Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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The human renal Na-PO4 cotransporter gene NaPi-3 was expressed in human embryonic kidney HEK-293 cells, and the transport characteristics were measured in cells transfected with a vector containing NaPi-3 or with the vector alone (sham transfected). The initial rate of 32PO4 influx had saturation kinetics for external Na and PO4 with K Na1/2 of 128 mM (PO4 = 0.1 mM) and K PO41/2 of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfected cells expressing the transporter. Transfection had no effect on the Na-independent 32PO4 influx, but transfection increased Na-dependent 32PO4 influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenate inhibited flux with an inhibition constant of 0.4 mM. The phosphate transport in sham- and NaPi-3-transfected cells has nearly the same temperature dependence in the absence and presence of extracellular Na. The Na-dependent phosphate flux decreased with pH in sham-transfected cells but was pH independent in transfected cells. The Na-dependent 32PO4 influx was inhibited by p-chloromercuriphenylsulfonate, phosphonoformate, phloretin, vanadate, and 5-(N-methyl-N-isobutyl)-amiloride but not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior of NaPi-3 homologues in the renal tubule of other species and, thus, demonstrate the fidelity of this transfection system for the study of this protein. Commensurate with the increased functional expression, there was an increase in the amount of NaPi-3 protein by Western analysis.
kinetics; sodium activation; membrane transport; pharmacology
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INTRODUCTION |
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SODIUM-PHOSPHATE COTRANSPORT is important for intestinal absorption of dietary phosphate, renal reabsorption of the filtered load of phosphate, and maintenance of intracellular phosphate concentration in all cells above electrochemical equilibrium. The red blood cell is an exception, because phosphate is near electrochemical equilibrium owing to the fourfold larger exchange flux of 32PO4 on AE1 (band 3) than the influx on the erythrocyte Na-phosphate cotransporter (23). The apical membrane transport in renal and gastrointestinal cells is mostly Na dependent, but the relative amounts of exchange and cotransport in the basolateral membranes are unknown. Several laboratories have investigated the Na-phosphate cotransporter in renal brush-border vesicles, but detailed kinetics have been difficult to establish (for review see Ref. 18). Several mammalian Na-phosphate (Na-Pi) cotransporters have been cloned, some (e.g., NaPi-1, NaPi-2, and NaPi-3, Genbank accession numbers M76466, L13257, and L13258, respectively) by expression cloning in oocytes (16, 28) and others by homology screening based on the former (6, 24, 29). Most recently, two cloned retrovirus receptors (19) were identified as homologous to a phosphate permease in Neurospora crassa (9) and were shown to be Na-dependent phosphate cotransporters (10, 20). Expression and kinetic characteristics of these proteins have been principally determined in Xenopus oocytes measuring 32PO4 uptake. The Na-PO4 transport activity expressed from size-fractionated poly(A)+ RNA from rabbit kidney cortex had apparent Michaelis-Menten constants for PO4 and Na (K PO4m and K Nam) of 0.19 ± 0.06 and 63 ± 19 mM, respectively, when Na replaced choline (27). The phosphate activation followed the Michaelis-Menten equation [Hill coefficient (n) = 1], whereas the Na activation had an n of 1.5 ± 0.3 (27). K Nam of NaPi-1 from cRNA was 50-60 mM. K PO4m of NaPi-2 expressed from cloned cRNA was 0.130 ± 0.015 mM and K Nam was 42 ± 7 mM (n = 2.5). K PO4m of NaPi-3 cRNA expressed in oocytes was 0.170 ± 0.023 mM and K Nam was 57 ± 13 mM (n = 2.1) (16). Quabius et al. (22) recently reported Na-PO4 cotransport expression by rat NaPi-2 in Madin-Darby canine kidney (MDCK) cells and a pig kidney cell line (LLC-PK1). In the study of Quabius et al., controlled expression of NaPi-2 by a dexamethasone-inducible element increased Na-dependent 32PO4 uptake 5- to 10-fold in MCDK cells but very little in LLC-PK1 cells. The transport kinetics were characterized only with regard to the pH dependence, which showed a maximum flux symmetrical about pH 7.5 and a four- to fivefold lower flux at pH 6.5.
