Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney

C. Graham1, P. Nalbant2, B. Schölermann2, H. Hentschel2, R. K. H. Kinne2, and A. Werner1

1 School of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom; and 2 Max-Planck-Institut für molekulare Physiologie, 44227 Dortmund, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent Pi cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number AW423104). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.

inorganic phosphate; electrophysiology


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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THE HOMEOSTASIS OF INORGANIC PHOSPHATE (Pi) is maintained by intestinal absorption and tightly controlled renal excretion (1). A family of Na-dependent Pi transport systems, denoted NaPi-II, is involved in the apical translocation steps in both renal and intestinal epithelia (20). NaPi-II homologs from several species have been characterized with respect to their structure, function, and tissue distribution (3, 19).

The type II transporter family can be subdivided into the functionally divergent isoforms NaPi-IIa and NaPi-IIb (27). In mammals, NaPi-IIa is expressed mainly in the apical membrane of the renal proximal tubule cells and is vital for controlled Pi reabsorption (19, 28). NaPi-IIb is expressed in the apical membrane of the small intestine and mediates Pi absorption from the lumen (15). The IIb transporter is also expressed in lung and secretory tissues (9).

Pi transport in fish is of interest because carp and winter flounder (Pleuronectes americanus) show renal Pi secretion and reabsorption with NaPi-II transporters likely to be involved in both translocation steps (13, 23, 27). The cloning and functional characterization of a NaPi-IIb (NaPi-IIb1) isoform from the intestine of the zebrafish (Danio rerio), a stenohaline freshwater teleost, has been reported (21). After expression in Xenopus laevis oocytes, the protein transported Pi in a sodium-, voltage-, and pH-dependent manner with functional properties comparable to the mammalian NaPi-IIa and flounder NaPi-IIb isoforms. A second NaPi-IIb-related mRNA, denoted NaPi-IIb2, was detected in zebrafish kidney, but the entire sequence has not yet been determined.

The tissue distribution of the two NaPi-IIb isoforms has previously been investigated by RT-PCR (21). However, the intracellular localization of the transporters is of prime importance in defining the physiological role of the proteins. Basolateral expression of NaPi-IIb indicates Pi secretion whereas apical expression would represent sites of Pi reabsorption. Accordingly, a single NaPi-IIb isoform is expressed in the apical and the basolateral membrane of different renal tubular segments in winter flounder (8, 16).

The aim of this study was to obtain a functional NaPi-IIb2 clone and to determine the intracellular localization and functional characteristics.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Animals

Zebrafish (Danio rerio) were bred in house. For the extraction of RNA, adult animals were anesthetized on ice and decapitated, and organs of interest were isolated.

Female clawed frogs (X. laevis) were purchased from H. Kähler (Hamburg, Germany). The removal and preparation of oocytes has previously been described in detail (26).

The care and use of experimental animals was carried out in accordance with the guidelines of the Department of Animal Care, Nordrhein-Westfalen, Germany.

Rapid Amplification of cDNA Ends

To complete the cDNA sequence of NaPi-IIb2 first 3'- and then 5'-rapid amplification of cDNA ends (RACE) was performed. For 3' RACE, the reverse transcription was primed with an adaptor primer containing 18 T residues and a NotI adaptor (Pharmacia, Freiburg, Germany). The following PCR included the adaptor primer and the forward primer 5'-CCGTTTTCACCTCCGCC-3'. Because no distinct fragment was amplified, a nested reaction was done with the primer 5'-GCTGGTATCCTGCTGTGGT-3'. The fragment of ~900 bp was cloned and sequenced. A kit from Gibco BRL (Neu-Isenburg, Germany) was used to amplify the 5'-end of NaPi-IIb2. Total RNA (1 µg) from zebrafish kidney was used. Reverse transcription was primed with a gene-specific oligonucleotide (5'-GTCTGTAGAGCATCC-3'), and subsequently the supplier's protocol was followed. The first PCR using the G-rich adaptor primer and the primer 5'-ACAGAAGTGCCGATATTTGCAC-3' resulted in a specific fragment of 550 bp. The fragment was cloned (TA cloning, Invitrogen, Groningen, Netherlands) and sequenced.

