Characterization of a type IIb sodium-phosphate cotransporterfrom zebrafish (Da - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:35 |只看该作者 |倒序浏览 |打印

作者：C.Graham, P.Nalbant, B.Schölermann, H.Hentschel, R.K. H.Kinne,  A.Werner作者单位：1 School of Cell and Molecular Biosciences,University of Newcastle, Newcastle upon Tyne, NE2 4HH, UnitedKingdom; and Max-Planck-Institut fürmolekulare Physiologie, 44227 Dortmund, Germany



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      【摘要】- a9 r. L+ p0 Q2 L: ^: a
   Zebrafish (Daniorerio) express two isoforms of the type IIb Na-dependentP i cotransporter (NaPi). Type NaPi-IIb1 haspreviously been cloned and characterized. Here, we report the cloningof 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 thatcontained typical NaPi-IIb characteristics such as acysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zonaoccludens-1) binding motif. Several approaches wereunsuccessful at cloning the 5' end of the transcript; products lackedan in-frame start codon. The missing information was obtained from anEST (GenBank accession number AW423104 ). The combined clone displayed ahigh degree of homology with published type IIb cotransportersequences. Specific antibodies were raised against a COOH-terminalepitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemicalmapping revealed apical expression of both isoforms in zebrafish renaland intestinal epithelia, as well as in bile ducts. The novel clone wasexpressed in oocytes, and function was assayed by the two-electrodevoltage-clamp technique. The function of the new NaPi-IIb2 clone wasfound to be significantly different from NaPi-IIb1 despite strongstructural similarities. NaPi-IIb2 was found to be strongly voltagesensitive, with higher affinities for both sodium and phosphate thanNaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to externalpH 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 IINaPi-cotransporter family.
      【关键词】 inorganic phosphate electrophysiology6 t1 t" Y% y) Q1 n- j6 Z9 u
   INTRODUCTION8 B, {+ v  |  g2 F) U/ R" _

THE HOMEOSTASIS OF INORGANIC PHOSPHATE (P i ) is maintained byintestinal absorption and tightly controlled renal excretion ( 1 ). A family of Na-dependent P i transportsystems, denoted NaPi-II, is involved in the apical translocation stepsin both renal and intestinal epithelia ( 20 ). NaPi-IIhomologs from several species have been characterized with respect totheir structure, function, and tissue distribution ( 3, 19 ).  C* h0 ^# R+ {) a

The type II transporter family can be subdivided into the functionallydivergent isoforms NaPi-IIa and NaPi-IIb ( 27 ). In mammals,NaPi-IIa is expressed mainly in the apical membrane of the renalproximal tubule cells and is vital for controlled P i reabsorption ( 19, 28 ). NaPi-IIb is expressed in the apical membrane of the small intestine and mediates P i absorptionfrom the lumen ( 15 ). The IIb transporter is also expressedin lung and secretory tissues ( 9 )./ u4 X1 l$ Y& b8 i2 K

P i transport in fish is of interest because carp and winterflounder (Pleuronectes americanus) show renal P i secretion and reabsorption with NaPi-II transporters likely to beinvolved in both translocation steps ( 13, 23, 27 ). Thecloning and functional characterization of a NaPi-IIb (NaPi-IIb1)isoform from the intestine of the zebrafish (Danio rerio), astenohaline freshwater teleost, has been reported ( 21 ).After expression in Xenopus laevis oocytes, the proteintransported P i in a sodium-, voltage-, and pH-dependent manner with functional properties comparable to the mammalian NaPi-IIaand flounder NaPi-IIb isoforms. A second NaPi-IIb-related mRNA, denotedNaPi-IIb2, was detected in zebrafish kidney, but the entire sequencehas not yet been determined.; j+ a8 ?! t) Y0 C! T1 o  O, Y/ I+ C

The tissue distribution of the two NaPi-IIb isoforms has previouslybeen investigated by RT-PCR ( 21 ). However, theintracellular localization of the transporters is of prime importancein defining the physiological role of the proteins. Basolateralexpression of NaPi-IIb indicates P i secretion whereasapical expression would represent sites of P i reabsorption.Accordingly, a single NaPi-IIb isoform is expressed in the apical andthe basolateral membrane of different renal tubular segments in winterflounder ( 8, 16 ).3 G- B; u$ C9 U

The aim of this study was to obtain a functional NaPi-IIb2 clone and todetermine the intracellular localization and functional characteristics.
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MATERIALS AND METHODS3 i; Q! w! e, `+ R: \
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Zebrafish (Danio rerio) were bred in house. For theextraction of RNA, adult animals were anesthetized on ice anddecapitated, 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 accordancewith the guidelines of the Department of Animal Care, Nordrhein-Westfalen, Germany.

