-3, the terminal 26 amino acids of E3KARP are absolutely a prerequisite for it to bind to ezrin (140). Lamprecht and Yun have proposed a model whereby the NHERF/ezrin complex functions as the structural link not only between NHE-3 and the actin filament, but also between NHE-3 and PKA based on ezrin's ability to bind the RII regulatory subunit of PKA (169,177). In essence, NHERF/ezrin acts as a functional AKAP (A kinase anchoring protein) for NHE-3. AKAP belong to a broad family of targeting proteins that contain structural moieties that bring specific kinases or phosphatases to the proximity of their cognate substrates to permit spatially restricted target-specific catalytic reactions (178,179). Close to 40 proteins have been identified to date that qualify functionally to be AKAP (179). Thus far, the model of NHERF/ezrin as an AKAP is compatible with a large body of data from several laboratories. However, some issues remain unclear. The purification of NHERF-1 was founded on its ability to restore PKA responsiveness to solubilized NHE-3 that was presumably devoid of native NHERF by limited trypsin digestion, size fractionation, or immunodepletion (163, 164, 165, 166, 167). In all of these studies, purified catalytic subunit of PKA was used to phosphorylate a protein mixture containing NHE-3. The catalytic subunit of PKA can usually phosphorylate substrates in solution quite efficiently without AKAP even though the same substrate may require AKAP for in vivo phosphorylation by PKA. Immuno- or affinity-purified recombinant NHE-3 from bacteria, insect cells, or mammalian cells are efficiently phosphorylated by purified PKA catalytic subunit in solution and PKCα in lipid micelles (37,117,118). If NHERF/ezrin is the functional AKAP for NHE-3, it is unclear how it performs its function in all of the reconstitution studies in the absence of the very moiety that ezrin binds, the RII regulatory subunit of PKA (163, 164, 165, 166, 167). If the sole function of the NHERF is to collude with ezrin to bring about NHE-3 phosphorylation, NHERF will be a "pre-phosphorylation" regulator. However, the current data do not rule out a "post-phosphorylation" role for the NHERF. If NHE-3 is actually "adequately" phosphorylated in solution by purified PKA catalytic subunit, the data from the reconstituted proteoliposomes experiments actually suggest an additional "post-phosphorylation" role for NHERF. One way NHERF can play a role in compartmentalization was recently defined by Hall and coworkers. cAMP addition universally inhibits NHE-3. β-Adrenergic agonists are coupled to adenylyl cyclase and PKA activation, yet β-adrenergic agonists fail to inhibit NHE-3 activity. Hall and coworkers showed that the key intermediate mechanism at play appears to be agonist-dependent sequestration of NHERF (180). Activation of the β-adrenergic receptor induces binding of NHERF to the carboxy-terminal tail of the receptor and renders it unavailable to facilitate regulation of NHE-3 by PKA. When the carboxy terminus of the β-adrenergic receptor was mutated to render it incompetent to bind NHERF, the β-adrenergic agonist can then inhibit NHE-3 via PKA activation. This mechanism can account for the fact that not all hormones that activate PKA end up with the same effect on NHE-3. One key data point that can potentially resolve whether NHERF is purely a "pre-phosphorylation" factor or if NHERF has a "post-phosphorylation" is whether NHE-3 is phosphorylated by β-adrenergic agonists acting on the wild-type versus NHERF-binding defective β-adrenergic receptor. The NHERF will likely turn out to be generic proteins that serve multiple functions via docking through their PDZ domains. In addition to NHE-3 and the β-adrenergic receptor, NHERF binding to the purinergic P2Y1 receptor (181) and the cystic fibrosis transmembrane regulator CFTR (181,182) has been described. In summary, it is likely that the NHERF family of proteins serve as anchors to bring the PKA catalytic subunit to the vicinity of NHE-3 (Figure 4). It is also likely that there are redundancies in the system and that other functional AKAP for NHE-3 exist. In addition, NHERF may have additional roles, in that they may facilitate the functional regulation of NHE-3 after changes in phosphorylation have already occurred. This notion is purely conjectural. No contenders have yet surfaced as "post-phosphorylation" regulators to account for why NHE-3 phosphorylation is insufficient for its functional regulation. It is almost certain that an increasing number of binding partners for NHE-3, in addition to the NHERF and megalin, will be identified.Figure 4: . Possible sites and pathways of regulated NHE-3 trafficking. Based on the current body of data on acute alterations of NHE-3 activity and cell surface antigen in response to certain agonists, a mostly conjectural paradigm is constructed of the mechanisms of regulation of plasma membrane NHE-3 protein abundance. Activity of surface NHE-3 can be altered through mechanisms involving NHE-3 phosphorylation plus other regulatory factors. Surface NHE-3 abundance can be modulated through regulation of insertion and/or retrieval of NHE-3.Acknowledgments The author's research program was supported by National Institutes of Health (DK48482), the Department of Veterans Affairs Research Service, the American Heart Association Texas Affiliate (98G052), and the National Kidney Foundation of Texas. The author acknowledges the invaluable contribution from his laboratory associates Michael Wiederkehr, Hui Zhao, Roberto Collazo, Lingzhi Fan, and Ladonna Crowder. The author is also grateful for helpful discussions with Robert Alpern and Michel Baum.