This is the first report of human NaPi-3 expression in cultured mammalian cells. We have measured the initial 32PO4 influx in human embryonic kidney (HEK-293) cells transfected with the vector alone (sham) or with the human renal NaPi-3 transporter expressed transiently in these cells. The kinetics have been characterized with respect to cation specificity, Na activation, phosphate activation, arsenate inhibition, pH dependence, temperature dependence, and the pharmacology of amiloride analogs and other compounds. We have found by reverse transcription-polymerase chain reaction (RT-PCR) analysis of HEK-293 cells that NaPi-3 (but not NaPi-1) is expressed in vector-only- and NaPi-3-transfected cells without significant differences in the amount of RT-PCR product, although functional expression increased 2.5-5 times. By Western analysis we have shown that functional expression was accompanied by a parallel increase in the amount of NaPi-3 protein that was of the proper molecular weight and glycosylation.
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METHODS |
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Materials and general methods. The cDNA construct for human Na-Pi cotransporter (NaPi-3) was a kind gift of Dr. Heini Murer (University of Zurich, Zurich, Switzerland). Fetal calf serum was obtained from Atlanta Biologicals (Norcross, GA); minimum essential medium (MEM), L-glutamine, and other media components were obtained from Life Technologies (Gaithersburg, MD). Disposable plastic culture flasks and dishes were obtained from Corning (Cambridge, MA). All chemicals were reagent grade or better and were obtained from Fisher Scientific (Norcross, GA) or Sigma Chemical (St. Louis, MO). Enzymes used in molecular biology manipulations were obtained from Life Technologies or New England Biolabs (Beverly, MA). Plasmids were purified using reagents and purification matrix (BIGPrep system) from 5 Prime-3 Prime (Boulder, CO) according to the manufacturer's protocol. Plasmid prepared by this method was free of RNA and typically was 80-90% supercoiled, as judged by agarose gel electrophoresis. Isotopes were purchased from New England Nuclear (Boston, MA).
Subcloning of NaPi-3 into mammalian expression vector. The plasmid (pSPORT-NaPi3) provided by Dr. H. Murer (16) contained the NaPi-3 cDNA [2,573 base pairs (bp)] in pSPORT-1 (Life Technologies). The cDNA contained the coding region (bp 82-1998 of insert), partial 5' untranslated region (UTR; bp 1-81 of insert), and the 3' UTR (bp 1999-2573 of insert) of NaPi-3. The plasmid containing NaPi-3 (pSPORT-NaPi3) was treated with EcoR I and Hind III, and the 2,186-bp fragment was gel isolated. This fragment contained the complete coding region for NaPi-3, 56 bp of 5' UTR, and 211 bp of 3' UTR. The expression vector, pRBG4, has been described previously (13) and has a multiple cloning site flanked by the cytomegalovirus transcriptional promoter and the SV40 polyadenylation signal sequence. The expression vector was linearized by treatment with EcoR I and Hind III and then gel isolated. Ligation of the isolated NaPi-3 coding region with the pRBG4 yielded a construct pRBG4-NaPi3, in which the NaPi-3 was in the correct orientation for transcription from the cytomegalovirus promoter (Fig. 1).
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Transfection of HEK-293 cells. HEK-293 cells were obtained from American Type Culture Collection at passage 31. These cells were used to prepare seed stocks at passage 32. Cells were used until passage 45, and then fresh cultures were started from frozen cells from passage 32. The cells were grown in MEM with Hanks' salts and supplemented with L-glutamine and 5% fetal calf serum at 37°C in 5% CO2-95% air. Transfection was carried out using standard calcium phosphate precipitation methods. Specifically, 5 days before the transfection, cells were plated in T75 flasks (75 cm2) at 2.5 × 104/cm2. On the day of the transfection the cell density was usually 2-3 × 105/cm2. The cells were washed, and fresh medium (20 ml) was placed in each culture flask. A 1.0-ml suspension containing the calcium phosphate-DNA precipitate from 40 µg of plasmid DNA was added dropwise with mixing to the media overlaying the cells. The cells were returned to the incubator for 4 h, and then 2 ml of 18% (vol/vol) glycerol were added to the media ("glycerol shocked"), and the cells were incubated for an additional 2 min at room temperature. The medium was then quickly aspirated from the flask, the cells were washed once with 25 ml of Dulbecco's phosphate-buffered saline (PBS), fresh medium was added to the cells (25 ml), and the cells were incubated overnight. On the next morning the cells were trypsinized by standard methods and resuspended to a final density of 1.5-1.7 × 105/ml, and 1.0 ml of the cell suspension was used to replate the cells in 24-well plates (16-mm-diameter wells; model 25820, Corning) at a density of 8.0 × 104 cells/cm2 (1.6 × 105 cells/well). Flux measurements were carried out at 48 ± 6 h posttransfection.