Rapid Amplification of cDNA Ends# n/ a% t" v( O. H" n

To complete the cDNA sequence of NaPi-IIb2 first 3'- and then5'-rapid amplification of cDNA ends (RACE) was performed. For 3' RACE,the reverse transcription was primed with an adaptor primer containing18 T residues and a NotI adaptor (Pharmacia, Freiburg,Germany). The following PCR included the adaptor primer and the forwardprimer 5'-CCGTTTTCACCTCCGCC-3'. Because no distinct fragment wasamplified, a nested reaction was done with the primer 5'-GCTGGTATCCTGCTGTGGT-3'. The fragment of ~900 bp was cloned andsequenced. A kit from Gibco BRL (Neu-Isenburg, Germany) was used toamplify the 5'-end of NaPi-IIb2. Total RNA (1 µg) from zebrafishkidney was used. Reverse transcription was primed with a gene-specificoligonucleotide (5'-GTCTGTAGAGCATCC-3'), and subsequently thesupplier's protocol was followed. The first PCR using the G-richadaptor primer and the primer 5'-ACAGAAGTGCCGATATTTGCAC-3' resulted ina 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) wasscreened 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 primers5'-GGATGCTCTACAGACTCACC-3' and 5'-TCCCGGCTCCCACGAGGATG-3'. In total,1.8 × 10 5 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 atlow stringency (0.2× SSC at 42°C). Positive clones were visualizedby using antidigoxygenin antibodies coupled to alkaline phosphataseaccording to the manufacturer's manual (Roche). Candidate areas wereexcised from the mother plate, and the phages were rescreened. Positiveclones were eluted (Qiagen DNA-extraction kit), and the inserts were sequenced.3 I, R& z  b! D

Immunocytochemistry6 M: C1 R3 l! s( J$ U1 V
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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 wereNH 2 -YDNPALGIEDEAKVT-COOH (NaPi-IIb2) andNH 2 -IIEPKKTVDSCEILK-COOH (NaPi-IIb1). Epitopes arehighlighted in Fig. 2 (green shading)./ ~* r, }% ?" y! E8 M9 E
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Cryosections from X. laevis oocytes were prepared accordingto Terada et al. ( 25 ). Briefly, cRNA-injected oocytes werefixed in 3% paraformaldehyde in PBS for 1 h, rinsed with PBS, andincubated in 30% sucrose for at least 16 h. Oocytes were embeddedin TissueTec (Miles Scientific, Naperville, IL) and frozen, and 5-µmsections were cut. Sections were rinsed twice with PBS and blocked with 10% normal serum for 10 min. Sections were washed three times withPBS, incubated with the first antibody for 1 h (17.5 µg in PBS,3% dry nonfat milk, 1% saponin), washed with PBS, and incubated withthe secondary Cy3-labeled antibody (Dianova, Hamburg, Germany) for1 h.
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Adult female zebrafish tissue was fixed for 10-15 min in ice-coldfixative (2% paraformaldehyde and 0.5% picric acid in 80% ethanol),rinsed with 0.1 mol/l cacodylate buffer, and embedded in paraffin forsectioning. Sections of 4-10 µm were rinsed in PBS andpreincubated with NH 4 Cl for 10 min. Nonspecific binding wasblocked 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 (diluted1:2,000), washing with PBS, and then incubating with Cy-3-conjugatedsecondary antibody. Nuclei were counterstained by addition of4',6-diamidino-2-phenylindole dihydrochloride (Sigma) to the lastincubation step.
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Two-Electrode Voltage Clamp
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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 standardprotocols ( 26 ). Transport activity was evaluated by thetwo-electrode voltage-clamp technique 2-3 days after cRNA injection.
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Microelectrodes were pulled from borosilicate glass capillaries (ClarkElectromedical Instruments) on a horizontal puller (model P-97,Sutter Instruments) and filled with 4 M potassium acetate. Electrodesshowed resistance in the range of 0.5-1.5 M when immersed incontrol ND96 solution containing (in mmol/l) 96 NaCl, 2 KCl,1.8 CaCl 2, 1 MgCl 2, 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 wasinduced by addition of P i to the bath medium. Currentrecordings were made either with membrane potential clamped (at 50mV) or with voltage step protocols [from V test = 120 to 20 mV, holding potential( V h ) = 50 mV, V test = 10 mV]. Experiments wererepeated with at least three oocytes per batch, and at least threebatches were used.# a; Z' s3 c  h8 |( H  i# l# i2 \