Library screening. A zebrafish genomic DNA library (Clontech, Heidelberg, Germany) was screened to identify the 5' end of the NaPi-IIb2 gene. A probe of 560 bp was generated by using PCR including digoxigenin-labeled nucleotides (Roche, Mannheim, Germany) using the primers 5'-GGATGCTCTACAGACTCACC-3' and 5'-TCCCGGCTCCCACGAGGATG-3'. In total, 1.8 × 105 clones on six plates were screened. Hybridization was performed overnight in DIG Easy Hyb (Roche) with 10 ng/ml probe added. The nylon membranes (Roche) were washed at low stringency (0.2× SSC at 42°C). Positive clones were visualized by using antidigoxygenin antibodies coupled to alkaline phosphatase according to the manufacturer's manual (Roche). Candidate areas were excised from the mother plate, and the phages were rescreened. Positive clones were eluted (Qiagen DNA-extraction kit), and the inserts were sequenced.

Immunocytochemistry

Custom antisera were purchased from Eurogentec (Cologne, Germany), including peptide synthesis, coupling to keyhole limpet hemocyanin, and the immunization of two rabbits per antigen. Standard protocols were followed. The peptide sequences were NH2-YDNPALGIEDEAKVT-COOH (NaPi-IIb2) and NH2-IIEPKKTVDSCEILK-COOH (NaPi-IIb1). Epitopes are highlighted in Fig. 2 (green shading).

Cryosections from X. laevis oocytes were prepared according to Terada et al. (25). Briefly, cRNA-injected oocytes were fixed in 3% paraformaldehyde in PBS for 1 h, rinsed with PBS, and incubated in 30% sucrose for at least 16 h. Oocytes were embedded in TissueTec (Miles Scientific, Naperville, IL) and frozen, and 5-µm sections were cut. Sections were rinsed twice with PBS and blocked with 10% normal serum for 10 min. Sections were washed three times with PBS, incubated with the first antibody for 1 h (17.5 µg in PBS, 3% dry nonfat milk, 1% saponin), washed with PBS, and incubated with the secondary Cy3-labeled antibody (Dianova, Hamburg, Germany) for 1 h.

Adult female zebrafish tissue was fixed for 10-15 min in ice-cold fixative (2% paraformaldehyde and 0.5% picric acid in 80% ethanol), rinsed with 0.1 mol/l cacodylate buffer, and embedded in paraffin for sectioning. Sections of 4-10 µm were rinsed in PBS and preincubated with NH4Cl for 10 min. Nonspecific binding was blocked with fish-gel mix (2% fetal calf serum; 2% BSA; and 0.2% fish-gelatin 45%, Sigma, Steinheim, Germany; in PBS) for 45 min. Sections were labeled by incubating with primary antibody (diluted 1:2,000), washing with PBS, and then incubating with Cy-3-conjugated secondary antibody. Nuclei were counterstained by addition of 4',6-diamidino-2-phenylindole dihydrochloride (Sigma) to the last incubation step.

Two-Electrode Voltage Clamp

cRNA was synthesized by using the mMESSAGEmMACHINE T7 kit (Ambion, Austin, TX) according to the supplier's protocol. Typically, X. laevis oocytes were injected with 10 ng cRNA by standard protocols (26). Transport activity was evaluated by the two-electrode voltage-clamp technique 2-3 days after cRNA injection.

Microelectrodes were pulled from borosilicate glass capillaries (Clark Electromedical Instruments) on a horizontal puller (model P-97, Sutter Instruments) and filled with 4 M potassium acetate. Electrodes showed resistance in the range of 0.5-1.5 MOmega when immersed in control ND96 solution containing (in mmol/l) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES; pH adjusted to 7.4 by titration with KOH. Steady-state membrane potentials were typically between -40 and -60 mV in control solution. Current was induced by addition of Pi to the bath medium. Current recordings were made either with membrane potential clamped (at -50 mV) or with voltage step protocols [from Vtest = -120 to 20 mV, holding potential (Vh) = -50 mV, Delta Vtest = 10 mV]. Experiments were repeated with at least three oocytes per batch, and at least three batches were used.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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We have previously identified two NaPi-IIb-related cDNA fragments originally isolated from zebrafish intestinal and renal tissues, named NaPi-IIb1 and NaPi-IIb2, respectively (21). The NaPi-IIb1 isoform was readily cloned and characterized; however, NaPi-IIb2 resisted several attempts of full-length cloning. Here, we report the cloning of NaPi-IIb2 and compare the two transcripts in terms of their localization and functional properties.

