Aquaporin-2, a regulated water channel, is expressed in apical membranes of rat distal colon epithelium

Aquaporin-2 (AQP-2) is the vasopressin-regulated water channel expressed in the apical membrane of principal cells in the collecting duct and is involved in the urinary concentrating mechanism. In the rat distal colon, vasopressin stimulates water absorption through an unknown mechanism. With the hypothesis that AQP-2 could contribute to this vasopressin effect, we studied its presence in rat colonic epithelium. We used RT-PCR, in situ hybridization, immunoblotting, and immunocytochemistry to probe for AQP-2 expression. An AQP-2 amplicon was obtained through RT-PCR of colon epithelium RNA, and in situ hybridization revealed AQP-2 mRNA in colonic crypts and, to a lesser extent, in surface absorptive epithelial cells. AQP-2 protein was localized to the apical membrane of surface absorptive epithelial cells, where it colocalized with H(+)-K(+)-ATPase but not with Na(+)-K(+)-ATPase. AQP-2 was absent from the small intestine, stomach, and liver. Water deprivation increased the hybridization signal and the protein level (assessed by Western blot analysis) for AQP-2 in distal colon. This was accompanied by increased p-chloromercuriphenylsulfonic acid-sensitive water absorption. These results indicate that AQP-2 is present in the rat distal colon, where it might be involved in a water-sparing mechanism. In addition, these results support the idea that AQP-2, and probably other aquaporins, are involved in water absorption in the colon.     

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump

Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.     

Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na(+)/H(+) exchanger

The present study was aimed at evaluating the role of D(1)- and D(2)-like receptors and investigating whether inhibition of Na(+) transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na(+)/H(+) exchanger, inhibition of the basolateral Na(+)-K(+)-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D(1)- and D(2)-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC(50) = 220 +/- 2 nM), marked intracellular acidification (IC(50) = 58 +/- 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 +/- 4.5% reduction) without affecting intracellular pH recovery after CO(2) removal. These results agree with the view that dopamine, through the activation of D(1)- but not D(2)-like receptors, inhibits both the Na(+)/H(+) exchanger (0.001933 +/- 0.000121 vs. 0.000887 +/- 0.000073 pH unit/s) and Na(+)-K(+)-ATPase without interfering with the Na(+)-independent HCO transporter. It is concluded that dopamine, through the action of D(1)-like receptors, inhibits both the Na(+)/H(+) exchanger and Na(+)-K(+)-ATPase, but its marked acidifying effects result from inhibition of the Na(+)/H(+) exchanger only, without interfering with the Na(+)-independent HCO transporter and Na(+)-K(+)-ATPase.     

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.     

Intestinal carbonic anhydrase, bicarbonate, and proton carriers play a role in the acclimation of rainbow trout to seawater

Abrupt transfer of rainbow trout from freshwater to 65% seawater caused transient disturbances in extracellular fluid ionic composition, but homeostasis was reestablished 48 h posttransfer. Intestinal fluid chemistry revealed early onset of drinking and slightly delayed intestinal water absorption that coincided with initiation of NaCl absorption and HCO(3)(-) secretion. Suggestive of involvement in osmoregulation, relative mRNA levels for vacuolar H(+)-ATPase (V-ATPase), Na(+)-K(+)-ATPase, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-HCO(3)(-) cotransporter 1, and two carbonic anhydrase (CA) isoforms [a general cytosolic isoform trout cytoplasmic CA (tCAc) and an extracellular isoform trout membrane-bound CA type IV (tCAIV)], were increased transiently in the intestine following exposure to 65% seawater. Both tCAc and tCAIV proteins were localized to apical regions of the intestinal epithelium and exhibited elevated enzymatic activity after acclimation to 65% seawater. The V-ATPase was localized to both basolateral and apical regions and exhibited a 10-fold increase in enzymatic activity in fish acclimated to 65% seawater, suggesting a role in marine osmoregulation. The intestinal epithelium of rainbow trout acclimated to 65% seawater appears to be capable of both basolateral and apical H(+) extrusion, likely depending on osmoregulatory status and intestinal fluid chemistry.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

Characteristics of the K(+)-ion transport in the rat intestine following the use of natural zeolites as food additives

Effects of zeolites as a food supplement have been studied on Wistar rats both in vivo perfusion experiments in the jejunum and distal colon and Rb fluxes through intestinal wall in the Ussing chamber. It has been found that zeolites decrease the K+ absorption and stimulate K+ secretion in the gut. This effect was due to inhibition of the apical N(+)-K(+)-ATPase and ouabain-sensitive Na(+)-independent K(+)-ATPase as well as the activation of the basolateral N(+)-K(+)-ATPase.     