RESULTS& n3 v1 B: [+ w6 c

We have previously identified two NaPi-IIb-related cDNA fragmentsoriginally isolated from zebrafish intestinal and renal tissues, namedNaPi-IIb1 and NaPi-IIb2, respectively ( 21 ). The NaPi-IIb1isoform was readily cloned and characterized; however, NaPi-IIb2resisted several attempts of full-length cloning. Here, we report thecloning of NaPi-IIb2 and compare the two transcripts in terms of theirlocalization and functional properties.3 V9 f0 [& A4 V1 \1 K5 b5 u
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Cloning of NaPi-IIb26 g8 Z( B3 d) n( n& F$ V: {
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A PCR-related strategy was envisaged to clone NaPi-IIb2. The COOHterminus deduced from the 3'-RACE product showed the cysteine clustercharacteristic of NaPi-IIb isoforms as well as the PDZ (PSD95-Dlg-zonaoccludens-1-domain) binding motif ( 27 ). Several differentapproaches were performed to clone the 5'-end of the cDNA withunsatisfactory results. Either truncated fragments were amplified or astop codon interrupted the open reading frame. The DNA sequence,corresponding to a 3'-fragment of NaPi-IIb2, was deposited as anexpressed sequence tag (accession number AF297180 ). We isolated andsequenced two phage clones encompassing most of the zebrafishNaPi-IIb2 clone including the 5'-nontranslated area (3,800 bp). Thisgenomic sequence information allowed us to assign an expressed sequencetag (accession number AW423104 ) from the gene bank to the 5'-end of theNaPI-IIb2 gene. It represented an alternative splice product andgenerated an open reading frame coding for the putative P i transporter. The full-length clone could then be amplified byoverlapping PCR. Figure 1 shows the structure of the NaPi-IIb2 gene.) ^8 b9 @' H) k2 E/ J1 D$ a- ]' L

Fig. 1. Structure of the NaPi-IIb2 gene. Structure was determinedfrom the sequence data of 2 clones. Exons and introns are to size.The gray area was not sequenced.
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Zebrafish NaPi-IIb1 and NaPi-IIb2 are 66% identical at the amino acidlevel (Fig. 2 ). The membrane-spanningdomains 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 andthe NH 2 and COOH termini.6 P3 V% j0 a6 e( c% e( l2 h: ~
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Fig. 2. ClustalW alignment of NaPi-IIb amino acid sequences. NaPi-IIbsequences 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-IIb2isoforms show 66% sequence identity. NaPi cotransporters are reportedto have 8 putative transmembrane-spanning domains. Membrane-spanningdomains (blue) were fitted by comparison with topological data fromother NaPi sequences ( 18, 22 ) and amino acid sequenceanalysis programs (TMHMM v2.0; http://www.cbs.dtu.dk/services/TMHMM/ ).A hydrophobic domain that does not span the membrane is shown byblack overscore. The N- and C-termini are both predicted to beintracellular. Intracellular loop 1 and extracellular loop 3 (yellow)are reported to be functionally significant ( 17 ). Motifsreported to be responsible for pH- (red overscore) and sodium-dependent(green overscore) ion transport are also highlighted. NaPi-IIb1 andNaPi-IIb2 epitopes are highlighted in green.5 L6 i2 I* ?9 Q+ ^& v! g