32PO4 influx measurements in HEK-293 cells. One milliliter of MEM from each well was aspirated and replaced with 0.5 ml of Na-free MEM-like medium for 5-6 min; other wells on all the 24-well plates were similarly treated. The Na-free MEM-like medium contained (mM) 143 N-methyl-D-glucamine chloride (NMDG-Cl), 4.36 KCl, 1.8 CaCl2, 0.81 MgCl2, 5.55 D-glucose, and 25 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and was titrated with KOH to pH 7.4 (at 37°C) and 7.65 (at 20°C). The wash medium was then aspirated and replaced with 0.5 ml of the same fresh Na-free medium that would remain on the cells until it was replaced by the influx medium. As a consequence, in the cells that normally have 73 ± 14 µmol Na/g protein, Na content (2.5 ± 1.0 µmol/g protein) and concentration are very low at the initiation of the flux. During all the influx measurements in Na-containing media, the Na content of the cell rises to steady state within the first 5 min (data not shown). The cells were incubated for 30-40 min at 37°C in room air and then placed on a water-thermostated platform at the desired temperature (21 ± 2°C), except where noted. The influx was initiated within 5 min after transfer to the thermostated platform. At known times the Na-free supernatant in individual wells was aspirated and the 32PO4-containing medium with or without Na was added. The flux medium was the same as the Na-free MEM medium, except it contained phosphate, usually 0.1 mM K2HPO4/KH2PO4, except as noted, and 2 µCi of 32PO4/ml, and 143 mM NaCl replaced the NMDG-Cl. The plate was intermittently and irregularly shaken during the influx. In general, we began the influx in a row of six wells at 30, 15, 10, 5, 2, and 1 min before termination of the influx, and we measured duplicate fluxes for each condition on the same plate. The influx was terminated by flicking the liquid contents of the wells into a radioactive waste container and washing all 24 wells simultaneously in ice-cold stopping solution containing (mM) 150 NaCl, 1 CaCl2, and 1 MgCl2. The wells were washed three times within 15 s. Washing was accomplished using a washing manifold constructed from culture plate tops with 24 holes into which were glued 5-ml plastic test tubes (15). The washing manifold was filled with stop solution and precooled on ice. Three separate manifolds were used to wash each plate to terminate the flux. After the plates were washed, the residual wash solution in each well was quickly aspirated. To each of the wells, 200 µl of 2% (wt/wt) sodium dodecyl sulfate (SDS) solution were added to lyse the cells; 100 µl of this solution were counted with 3 ml of Optifluor (Packard Instruments, Meriden, CT) and counted in a liquid scintillation counter. The remaining 100 µl in the well were used to measure the amount of protein per well. An equally good method was used in later experiments, where we added 500 µl of 25 mM NaOH and 0.1% sodium deoxycholate to lyse the cells and then used 50 µl to measure protein and 350 µl for liquid scintillation counting. Triplicate 10-µl samples of the influx solution were counted contemporaneously for the determination of specific activity. Protein was determined using a standard bicinchoninic acid (BCA) protein assay (30), in which bovine serum albumin (Sigma Chemical) was used as a standard [extinction coefficient at 280 nm for a 1% (wt/vol) solution in 0.9% NaCl = 6.10]. The assay was carried out in 96-well microtiter plates (model 25880-96, Corning). Briefly, 50 µl of distilled water, followed by 100 µl of complete BCA reagent (prepared according to manufacturer's protocol, Pierce Chemical, Rockford, IL), were added to each well containing 50 µl of cell lysate. The microtiter plate was then covered with adhesive film, samples were incubated at 55-60°C for 15 min and cooled to room temperature, and the absorbance at 595 nm was determined using a Titertek Multiskan (ICN, Costa Mesa, CA) plate reader. The standard curve on each plate was prepared by making dilutions (0.01-0.40 mg/ml) of a stock bovine serum albumin solution (2 mg/ml in 0.9% NaCl) in 1% SDS or 12.5 mM NaOH and 0.05% sodium deoxycholate. The data for the standard curve were fit to a quadratic equation, and the amount of protein in the samples was calculated from the fit parameters. The net counts per minute (cpm) per microgram of protein were graphed against the elapsed time of exposure to the radioactive influx solution. The six data points were fit to a straight line by the least-squares method (Fig. 2). The slope of the fit line divided by the specific activity (cpm/nmol) was the calculated flux. The standard error of each flux value was calculated by dividing the standard error of the slope by the specific activity. The influx was usually linear for at least 30 min for 32PO4 influx measurements.1 Hyperbolic activation curves were fit by nonlinear regression with the Levenberg-Marquardt algorithm using the XLMath dynamic link library add-in (version 3.0, Roy Kari) for Microsoft Excel (version 5.0, Microsoft, Redmond, WA).