Cloning of NaPi-IIb2

A PCR-related strategy was envisaged to clone NaPi-IIb2. The COOH terminus deduced from the 3'-RACE product showed the cysteine cluster characteristic of NaPi-IIb isoforms as well as the PDZ (PSD95-Dlg-zona occludens-1-domain) binding motif (27). Several different approaches were performed to clone the 5'-end of the cDNA with unsatisfactory results. Either truncated fragments were amplified or a stop codon interrupted the open reading frame. The DNA sequence, corresponding to a 3'-fragment of NaPi-IIb2, was deposited as an expressed sequence tag (accession number AF297180). We isolated and sequenced two lambda  phage clones encompassing most of the zebrafish NaPi-IIb2 clone including the 5'-nontranslated area (3,800 bp). This genomic sequence information allowed us to assign an expressed sequence tag (accession number AW423104) from the gene bank to the 5'-end of the NaPI-IIb2 gene. It represented an alternative splice product and generated an open reading frame coding for the putative Pi transporter. The full-length clone could then be amplified by overlapping PCR. Figure 1 shows the structure of the NaPi-IIb2 gene.


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Fig. 1.   Structure of the NaPi-IIb2 gene. Structure was determined from the sequence data of 2 lambda  clones. Exons and introns are to size. The gray area was not sequenced.

Zebrafish NaPi-IIb1 and NaPi-IIb2 are 66% identical at the amino acid level (Fig. 2). The membrane-spanning domains and intracellular loop 1 (ICL1) and extracellular loop 3 (ECL3) are well conserved between different members of the NaPi-IIb family, with major differences localized to the large extracellular loop 2 and the NH2 and COOH termini.


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Fig. 2.   ClustalW alignment of NaPi-IIb amino acid sequences. NaPi-IIb sequences from zebrafish (zfIIb1 and zfIIb2), flounder (fIIb), mouse (mIIb), and rat (rIIb) were aligned with the ClustalW analysis program (http://clustalw.genome.ad.jp/). The zebrafish NaPi-IIb1 and NaPi-IIb2 isoforms show 66% sequence identity. NaPi cotransporters are reported to have 8 putative transmembrane-spanning domains. Membrane-spanning domains (blue) were fitted by comparison with topological data from other NaPi sequences (18, 22) and amino acid sequence analysis programs (TMHMM v2.0; http://www.cbs.dtu.dk/services/TMHMM/). A hydrophobic domain that does not span the membrane is shown by black overscore. The N- and C-termini are both predicted to be intracellular. Intracellular loop 1 and extracellular loop 3 (yellow) are reported to be functionally significant (17). Motifs reported to be responsible for pH- (red overscore) and sodium-dependent (green overscore) ion transport are also highlighted. NaPi-IIb1 and NaPi-IIb2 epitopes are highlighted in green.

NaPi-IIb1 and -IIb2 have an overlapping expression pattern: RT-PCR experiments revealed a rather broad expression pattern of the two isoforms. NaPi-IIb1 is expressed in intestine, eye, and kidney, and NaPi-IIb2 is expressed in intestine, eye, kidney, brain, liver, heart, and testis (21). Here we have used immunocytochemistry to further elucidate the cellular location of NaPi-IIb1 and NaPi-IIb2.

Immunocytochemistry

The COOH terminus of various NaPi-II proteins has proven suitable for antiserum production (4); consequently, we chose comparable epitopes in zebrafish NaPi-IIb1 and NaPi-IIb2. The specificity of the two antisera were tested by using thin sections of cRNA-injected X. laevis oocytes expressing the zebrafish NaPi-IIb proteins. Both antisera recognized the cognate epitope without cross-reacting with the other isoform (Fig. 3).


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Fig. 3.   Testing the NaPi-IIb1 and NaPi-IIb2 specific antisera. NaPi-IIb1 and NaPi-IIb2 antisera specificity were tested by incubating NaPi-IIb1 and -IIb2 cRNA-injected oocytes with the antisera. Both antisera detected the target epitope with no cross-detection of the other isoform.

Figure 4, A and B, shows serial sections of zebrafish intestine labeled with NaPi-IIb1- and NaPi-IIb2-specific antisera, respectively. The sections show a villus (longitudinal axis denoted by dashed line) with the luminal space marked L. Both NaPi-IIb1 and NaPi-IIb2 are strongly localized to the apical membrane of the enterocytes with no signal detected intracellularly or in basolateral membranes. Incubation of serial sections with the cognate preimmune sera did not yield a specific signal (Fig. 4, C and D).