NaPi-IIb1 and -IIb2 have an overlapping expression pattern: RT-PCRexperiments revealed a rather broad expression pattern of the twoisoforms. NaPi-IIb1 is expressed in intestine, eye, and kidney, andNaPi-IIb2 is expressed in intestine, eye, kidney, brain, liver, heart,and testis ( 21 ). Here we have used immunocytochemistry tofurther elucidate the cellular location of NaPi-IIb1 and NaPi-IIb2.4 l# _& f. u9 e* J) J

Immunocytochemistry$ n# p7 z5 o4 b3 r% ]$ [

The COOH terminus of various NaPi-II proteins has proven suitablefor 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 ofcRNA-injected X. laevis oocytes expressing the zebrafishNaPi-IIb proteins. Both antisera recognized the cognate epitope without cross-reacting with the other isoform (Fig. 3 ).

Fig. 3. Testing the NaPi-IIb1 and NaPi-IIb2 specific antisera.NaPi-IIb1 and NaPi-IIb2 antisera specificity were tested by incubatingNaPi-IIb1 and -IIb2 cRNA-injected oocytes with the antisera. Bothantisera detected the target epitope with no cross-detection of theother isoform.% i5 v5 e+ e0 k1 Y2 ?6 U

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

Fig. 4. Immunocytochemical mapping of NaPi-IIb1 and -IIb2isoforms. Serial sections of zebrafish tissues were labeled with eitherNaPi-IIb1 ( A, E, G )- or NaPi-IIb2( B, F, H )-specific antisera anddetected by fluorescence microscopy. A and B :both isoforms are detected exclusively in the apical membrane ofintestinal enterocytes. A single villus is shown with the longitudinalaxis marked with a dashed line and lumen marked L. C and D : sections incubated with preimmune seralacked specific staining. E and F : NaPi-IIb1 andNaPi-IIb2 exhibit a partially overlapping expression pattern in thekidney, with apical membranes showing strong staining. Dashed linedenotes the longitudinal axis of a nephron segment. Both isoforms arepresent 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-IIb2probes (red) and then counterstained with lens culinaris lectin (green)and DAPI (blue). Both isoforms are strongly expressed in the luminalmembrane.+ q1 Z7 d; T! a+ R7 h$ }  S

Figure 4, E and F, shows serial sections ofzebrafish kidney labeled with NaPi-IIb1- and NaPi-IIb2-specificantisera, respectively. The longitudinal axis of a specific nephronfragment is marked by dashed lines. Expression of NaPi-IIb1 andNaPi-IIb2 was largely apical (arrows), and a high degree of overlapbetween the two proteins was evident. However, NaPi-IIb2 was detectedin a larger number of nephron segments, suggesting that a morewidespread expression than NaPi-IIb1 although differences in antiseraaffinity cannot be ruled out.0 S4 v; @7 Q" H

In addition to the expression of NaPi-IIb transcripts in renal andintestinal epithelium, a distinct signal was also exclusively detectedin the apical membrane of bile ducts (Fig. 4, G and H ).$ @( Y0 u" g5 q! [6 c& Y: \
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Functional Characterization of NaPi-IIb2# y# P. }0 N5 f) o3 R$ i
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Previous experiments have demonstrated that the NaPi-IIb1 isoformoperates 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 bein accordance with previously reported findings.
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Phosphate dependence. Addition of phosphate to the external medium triggered an inwardcurrent in a dose-dependent manner (Fig. 5, A and B ). Water-injected controloocytes did not exhibit phosphate-induced currents (data not shown).

Fig. 5. Analysis of NaPi-IIb2 transport activity:phosphate-dependent transport. Xenopus laevis oocytes wereinjected with NaPi-IIb2 cRNA, and phosphate-induced currents wereanalyzed by 2-electrode voltage clamp 2-3 days after injection.Increasing bath P i concentration ()induced an inward current that saturated at ~1 mmol/l. A :typical P i -induced currents recorded during a voltage stepprotocol. B : currents ( I ) induced by bathP i at holding potential = 50 mV were expressed as afraction of maximum current ( I max ). Mean data(20 oocytes) were fitted by the Michaelis-Menten equation. Thedetermined relative affinity for phosphate( K m Pi ) was 29.3 ± 5.5 µmol/l. C : K m Pi wasfound to be influenced by membrane potential with a significantdecrease in K m Pi on depolarizing themembrane from 120 to 60 mV. * P t -test.0 ^+ r( k9 z; l