Mechanism of acid adaptation of a fish living in a pH 3.5 lake

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.     

Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.     

Recent experiments towards a model for fluid secretion in Rhodnius Upper Malpighian Tubules (UMT)

Three different methods have been used to improve a model for fluid secretion in Upper Malpighian Tubules (UMT) of the blood sucking insect Rhodnius prolixus. (I) In the first, UMT double perfusions in 5th instar Rhodnius were used to measure their fluid secretion rate. They were stimulated to secrete with 5-HT. Double perfusions allowed access separately to the basolateral and the apical cell membranes with pharmacological agents known to block different ion transport functions, namely ATPases, cotransporters and/or countertransporters and ion and water channels: ouabain, bafilomycin A1, furosemide, bumetanide, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. The basic assumption is that changes in water movement reflect changes in ion transport mechanisms. (II) Intracellular Na(+) concentrations were measured with a fluorometric method in dissected R. prolixus UMT, under several experimental conditions. (III) ATPase activities were measured in R. prolixus UMT. A tentative model for the function of the UMT cell is presented. We find that (a) at the basolateral cell membrane, fundamental is a Na(+)-K(+)-2Cl(-) cotransporter; of intermediate importance are the Na(+)-K(+)-ATPase and a ouabain-insensitive Na(+)-ATPase, ion channels and Rp-MIP water channels. (b) At the apical cell membrane, most important are a V-H(+)-ATPase; and a K(+) and/or Na(+)-H(+) exchanger.     

Modification of sarcolemmal Na+-K+-ATPase and Na+/Ca2+ exchanger expression in heart failure by blockade of renin-angiotensin system

The activities of both sarcolemmal (SL) Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger, which maintain the intracellular cation homeostasis, have been shown to be depressed in heart failure due to myocardial infarction (MI). Because the renin-angiotensin system (RAS) is activated in heart failure, this study tested the hypothesis that attenuation of cardiac SL changes in congestive heart failure (CHF) by angiotensin-converting enzyme (ACE) inhibitors is associated with prevention of alterations in gene expression for SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger. CHF in rats due to MI was induced by occluding the coronary artery, and 3 wk later the animals were treated with an ACE inhibitor, imidapril (1 mg.kg(-1).day(-1)), for 4 wk. Heart dysfunction and cardiac hypertrophy in the infarcted animals were associated with depressed SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities. Protein content and mRNA levels for Na(+)/Ca(2+) exchanger as well as Na(+)-K(+)-ATPase alpha(1)-, alpha(2)- and beta(1)-isoforms were depressed, whereas those for alpha(3)-isoform were increased in the failing heart. These changes in SL activities, protein content, and gene expression were attenuated by treating the infarcted animals with imidapril. The beneficial effects of imidapril treatment on heart function and cardiac hypertrophy as well as SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the infarcted animals were simulated by enalapril, an ACE inhibitor, and losartan, an angiotensin receptor antagonist. These results suggest that blockade of RAS in CHF improves SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the failing heart by preventing changes in gene expression for SL proteins.     

V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 immunoreactivity in the gill epithelium of the Pacific hagfish (Epatretus stoutii)

We report the presence of the ion transporting proteins V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in the gill epithelium of the Pacific hagfish Epatretus stoutii. Heterologous antibodies recognized single bands of the appropriate sizes for the three transporters in western blots. Immunohistochemical staining revealed that the distribution of labeled cells in the gill epithelium was identical for the three proteins. Immunopositive cells were most abundant in the primary filament from the afferent side of the gill pouch, and their number diminished towards the lamella. Na(+)/K(+)-ATPase-like immunoreactivity (L-IR) occurred throughout the cell cytoplasm, probably associated to the basolateral tubular system. V-H(+)-ATPase L-IR was similar to Na(+)/K(+)-ATPase, although some cells had slightly heavier staining in either the supra- or infra-nuclear region. NHE2 L-IR was also generally cytoplasmic, but a minority of the cells had stronger immunoreactivity in the apical region. In general, all three ion transporting proteins were localized in the same cells, as estimated from 4-microm immunostained consecutive sections. We hypothesize that these putative ion-transporting cells are involved in systemic acid/base regulation and discuss other possible roles. This is the first report of V-H(+)-ATPase in myxinoids, and the first NHE2 report in the Pacific hagfish.     