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Fig. 4.   Immunocytochemical mapping of NaPi-IIb1 and -IIb2 isoforms. Serial sections of zebrafish tissues were labeled with either NaPi-IIb1 (A, E, G)- or NaPi-IIb2 (B, F, H)-specific antisera and detected by fluorescence microscopy. A and B: both isoforms are detected exclusively in the apical membrane of intestinal enterocytes. A single villus is shown with the longitudinal axis marked with a dashed line and lumen marked L. C and D: sections incubated with preimmune sera lacked specific staining. E and F: NaPi-IIb1 and NaPi-IIb2 exhibit a partially overlapping expression pattern in the kidney, with apical membranes showing strong staining. Dashed line denotes the longitudinal axis of a nephron segment. Both isoforms are present at high levels in the apical membranes (arrows). However, NaPi-IIb2 appears more abundant than NaPi-IIb1. G, H: bile ducts were labeled with NaPi-IIb1 or NaPi-IIb2 probes (red) and then counterstained with lens culinaris lectin (green) and DAPI (blue). Both isoforms are strongly expressed in the luminal membrane.

Figure 4, E and F, shows serial sections of zebrafish kidney labeled with NaPi-IIb1- and NaPi-IIb2-specific antisera, respectively. The longitudinal axis of a specific nephron fragment is marked by dashed lines. Expression of NaPi-IIb1 and NaPi-IIb2 was largely apical (arrows), and a high degree of overlap between the two proteins was evident. However, NaPi-IIb2 was detected in a larger number of nephron segments, suggesting that a more widespread expression than NaPi-IIb1 although differences in antisera affinity cannot be ruled out.

In addition to the expression of NaPi-IIb transcripts in renal and intestinal epithelium, a distinct signal was also exclusively detected in the apical membrane of bile ducts (Fig. 4, G and H).

Functional Characterization of NaPi-IIb2

Previous experiments have demonstrated that the NaPi-IIb1 isoform operates in a sodium-, phosphate-, pH-, and voltage-dependent manner (21). We used NaPi-IIb1-injected oocytes as positive controls. These data are not shown in this paper but were found to be in accordance with previously reported findings.

Phosphate dependence. Addition of phosphate to the external medium triggered an inward current in a dose-dependent manner (Fig. 5, A and B). Water-injected control oocytes did not exhibit phosphate-induced currents (data not shown).


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Fig. 5.   Analysis of NaPi-IIb2 transport activity: phosphate-dependent transport. Xenopus laevis oocytes were injected with NaPi-IIb2 cRNA, and phosphate-induced currents were analyzed by 2-electrode voltage clamp 2-3 days after injection. Increasing bath Pi concentration ([Pi]) induced an inward current that saturated at ~1 mmol/l. A: typical Pi-induced currents recorded during a voltage step protocol. B: currents (I) induced by bath Pi at holding potential = -50 mV were expressed as a fraction of maximum current (Imax). Mean data (20 oocytes) were fitted by the Michaelis-Menten equation. The determined relative affinity for phosphate (K<UP><SUB>m</SUB><SUP>Pi</SUP></UP>) was 29.3 ± 5.5 µmol/l. C: K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> was found to be influenced by membrane potential with a significant decrease in K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> on depolarizing the membrane from -120 to -60 mV. * P < 0.05 paired t-test.

Maximal current was obtained between 1 and 3 mmol/l external Pi, consistent with other sodium-phosphate cotransport systems (2, 12, 21). The relative affinity for phosphate (K<UP><SUB>m</SUB><SUP>Pi</SUP></UP>) was determined for oocytes clamped at -50 mV by fitting data with the Michaelis-Menten equation (Fig. 5B). NaPi-IIb2 was calculated to have a K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> of 29.28 ± 5.47 µmol/l, which suggests that NaPi-IIb2 has a greater affinity for Pi than NaPi-IIb1 (K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> = 250 µmol/l). The affinity for phosphate was influenced by the membrane potential with a significant decrease in K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> observed on membrane depolarization (Fig. 5C).

Sodium dependence. Like NaPi-IIb1, it was found that the NaPi-IIb2 isoform functions in a sodium-dependent manner (Fig. 6, A and B). Mean current was plotted as a function of sodium concentration and fitted with the Hill equation (Fig. 6C). The relative affinity for sodium, K<UP><SUB>m</SUB><SUP>Na</SUP></UP>, and the Hill coefficient (n) was 42.3 ± 1.90 mmol/l and 2.16 ± 0.089, respectively. The previously reported values for NaPi-IIb1 were 67.1 mmol/l and 2.1, respectively (21). These data suggest that NaPi-IIb2 has a higher affinity for sodium than NaPi-IIb1 but the stoichiometry of Na:Pi binding seems to be conserved between isoforms. Both K<UP><SUB>m</SUB><SUP>Na</SUP></UP> and the calculated Hill coefficient are unaffected by membrane potential (Fig. 6, D and E, respectively).