Maximal current was obtained between 1 and 3 mmol/l externalP i, consistent with other sodium-phosphate cotransportsystems ( 2, 12, 21 ). The relative affinity for phosphate( K m Pi ) was determined foroocytes clamped at 50 mV by fitting data with the Michaelis-Mentenequation (Fig. 5 B ). NaPi-IIb2 was calculated to havea K m Pi of 29.28 ± 5.47 µmol/l, which suggests that NaPi-IIb2 has a greater affinity forP i than NaPi-IIb1 ( K m Pi = 250 µmol/l). The affinity for phosphate was influenced by themembrane potential with a significant decrease in K m Pi observed on membranedepolarization (Fig. 5 C ).' R* z  e5 ^# A
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Sodium dependence. Like NaPi-IIb1, it was found that the NaPi-IIb2 isoform functions in asodium-dependent manner (Fig. 6, A and B ). Mean current was plottedas a function of sodium concentration and fitted with the Hill equation(Fig. 6 C ). The relative affinity for sodium, K m Na, 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 thatNaPi-IIb2 has a higher affinity for sodium than NaPi-IIb1 but thestoichiometry of Na

i binding seems to be conserved between isoforms.Both K m Na and the calculated Hillcoefficient are unaffected by membrane potential (Fig. 6, D and E, respectively)." ~7 _9 f8 B9 L9 X2 u8 o

Fig. 6. Analysis of NaPi-IIb2 transport activity: sodiumdependent transport. Oocytes were bathed in media containing 1 mmol/lphosphate, and currents were measured at different sodiumconcentrations (). A : typical current trace at V test = 50 mV. B : typicalcurrent-voltage relationship from a single NaPi-IIb2-injected oocytesubjected to a voltage step protocol. C : mean data (10 oocytes) were fitted with the Hill equation. The determined relativeaffinity for sodium ( K m Na ) was 42.3 ± 1.9 mmol/l, and the Hill coefficient was 2.2 ± 0.09. Membranepotential 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 ratesin basic conditions whereas the intestinal isoform NaPi-IIb showsmaximal transport rates in acidic conditions, although the effect of pHon function is less pronounced ( 15 ). The fish intestine isthought to be less acidic than the mammalian intestine; therefore, theeffect of pH on fish NaPi cotransporter function was of interest.Flounder NaPi-IIb is expressed in both renal and intestinal tissues anddisplays pH-dependent function similar to mammalian NaPi-IIa (transportrate at pH 8 is greater than at pH 6.5) ( 12 ). ZebrafishNaPi-IIb1 function exhibits a similar pH profile ( 21 );therefore, we investigated the role of pH on NaPi-IIb2 transporter function.
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Figure 7 A displays typicalcurrent-voltage relationships for a NaPi-IIb2-injected oocyte bathed in1 mmol/l P i at pH 6.0-8.0. Note that at V test = 120 mV the transport rate was maximalat 6.0). However, at V test = 50 mV, maximal transport ratesoccurred at pH 7.0, although pH had a negligible effect on NaPi-IIb2transport over the pH range 6.5-8.0. This is in contrast to thefindings from NaPi-IIb1, for which it was reported that transport wasstrongly pH dependent and could be stimulated by an alkaline externalmedium ( 21 ).& |  N2 J7 l7 h/ ]7 A% X
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Fig. 7. Analysis of NaPi-IIb2 transport activity: pH-dependenttransport. The effect of extracellular pH on currents induced by 1 mmol/l phosphate is shown for a typical oocyte ( A ) and meandata (11 oocytes) are expressed as a fraction of maximal current at V test = 50 mV ( B ).