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Cell protein content. The amount of protein per 105 cells was determined in several aliquots of freshly dissociated and washed cells at different cell concentrations as determined by counting in a hemocytometer and trypan blue exclusion from the cells (>90% viable) and measuring the protein by the BCA assay. In our hands, HEK-293 cells have 9.8 µg protein/105 cells when grown at a density of 1-5 × 105 cells/cm2.
Assay of Pi. Pi was measured using a modification of a published method (7). Briefly, a fresh solution of 47 mg of malachite green (Sigma Chemical) in 100 ml of 0.1 N HCl was prepared and filtered if necessary through Whatman no. 2 filter paper. Stock solutions were made of molybdic acid (5.72%, wt/vol) or sodium molybdate (4.2%, wt/vol) in 4 N HCl and a 1:50 dilution (vol/vol) of Tween 20 (Sigma Chemical) in water, and an oxidizing solution was made of 5 ml of concentrated 36 N H2SO4 and 10 ml of 70% perchloric acid added to 500 ml of water. A dilution mix of 180 ml of malachite green stock, 60 ml of molybdic acid stock, and 1.5 ml of diluted Tween 20 was made. In 13-ml plastic test tubes we placed 100 µl of sample or standard (0.129 mM K2HPO4, 4 mg P/l), 100 µl of oxidizing stock solution, and 1 ml of dilution mix. The optical density at 650 nm was measured in a Novaspec II spectrophotometer (Pharmacia-LKB, Piscataway, NJ) against a control of glass-distilled water.
RT-PCR analysis of Na-phosphate cotransporter isoforms in HEK-293 cells. Oligo deoxynucleotide primers were designed to specifically amplify the NPT1 or NaPi-3 Na-phosphate cotransporters. NPT1 and NaPi-3 are the two Na-phosphate cotransporter isoforms that have been described for human kidney. The primers specific for NPT1 are predicted to amplify a 900-bp product from a cDNA template; the sequences for these primers are as follows: 5'-GAGAAT(A/C)TGGGT(C/G)AA(A/G)TGGGC-3' (forward) and 5'-(C/T)TT(G/A)GCCCAGTCCTGGATCTC-3' (reverse). The primers specific for NaPi-3 are predicted to amplify a 450-bp product from a cDNA template; the sequences for these primers are as follows: 5'-AGTCTCAT(C/T)C(A/G)GAT(C/T)TGGTG-3' (forward) and 5'-GGCCAG(G/T)GC(A/T)GCCAGGAT-3' (reverse). Total RNA was isolated from HEK-293 cells transfected with vector alone (pRBG4) or vector containing NaPi-3 cDNA (pRBG4-NaPi3) using TRIzol LS (Life Technologies). RT-PCR was carried out by means of a single-tube reaction for the RT and the PCR reactions using recombinant Thermus thermophilus DNA polymerase and reagents essentially as recommended by the manufacturer's protocol (EZ rTth RNA PCR kit, Applied Biosystems, Foster City, CA). The RT-PCR reaction was carried out in a volume of 50 µl and contained 2.0 mM manganese acetate, 1 µM each primer, 300 µM each dNTP, 5 U of rTth DNA polymerase, and 100 ng of total RNA. The reaction was carried out in 500-µl microtubes and overlaid with paraffin wax. An Ericomp EZ Cycler TwinBlock thermocyler was used for temperature cycling, and the program was as follows: RT for 1 cycle at 60°C for 30 min, then an initial step at 94°C for 5 min, 40 cycles at 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, and, finally, 1 cycle at 72°C for 10 min. After the RT-PCR reaction, 10 µl of the reaction were removed and analyzed by gel electrophoresis [1% agarose in 0.2 M tris(hydroxymethyl)aminomethane (Tris) base, 0.18 boric acid, and 0.004 M Na2-EDTA and electrophoresis at 0.5 V direct current (DC)/cm gel]. The size of the reaction products was estimated by comparison to known molecular weight standards (1-kb ladder, Life Technologies).