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Fig. 6.   Analysis of NaPi-IIb2 transport activity: sodium dependent transport. Oocytes were bathed in media containing 1 mmol/l phosphate, and currents were measured at different sodium concentrations ([Na]). A: typical current trace at Vtest = -50 mV. B: typical current-voltage relationship from a single NaPi-IIb2-injected oocyte subjected to a voltage step protocol. C: mean data (10 oocytes) were fitted with the Hill equation. The determined relative affinity for sodium (K<UP><SUB>m</SUB><SUP>Na</SUP></UP>) was 42.3 ± 1.9 mmol/l, and the Hill coefficient was 2.2 ± 0.09. Membrane potential was found to have no effect on the affinity for sodium (D) and the Hill coefficient (n; E).

pH dependence. In mammals, the NaPi-IIa cotransporters display maximal transport rates in basic conditions whereas the intestinal isoform NaPi-IIb shows maximal transport rates in acidic conditions, although the effect of pH on function is less pronounced (15). The fish intestine is thought to be less acidic than the mammalian intestine; therefore, the effect of pH on fish NaPi cotransporter function was of interest. Flounder NaPi-IIb is expressed in both renal and intestinal tissues and displays pH-dependent function similar to mammalian NaPi-IIa (transport rate at pH 8 is greater than at pH 6.5) (12). Zebrafish NaPi-IIb1 function exhibits a similar pH profile (21); therefore, we investigated the role of pH on NaPi-IIb2 transporter function.

Figure 7A displays typical current-voltage relationships for a NaPi-IIb2-injected oocyte bathed in 1 mmol/l Pi at pH 6.0-8.0. Note that at Vtest = -120 mV the transport rate was maximal at pH 8.0 (function at pH 8.0 > 6.0). However, at Vtest = -50 mV, maximal transport rates occurred at pH 7.0, although pH had a negligible effect on NaPi-IIb2 transport over the pH range 6.5-8.0. This is in contrast to the findings from NaPi-IIb1, for which it was reported that transport was strongly pH dependent and could be stimulated by an alkaline external medium (21).


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Fig. 7.   Analysis of NaPi-IIb2 transport activity: pH-dependent transport. The effect of extracellular pH on currents induced by 1 mmol/l phosphate is shown for a typical oocyte (A) and mean data (11 oocytes) are expressed as a fraction of maximal current at Vtest = -50 mV (B).

Voltage dependence. Figure 8 shows that there is a marked functional difference of NaPi-IIb1 and NaPi-IIb2 with respect to membrane potential. NaPi-IIb1 function is relatively insensitive to membrane potential with current ~75% of maximum at a membrane potential of 20 mV. However, as the membrane potential increases, NaPi-IIb2 function decreases rapidly to ~20% of maximal current at Vtest = 20 mV. Therefore, NaPi-IIb2 transporter function is more dependent on membrane potential, a characteristic similar to that of winter flounder (12, 21) and mammalian sodium-phosphate cotransporters (10).


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Fig. 8.   Voltage dependence of NaPi-IIb1- and NaPi-IIb2-mediated Pi transport. Currents were induced with 1 mmol/l Pi in the bath. At steady state, currents were recorded during a voltage step protocol (Vtest = -120 to 20 mV, Delta Vtest = 10 mV). Recorded currents (I) were normalized to Imax and expressed as means ± SE of 5 and 20 oocytes injected with NaPi-IIb1 and NaPi-IIb2 cRNA, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mammalian phosphate homeostasis is maintained by uptake from the small intestine followed by tightly regulated renal reabsorption (1). The sodium-phosphate cotransporters involved in these processes are structurally related but functionally distinct (20). NaPi-IIa is expressed in the kidney and exhibits maximal transport rates under alkaline conditions, whereas the intestinal NaPi-IIb function is less sensitive to external pH (15). This ensures that intestinal reabsorption of Pi occurs over a wide pH range, for example, when challenged by acidic output from the stomach and by neutralization in the duodenum.