Demonstration of a Na(+)/H(+) exchanger NHE1 in fresh bovine corneal endothelial cell basolateral plasma membrane.

Apical and basolateral plasma membranes of fresh bovine corneal endothelial cells were isolated using positively charged polyacrylamide beads. Marker enzyme assays demonstrated that the isolated apical and basolateral plasma membrane domains could be isolated and separated with relative purity. Western blotting with a polyclonal anti-NHE1 antibody detected a protein of 70 kDa in the basolateral plasma membrane isolate. NHE1 immunoreactivity was not detected in the apical membrane sample. This suggests that the Na(+)/H(+) exchanger, NHE1, is strictly localised to the basolateral membrane of fresh bovine corneal endothelial cells.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation

Short-term exposure of coho salmon smolts (Oncorhynchus kisutch) to a gradual increase in salinity over 2 d (0 per thousand -32 per thousand ) resulted in a decrease in proton pump abundance, detected as changes in immunoreactivity with a polyclonal antibody against subunit A of bovine brain vacuolar H(+)-ATPase. N-ethylmaleimide (NEM)-sensitive H(+)-ATPase activities in gill homogenates remained unchanged over 8 d to coincide with a 3.5-fold increase in Na(+)/K(+)-ATPase activities. A transient increase in plasma [Na(+)] and [Cl(-)] levels over the 8-d period was preceded by a 10-fold increase in plasma cortisol levels, which peaked after 12 h. Long-term (1 mo) acclimation to seawater resulted in the loss of apical immunoreactivity for vH(+)-ATPase and band 3-like anion exchanger in the mitochondria-rich cells identified by high levels of Na(+)/K(+)-ATPase immunoreactivity. The polyclonal antibody Ab597 recognized a Na(+)/H(+) exchanger (NHE-2)-like protein in what appears to be an accessory cell (AC) type. Populations of these ACs were found associated with Na(+)/K(+)-ATPase rich chloride cells in both freshwater- and seawater-acclimated animals.     

Chloride and potassium channel function in alveolar epithelial cells

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na(+) transport and, in particular, the role of amiloride-sensitive Na(+) channels in the apical membrane and the Na(+)-K(+)-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl(-) and K(+) channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl(-) and K(+) channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.     

Dopamine regulation of Na+-K+-ATPase requires the PDZ-2 domain of sodium hydrogen regulatory factor-1 (NHERF-1) in opossum kidney cells

Na(+)-K(+)-ATPase activity in renal proximal tubule is regulated by several hormones including parathyroid hormone (PTH) and dopamine. The current experiments explore the role of Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) in dopamine-mediated regulation of Na(+)-K(+)-ATPase. We measured dopamine regulation of ouabain-sensitive (86)Rb uptake and Na(+)-K(+)-ATPase α1 subunit phosphorylation in wild-type opossum kidney (OK) (OK-WT) cells, OKH cells (NHERF-1-deficient), and OKH cells stably transfected with full-length human NHERF-1 (NF) or NHERF-1 constructs with mutated PDZ-1 (Z1) or PDZ-2 (Z2) domains. Treatment with 1 μM dopamine decreased ouabain-sensitive (86)Rb uptake, increased phosphorylation of Na(+)-K(+)-ATPase α1-subunit, and enhanced association of NHERF-1 with D1 receptor in OK-WT cells but not in OKH cells. Transfection with wild-type, full-length, or PDZ-1 domain-mutated NHERF-1 into OKH cells restored dopamine-mediated regulation of Na(+)-K(+)-ATPase and D1-like receptor association with NHERF-1. Dopamine did not regulate Na(+)-K(+)-ATPase or increase D1-like receptor association with NHERF-1 in OKH cells transfected with mutated PDZ-2 domain. Dopamine stimulated association of PKC-ζ with NHERF-1 in OK-WT and OKH cells transfected with full-length or PDZ-1 domain-mutated NHERF-1 but not in PDZ-2 domain-mutated NHERF-1-transfected OKH cells. These results suggest that NHERF-1 mediates Na(+)-K(+)-ATPase regulation by dopamine through its PDZ-2 domain.