Voltage dependence. Figure 8 shows that there is a markedfunctional difference of NaPi-IIb1 and NaPi-IIb2 with respect tomembrane potential. NaPi-IIb1 function is relatively insensitive tomembrane potential with current ~75% of maximum at a membranepotential of 20 mV. However, as the membrane potential increases,NaPi-IIb2 function decreases rapidly to ~20% of maximal current at V test = 20 mV. Therefore, NaPi-IIb2 transporterfunction is more dependent on membrane potential, a characteristicsimilar to that of winter flounder ( 12, 21 ) and mammaliansodium-phosphate cotransporters ( 10 ).
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Fig. 8. Voltage dependence of NaPi-IIb1- and NaPi-IIb2-mediatedP i transport. Currents were induced with 1 mmol/lP i in the bath. At steady state, currents were recordedduring a voltage step protocol ( V test = 120 to20 mV, V test = 10 mV). Recorded currents( I ) were normalized to I max andexpressed as means ± SE of 5 and 20 oocytes injected withNaPi-IIb1 and NaPi-IIb2 cRNA, respectively.( X% k& a" F5 f& U7 x

DISCUSSION
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Mammalian phosphate homeostasis is maintained by uptake from thesmall 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 exhibitsmaximal transport rates under alkaline conditions, whereas theintestinal NaPi-IIb function is less sensitive to external pH( 15 ). This ensures that intestinal reabsorption ofP i occurs over a wide pH range, for example, whenchallenged by acidic output from the stomach and by neutralization inthe duodenum.$ i/ o  P3 F* p1 h* R; W
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The direction of renal transepithelial P i transport in fishis closely related to the glomerular filtration rate (GFR) and thus to the habitat of the species. Freshwater fish are hypertonic totheir surroundings and so water and ion balance is achieved by a highGFR followed by reabsorption of sodium, phosphate, and other ions.Marine fish are hypotonic to their surroundings; consequently, GFR isrelatively low to maintain water levels and controlled renal secretioncontributes to maintaining ionic balance. Therefore, in freshwaterteleosts net absorption of P i seems to prevail whereas netsecretion occurs in marine teleosts ( 5 ). Recently, itemerged that members of the NaPi-II protein family play a pivotal role in both P i secretion and reabsorption ( 8, 28 ).Micropuncture studies identified the proximal tubular segment PII ofwinter flounder ( P. americanus ) and skate ( Rajaerinacea ) as the major site of tubular secretion ( 5 ).Experiments carried out in flounder suggested that a basolaterallysorted NaPi-II homolog drives P i secretion in this segment.In adjacent collecting tubules, the same transporter was found in theapical membrane, possibly involved in the tuned reabsorption ofP i ( 8 ). In zebrafish, two distinct NaPi-IIhomologs have been reported; however, their physiological role wasunclear ( 21 ). Figure 9 summarizes the localization of NaPi-IIb cotransporters in fish andNaPi-IIa in the mammalian kidney. Here, we describe the functionalcharacterization of the second isoform NaPi-IIb2 as well as theimmunohistochemical localization of NaPi-IIb1 and NaPi-IIb2.

Fig. 9. Schematic summary of renal tubular phosphate reabsorptionin marine and freshwater teleosts and mammals. Representation andnomenclature of the different nephron segments are adapted fromDantzler ( 5 ). Isoforms of the Na/P i cotransporters are represented in different colors: zebrafish NaPi-IIb1in blue, NaPi-IIb2 in yellow, flounder NaPi-IIb in green, and mammalianNaPi-IIa in red. Phosphate movement is indicated by black arrows.Winter flounder, a marine teleost, exhibits a low glomerular filtrationrate (GFR). P i is secreted in the proximal tubular segmentPII 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 P i secretion.NaPi-IIb in the apical membrane prevents P i wasting and mayreduce the formation of precipitates in highly concentrated urine. Inzebrafish, NaPi-IIb1 and NaPi-IIb2 are both expressed in the apicaltubular membrane and mediate P i reabsorption. Indirectevidence suggests that NaPi-IIb1 and NaPi-IIb2 are expressed in laterparts of the renal tubule. This would indicate that P i excretion in fish is regulated in distal nephron segments regardless ofwhether P i reached the primary urine by glomerularfiltration or tubular secretion. In the mammalian kidney, NaPi-IIamediates P i reabsorption in the early part of the proximaltubule. According to its physiological function, the transporter islocated in the apical membrane. The fact that mammals express the"novel" isoform NaPi-IIa in kidney may reflect the shift fromdistal (fish) to proximal P i reabsorption(mammals).9 h: M- j7 Q- S6 I2 f1 [* R5 q6 Z