The direction of renal transepithelial Pi transport in fish is closely related to the glomerular filtration rate (GFR) and thus to the habitat of the species. Freshwater fish are hypertonic to their surroundings and so water and ion balance is achieved by a high GFR followed by reabsorption of sodium, phosphate, and other ions. Marine fish are hypotonic to their surroundings; consequently, GFR is relatively low to maintain water levels and controlled renal secretion contributes to maintaining ionic balance. Therefore, in freshwater teleosts net absorption of Pi seems to prevail whereas net secretion occurs in marine teleosts (5). Recently, it emerged that members of the NaPi-II protein family play a pivotal role in both Pi secretion and reabsorption (8, 28). Micropuncture studies identified the proximal tubular segment PII of winter flounder (P. americanus) and skate (Raja erinacea) as the major site of tubular secretion (5). Experiments carried out in flounder suggested that a basolaterally sorted NaPi-II homolog drives Pi secretion in this segment. In adjacent collecting tubules, the same transporter was found in the apical membrane, possibly involved in the tuned reabsorption of Pi (8). In zebrafish, two distinct NaPi-II homologs have been reported; however, their physiological role was unclear (21). Figure 9 summarizes the localization of NaPi-IIb cotransporters in fish and NaPi-IIa in the mammalian kidney. Here, we describe the functional characterization of the second isoform NaPi-IIb2 as well as the immunohistochemical localization of NaPi-IIb1 and NaPi-IIb2.


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Fig. 9.   Schematic summary of renal tubular phosphate reabsorption in marine and freshwater teleosts and mammals. Representation and nomenclature of the different nephron segments are adapted from Dantzler (5). Isoforms of the Na/Pi cotransporters are represented in different colors: zebrafish NaPi-IIb1 in blue, NaPi-IIb2 in yellow, flounder NaPi-IIb in green, and mammalian NaPi-IIa in red. Phosphate movement is indicated by black arrows. Winter flounder, a marine teleost, exhibits a low glomerular filtration rate (GFR). Pi is secreted in the proximal tubular segment PII followed by reabsorption in the adjacent collecting tubule (8) with a single NaPi-IIb isoform involved in both steps. NaPi-IIb in the basolateral membrane drives Pi secretion. NaPi-IIb in the apical membrane prevents Pi wasting and may reduce the formation of precipitates in highly concentrated urine. In zebrafish, NaPi-IIb1 and NaPi-IIb2 are both expressed in the apical tubular membrane and mediate Pi reabsorption. Indirect evidence suggests that NaPi-IIb1 and NaPi-IIb2 are expressed in later parts of the renal tubule. This would indicate that Pi excretion in fish is regulated in distal nephron segments regardless of whether Pi reached the primary urine by glomerular filtration or tubular secretion. In the mammalian kidney, NaPi-IIa mediates Pi reabsorption in the early part of the proximal tubule. According to its physiological function, the transporter is located in the apical membrane. The fact that mammals express the "novel" isoform NaPi-IIa in kidney may reflect the shift from distal (fish) to proximal Pi reabsorption (mammals).

NaPi-IIb1 and NaPi-IIb2 Have Overlapping Expression Patterns

The presence of two NaPi-IIb isoforms represents a common feature in both freshwater and marine species, although the physiological significance of the closely related transporters remains to be established (28). Our specific antisera localized both transporters in the apical membrane of renal tubular cells, intestinal enterocytes, and bile duct epithelia. The data suggest that both isoforms play a role in accumulating body Pi by mediating (renal) reabsorption and intestinal uptake. Expression of NaPi-II in bile ducts has not been reported previously.

Elevated concentrations of Pi and/or Ca2+ in urine or bile increase the risk of stone formation. We hypothesize that the physiological role of NaPi-IIb in bile ducts is to scavenge Pi to prevent formation of precipitates. Pi may be not only secreted or filtered into bile but also generated by the breakdown of hormones or other extracellular compounds.

The two NaPi-IIb isoforms are coexpressed in the same organs. The markedly different affinities for Pi indicate that the two transporters complement each other to function efficiently over a large range of extracellular Pi concentrations. Thus NaPi-IIb1 would represent a low-affinity, high-capacity system absorbing the bulk of Pi, whereas NaPi-IIb2 would be responsible for efficient transport at low external Pi concentrations (high affinity, low capacity). The renal reabsorption of glucose in mammals by the sodium-glucose cotransporters (SGLT) follows a similar strategy. That is, uptake of glucose is mediated predominantly in the proximal convoluted tubule by the low-affinity, high-capacity transporter SGLT2 with fine control of reabsorption in the straight proximal tubule by the high-affinity, low-capacity SGLT1 (29).