Peptide synthesis and production of antibodies. A peptide corresponding to NH2-terminal residues 77-93 (K-L-A-L-E-E-E-Q-K-P-E-S-R-L-V-P-K) was synthesized and purified by reverse-phase high-performance liquid chromatography (Emory University Microchemical Facility, Winship Cancer Center). An additional cysteine residue was added to the NH2 terminus of the peptide to facilitate conjugation of the peptide to carrier protein. The immunizing peptide was conjugated via its NH2-terminal cysteine to keyhole limpet hemocyanin (KLH; Pierce) with sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC; Pierce). Briefly, 2.0 ml of KLH (10 mg/ml in water) were first activated by addition of 1.0 ml of sulfo-SMCC (4.6 mM or 2 mg/ml) dissolved in conjugation buffer (83 mM Na2HPO4, 900 mM NaCl, and 100 mM EDTA). The activation reaction of KLH and sulfo-SMCC was allowed to proceed for 1 h at room temperature (18-22°C). The unreacted sulfo-SMCC was removed from KLH and sulfo-SMCC-activated KLH by gel filtration column chromatography. The reaction mixture was applied to a 10-ml column (EconoPac 10DG column, Bio-Rad, Hercules, CA) equilibrated with conjugation buffer, and fractions (2.5 ml) were collected. The protein concentration in each fraction was determined as described above, and the fractions containing the protein peak (fractions 4-7) were pooled. The activated protein (sulfo-SMCC-activated KLH) was incubated with 10 mg of the peptide dissolved in 2.5 ml of conjugation buffer. The conjugation reaction was allowed to proceed for 2 h at room temperature and then dialyzed overnight at 4°C against 500 ml of PBS containing (mM) 1.76 KH2PO4, 8.05 Na2HPO4, 136 NaCl, and 2.68 KCl (pH 7.2). The final solution contained 2.1 mg/ml peptide-KLH in PBS (~0.4 mg peptide/ml and 1.7 mg KLH/ml). The peptide conjugate (0.33 ml) was emulsified with Freund's complete adjuvant (0.67 ml), and 0.5 ml was injected subcutaneously and 0.5 ml intradermally into New Zealand White rabbits. Additional injections in incomplete Freund's adjuvant were administered at 1, 2, 4, 8, and 12 wk after the initial injection. Positive antiserum was obtained at 4 wk, before the third boost. Injections were carried out and antiserum was obtained according to standard protocols at Lampire Biological Laboratories (Pipersville, PA). Control sera were obtained from the same rabbits before the first injection. Three rabbits were injected according this protocol; however, only rabbit 4889 produced positive antiserum that was specifically competed by the immunizing peptide in Western analysis.
Preparation of cell extracts.
After transfection as described above, HEK-293 cells were plated in
35-mm culture dishes at 1.5 × 105
cells/cm2 (~1.5 × 106 cells/plate). Total cell
lysates were prepared at 48-72 h posttransfection. The cells were
first washed three times with Dulbecco's PBS containing calcium and
magnesium (in mM: 2.67 KCl, 1.47 KH2PO4,
138 NaCl, 8.1 Na2HPO4,
0.90 CaCl2, and 0.49 MgCl2). The cells were lysed directly in the culture dish by addition of 0.5 ml of lysis buffer containing 50 mM HEPES · KOH (pH 7.6), 200 mM NaCl,
5% SDS, 1.0 mM ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 2.0 mM Pefabloc (Boehringer-Mannheim, Indianapolis, IN), and 5 µg/ml leupeptin and incubated at room temperature for 5 min. Then the
plate was scraped with a disposable cell scraper, and a
positive-displacement pipette was used to remove the lysate to a
microcentrifuge tube (1.5 ml). The tube containing the lysate was
placed in a beaker with ice and sonicated with five 30-s pulses (50%
duty cycle, output setting = 1) using a 2.5-mm-diameter probe (Sonifier
350, Branson Ultrasonics, Danbury, CT). Samples were frozen in liquid
nitrogen and stored at
80°C until use. The protein concentration of diluted lysate samples (1:10-1:100 in 2% SDS) was determined using the BCA protein assay as described above. Human
kidney medulla and cortex were obtained from Dr. Jeff M. Sands (Renal
Div., Dept. of Medicine, Emory University School of Medicine). The
medulla and cortex were dissected from whole human kidney (free of
human immunodeficiency virus, hepatitis A, and hepatitis B) that was
generally healthy but was rejected for transplant use. After dissection
the medulla and cortex sections were frozen in liquid nitrogen and
stored at
80°C until use. The frozen kidney sections were
weighed and then powdered in a mortar and pestle in the presence of
liquid nitrogen. Lysis buffer (described above) was added to the
powdered tissue at 4.0 ml/g tissue, and the extract was further
homogenized by 10-12 up-down strokes in a Tenbroeck tissue grinder
(0.15-mm clearance). Protein concentration was determined as for
lysates prepared from cultured cells.