NaPi-IIb1 and NaPi-IIb2 Have Overlapping Expression Patterns
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The presence of two NaPi-IIb isoforms represents a common featurein both freshwater and marine species, although the physiological significance of the closely related transporters remains to be established ( 28 ). Our specific antisera localized bothtransporters in the apical membrane of renal tubular cells, intestinalenterocytes, and bile duct epithelia. The data suggest that bothisoforms play a role in accumulating body P i by mediating(renal) reabsorption and intestinal uptake. Expression of NaPi-II inbile ducts has not been reported previously.2 a" r( r7 R' |: ?
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Elevated concentrations of P i and/or Ca 2  inurine or bile increase the risk of stone formation. We hypothesize thatthe physiological role of NaPi-IIb in bile ducts is to scavengeP i to prevent formation of precipitates. P i maybe not only secreted or filtered into bile but also generated by thebreakdown of hormones or other extracellular compounds.
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The two NaPi-IIb isoforms are coexpressed in the same organs. Themarkedly different affinities for P i indicate that the two transporters complement each other to function efficiently over a largerange of extracellular P i concentrations. Thus NaPi-IIb1 would represent a low-affinity, high-capacity system absorbing the bulkof P i, whereas NaPi-IIb2 would be responsible for efficient transport at low external P i concentrations (high affinity,low capacity). The renal reabsorption of glucose in mammals by the sodium-glucose cotransporters (SGLT) follows a similarstrategy. That is, uptake of glucose is mediated predominantly in theproximal convoluted tubule by the low-affinity, high-capacitytransporter SGLT2 with fine control of reabsorption in the straightproximal 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 flankingregions expressing only one of the isoforms. Coexpression of the twoisoforms could prove beneficial if a relatively short tubular fragmenthad to cope with highly variable loads of P i.+ ]9 |* V5 e7 m8 W0 E- Y
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A second renal NaPi-II-related isoform (NaPi-IIc) has recently beenreported in rats and humans ( 24 ). NaPi-IIc ispredominantly expressed in weaning rats and to a lower level in adultanimals. In contrast to all other NaPi-II transporters, NaPi-IIcmediates electroneutral cotransport of sodium and P i.Comparison of NaPi-II protein sequences did not reveal a significantphylogenetic link between zebrafish NaPi-IIb2 and rat or human NaPi-IIc(not shown).
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Zebrafish NaPi-IIb2 Is Functionally Distinct From NaPi-IIb1
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Many cloned NaPi-II cotransporters have been functionallycharacterized by expressing the protein in X. laevis oocytesand 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 P i, 100 mmol/l sodium, and V test = 50 mV. A number of similaritiesbetween 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 m Pi and sodium K m Na are in the range of 43-70µmol/l and 33-46 mmol/l, respectively. The exception iszebrafish NaPi-IIb1, which is relatively voltage insensitive and has alower affinity for phosphate and sodium ( K m Pi and K m Na 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 ismuch less sensitive to external pH than other members of the family.The functional properties of the newly cloned zebrafish NaPi-IIb2 wereof interest to further elucidate the role of NaPi-IIb cotransporters infish P i handling.
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Phosphate dependence of the NaPi-II cotransporters. Zebrafish NaPi-IIb1 and NaPi-IIb2 exhibit significantly differentapparent affinities for external phosphate ions; K m Pi are 250 ( 21 ) and29.3 µmol/l, respectively.7 P" O4 ^; |0 q