The colocalization of the two transporters represents a puzzling fact. However, our experiments do not exclude a partial overlap with flanking regions expressing only one of the isoforms. Coexpression of the two isoforms could prove beneficial if a relatively short tubular fragment had to cope with highly variable loads of Pi.

A second renal NaPi-II-related isoform (NaPi-IIc) has recently been reported in rats and humans (24). NaPi-IIc is predominantly expressed in weaning rats and to a lower level in adult animals. In contrast to all other NaPi-II transporters, NaPi-IIc mediates electroneutral cotransport of sodium and Pi. Comparison of NaPi-II protein sequences did not reveal a significant phylogenetic link between zebrafish NaPi-IIb2 and rat or human NaPi-IIc (not shown).

Zebrafish NaPi-IIb2 Is Functionally Distinct From NaPi-IIb1

Many cloned NaPi-II cotransporters have been functionally characterized by expressing the protein in X. laevis oocytes and analyzing currents with the two-electrode voltage-clamp technique. All data quoted in this paper were acquired under standard conditions, i.e., 1 mmol/l Pi, 100 mmol/l sodium, and Vtest = -50 mV. A number of similarities between NaPi-II cotransporters have been determined. Typically, NaPi-II-mediated transport is voltage sensitive and electrogenic (usually 3:1 ratio of sodium to phosphate ions), and K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> and sodium K<UP><SUB>m</SUB><SUP>Na</SUP></UP> are in the range of 43-70 µmol/l and 33-46 mmol/l, respectively. The exception is zebrafish NaPi-IIb1, which is relatively voltage insensitive and has a lower affinity for phosphate and sodium (K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> and K<UP><SUB>m</SUB><SUP>Na</SUP></UP> are 250 µmol/l and 67.1 mmol/l, respectively). Transporter activity is generally pH dependent, showing strong stimulation by a neutral or alkaline environment. Mammalian NaPi-IIb is weakly stimulated by acidity, but transport is much less sensitive to external pH than other members of the family. The functional properties of the newly cloned zebrafish NaPi-IIb2 were of interest to further elucidate the role of NaPi-IIb cotransporters in fish Pi handling.

Phosphate dependence of the NaPi-II cotransporters. Zebrafish NaPi-IIb1 and NaPi-IIb2 exhibit significantly different apparent affinities for external phosphate ions; K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> are 250 (21) and 29.3 µmol/l, respectively.

The molecular basis of the wide range of reported affinities is as yet undetermined. All cloned NaPi-IIb cotransporters have a relatively high affinity for phosphate (K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> = 10-50 µmol/l) with the exception of zebrafish NaPi-IIb1 (K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> = 250 µmol/l) (17). ICL1 and ECL3 of rat NaPi-IIa show high degrees of homology and are suggested to be of functional importance (17). Comparison of the aligned sequences for the NaPi-IIb cotransporters (Fig. 2) shows that ECL3 is well conserved between all NaPi-IIb isoforms. In contrast, zebrafish NaPi-IIb1 has a number of divergent amino acids in ICL1 that may indicate the phosphate binding pocket of the NaPi-II cotransporter family.

Sodium dependence of the NaPi-II cotransporters. The reported values for the sodium affinity of NaPi-II cotransporters display a wide range. Reported values of K<UP><SUB>m</SUB><SUP>Na</SUP></UP> are in the range of 37 (14) to 62 mmol/l (6) for mammalian NaPi-IIa, 20 (15) to 38 mmol/l (6) for mammalian NaPi-IIb, 46 mmol/l (12) for flounder NaPi-IIb, and 67 mmol/l (21) for zebrafish NaPi-IIb1. Calculated values of K<UP><SUB>m</SUB><SUP>Na</SUP></UP> are likely to be subject to error because current recordings are made below saturating sodium concentrations. The calculated K<UP><SUB>m</SUB><SUP>Na</SUP></UP> (42 mmol/l) for zebrafish NaPi-IIb2 from this study is similar to published data for other NaPi-II transporters.

De la Horra et al. (6) reported differences between K<UP><SUB>m</SUB><SUP>Na</SUP></UP> for mammalian NaPi-IIa and NaPi-IIb transporters expressed in X. laevis oocytes. Mutagenesis studies indicated that a single amino acid switch from phenylalanine to leucine in NaPi-IIa caused an increase in affinity, similar to values reported for NaPi-IIb. This residue is well conserved between all NaPi-IIb isoforms (Fig. 2, green overscore) and so is unlikely to solely explain the decreased sodium affinity of NaPi-IIb1. Therefore, other regions are likely to play a role in sodium binding to NaPi-II transporters.

pH dependence of the NaPi-II cotransporters. The differences in pH sensitivity of mammalian NaPi-IIa and NaPi-IIb have been investigated at the molecular level. Site-directed mutagenesis experiments have nominated a candidate motif for the pH-dependent nature of NaPi-IIa function. It was found that switching the charged amino acids REK (in ECL3) of NaPi-IIa with the uncharged residues GNT of NaPi-IIb led to the loss of pH-dependent transport of mammalian NaPi-IIa and gain of pH sensitivity of NaPi-IIb (refer to Fig. 2, red overscore) (7). Flounder NaPi-IIb has only two charged amino acids in the same region (AEK) and is shown to exhibit pH dependence similar to mammalian NaPi-IIa (12). A similar phenomenon was reported for zebrafish NaPi-IIb1, which contains only one charged amino acid (GET). NaPi-IIb2 has this same motif (GET) and so was also predicted to exhibit pH dependence in a NaPi-IIa like manner. However, NaPi-IIb2 exhibits pH-dependent characteristics, unlike other members of the NaPi family. In the pH range 6.5-8.0, NaPi-IIb2 function is largely pH independent (a type IIb characteristic), but function was severely curtailed between pH 6.0 and 6.5 (a type IIa characteristic). Therefore, we suggest that another, as yet unidentified, motif must play a role in determining the pH sensitivity of transport in zebrafish NaPi-II cotransporters.

Voltage dependence. NaPi-IIb2 function was found to be strongly dependent on membrane potential (cotransport was reduced by 70% on depolarizing the oocyte membrane from -120 to 0 mV). This finding is conserved between all cloned NaPi cotransporters with the exception of zebrafish NaPi-IIb1, which is relatively voltage insensitive.

One surprising finding in our study was that the calculated affinities for phosphate and sodium displayed unexpected sensitivities to membrane potential. Forster et al. (10-12) proposed an eight-stage model of sodium phosphate cotransport with the orientation of the empty carrier and the binding of the first sodium ion dependent on membrane potential. The binding of phosphate to the carrier was thought to be independent of membrane potential. In accordance with this model, all cloned and characterized NaPi-II cotransporters have displayed voltage-sensitive K<UP><SUB>m</SUB><SUP>Na</SUP></UP> and voltage-insensitive K<UP><SUB>m</SUB><SUP>Pi</SUP></UP> . However, our data show the opposite trend, with sodium binding displaying no voltage dependence and phosphate binding inhibited at hyperpolarizing membrane potentials. These differences are likely to result from divergent kinetics of intramolecular charge movements within the NaPi-II cotransporter family.

In conclusion, zebrafish express two distinct NaPi-IIb-type cotransporters. These proteins have largely overlapping expression patterns, although NaPi-IIb2 is expressed more widely in the kidney. However, these cotransporters differ functionally in terms of their affinity for phosphate and sodium ions, sensitivity to membrane potential, and the effect of pH on transport rates. These properties are summarized in Table 1.

                              
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Table 1.   Comparison of zebrafish NaPi-IIb1 and NaPi-IIb2 structural and functional properties

These findings highlight the wide diversity in type II NaPi cotransporter function despite strong structural similarities between family members. Zebrafish NaPi-IIb1 and NaPi-IIb2 are closely related at the amino acid level but show different functional properties. These transporters are a useful model for the future investigation of amino acid motifs responsible for phosphate, sodium, and hydrogen ion interaction.


    ACKNOWLEDGEMENTS

The authors thank Ursula Strunck, Brian Burtle, and Heike Rimpel for excellent technical assistance.


    FOOTNOTES

This study was supported by the Wellcome Trust, The Max-Planck Society, and the University of Newcastle upon Tyne.

Address for reprint requests and other correspondence: A. Werner, School of Cellular and Molecular Biosciences, Univ. of Newcastle, Newcastle upon Tyne, NE2 4HH, UK (E-mail: andreas.werner{at}ncl.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 17, 2002;10.1152/ajprenal.00356.2002

Received 4 October 2002; accepted in final form 10 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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