The molecular basis of the wide range of reported affinities is as yetundetermined. All cloned NaPi-IIb cotransporters have a relatively highaffinity for phosphate ( K m Pi = 10-50µmol/l) with the exception of zebrafish NaPi-IIb1( K m Pi = 250 µmol/l) ( 17 ).ICL1 and ECL3 of rat NaPi-IIa show high degrees of homology and aresuggested to be of functional importance ( 17 ). Comparisonof the aligned sequences for the NaPi-IIb cotransporters (Fig. 2 ) showsthat ECL3 is well conserved between all NaPi-IIb isoforms. In contrast,zebrafish NaPi-IIb1 has a number of divergent amino acids in ICL1 thatmay indicate the phosphate binding pocket of the NaPi-II cotransporter family.( k: x% F/ |5 \3 X( M$ W
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Sodium dependence of the NaPi-II cotransporters. The reported values for the sodium affinity of NaPi-II cotransportersdisplay a wide range. Reported values of K m Na are in the range of 37 (14) to 62 mmol/l ( 6 ) for mammalian NaPi-IIa,20 (15) to 38 mmol/l ( 6 ) for mammalianNaPi-IIb, 46 mmol/l ( 12 ) for flounder NaPi-IIb, and 67 mmol/l ( 21 ) for zebrafish NaPi-IIb1. Calculated values of K m Na are likely to be subject toerror because current recordings are made below saturating sodiumconcentrations. The calculated K m Na (42 mmol/l) for zebrafish NaPi-IIb2 from this study is similar topublished data for other NaPi-II transporters.5 k& m7 q7 i, Z/ s4 R; S
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De la Horra et al. ( 6 ) reported differences between K m Na for mammalian NaPi-IIa andNaPi-IIb transporters expressed in X. laevis oocytes.Mutagenesis studies indicated that a single amino acid switch fromphenylalanine to leucine in NaPi-IIa caused an increase in affinity,similar to values reported for NaPi-IIb. This residue is well conservedbetween all NaPi-IIb isoforms (Fig. 2, green overscore) and so isunlikely to solely explain the decreased sodium affinity of NaPi-IIb1. Therefore, other regions are likely to play a role in sodium binding toNaPi-II transporters.
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pH dependence of the NaPi-II cotransporters. The differences in pH sensitivity of mammalian NaPi-IIa and NaPi-IIbhave 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 ofmammalian NaPi-IIa and gain of pH sensitivity of NaPi-IIb (refer toFig. 2, red overscore) ( 7 ). Flounder NaPi-IIb has only two charged amino acids in the same region (AEK) and is shown to exhibit pHdependence similar to mammalian NaPi-IIa ( 12 ). A similar phenomenon was reported for zebrafish NaPi-IIb1, which contains onlyone charged amino acid (GET). NaPi-IIb2 has this same motif (GET) andso was also predicted to exhibit pH dependence in a NaPi-IIa likemanner. 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 IIbcharacteristic), but function was severely curtailed between pH 6.0 and6.5 (a type IIa characteristic). Therefore, we suggest that another, asyet unidentified, motif must play a role in determining the pHsensitivity of transport in zebrafish NaPi-II cotransporters.
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Voltage dependence. NaPi-IIb2 function was found to be strongly dependent on membranepotential (cotransport was reduced by 70% on depolarizing the oocytemembrane from 120 to 0 mV). This finding is conserved between allcloned NaPi cotransporters with the exception of zebrafish NaPi-IIb1,which is relatively voltage insensitive.$ d! R( I! Y1 N

One surprising finding in our study was that the calculated affinitiesfor phosphate and sodium displayed unexpected sensitivities to membranepotential. Forster et al. ( 10-12 ) proposed aneight-stage model of sodium phosphate cotransport with the orientationof the empty carrier and the binding of the first sodium ion dependent on membrane potential. The binding of phosphate to the carrier wasthought to be independent of membrane potential. In accordance withthis model, all cloned and characterized NaPi-II cotransporters havedisplayed voltage-sensitive K m Na and voltage-insensitive K m Pi. However,our data show the opposite trend, with sodium binding displaying novoltage 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.7 j# t1 H; t+ c

In conclusion, zebrafish express two distinct NaPi-IIb-typecotransporters. 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 theiraffinity for phosphate and sodium ions, sensitivity to membrane potential, and the effect of pH on transport rates. These properties are summarized in Table 1.

Table 1. Comparison of zebrafish NaPi-IIb1 and NaPi-IIb2 structural andfunctional properties. p& Y( B6 ^3 |, s% f9 e! y& r4 s
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These findings highlight the wide diversity in type II NaPicotransporter function despite strong structural similarities betweenfamily members. Zebrafish NaPi-IIb1 and NaPi-IIb2 are closely relatedat the amino acid level but show different functional properties. Thesetransporters are a useful model for the future investigation of aminoacid motifs responsible for phosphate, sodium, and hydrogen ion interaction.1 r; z; _  ?% l2 ^' [  ^! `/ K

ACKNOWLEDGEMENTS8 h/ I  C  I, `: ?. s5 g1 ]" u
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The authors thank Ursula Strunck, Brian Burtle, and Heike Rimpelfor excellent technical assistance.
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