Western analysis.
SDS-polyacrylamide gel electrophoresis was carried out according to the
procedure of Laemmli (12) as modified by Staehelin et al. (25). The
stacking gel contained 5% acrylamide, 0.13% bisacrylamide, 0.125 M
Tris · HCl (pH 6.8), and 0.1% SDS. The resolving gel
contained 7.5% acrylamide, 1.33% bisacrylamide, 0.375 M
Tris · HCl (pH 8.8), and 0.1% SDS. Samples (10 µl)
were diluted with 10 µl of sample buffer (88 mM
Tris · HCl, pH 6.8, 25% glycerol, 8% SDS, 0.01%
bromphenol blue, and 715 mM -mercaptoethanol) and heated for 10 min
at 95°C; 10-20 µl were applied to the gel. The running
buffer contained 0.025 M Tris base, 0.192 M glycine, and 0.1% SDS at
pH 8.3. Electrophoresis was carried out at a constant voltage of 200 V
DC until the dye front was ~5 mm from the bottom of the gel (~40
min). The gel was then washed in transfer buffer (2 changes of buffer
in 30 min). Electrophoretic transfer (100 V DC, 250-350 mA for 1 h) onto nitrocellulose paper (type BA85, 0.45-µm pore size,
Schleicher and Schuell, Keene, NH) was performed essentially as
previously described (21, 26). After transfer the nitrocellulose was
blocked for 1 h at room temperature with a solution containing 5%
(wt/vol) nonfat dry milk (blotting grade blocker, Bio-Rad) and
0.05% (vol/vol) Tween 80 in HN medium (in mM: 10 HEPES · KOH, pH 7.6, and 150 NaCl). After the
blocking step the nitrocellulose was washed for 30 min at room
temperature with six changes of HN media. The blots were incubated for
1 h at room temperature with the primary antiserum at a dilution of 1:1,000 in a solution containing 0.5% nonfat dry milk in HN medium and
then washed as described above. In some experiments the immunizing peptide was included (1 µg/ml) with the primary antiserum. The blots
were then incubated for 1 h at room temperature with the second
antibody (goat anti-rabbit immunoglobulin G conjugated to horseradish
peroxidase; KPL, Gaithersburg, MD), diluted to 20 ng/ml in a solution
containing 0.5% nonfat dry milk in HN medium, and then washed as
described above. The primary antibody-secondary antibody complex was
visualized using an enhanced luminol-based reagent (SuperSignal,
Pierce). Briefly, the blots were incubated with SuperSignal reagent for
1 min, excess reagent was carefully drained from the blot, and then the
blot was placed in a clear polyvinyl envelope against autoradiography
film (Reflections film, New England Nuclear). Exposure to the film
varied from 15 to 60 s. Prestained molecular weight markers (Bio-Rad)
were run on all gels. The apparent molecular weight of the prestained
markers was always determined using unstained standard protein markers in separate experiments. Imaging and densitometry were carried out
using an IS1000 imaging system and software (Alpha Innotech, San
Leandro, CA). Where indicated, the density value is in arbitrary units
and is the sum of all pixel values after background correction. In some
experiments the samples were treated with recombinant endoglycosidase
Hf (New England Biolabs). Briefly,
25 µg (10-20 µl) of total protein in lysis buffer (as
described above) were diluted to a volume of 45 µl with a solution
containing 0.5% (wt/vol) SDS and 1% (vol/vol)
-mercaptoethanol. The sample was then heated at 100°C for 10 min
and cooled to 37°C, and then 5 µl of reaction buffer (0.5 M
sodium citrate, pH 5.5) and 1 µl of endoglycosidase Hf (1,000 U) were added to the
denatured protein. Incubation was continued at 37°C for 60 min, and
the reaction was terminated by addition of an equal volume of sample
buffer and heating at 95°C for 10 min.
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RESULTS |
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32PO4 uptake by HEK-293 cells.
To detect the NaPi-3-induced and background Na-phosphate cotransporter
activity, we have measured the
32PO4
influx. The transport activity of HEK-293 cells was determined from
cells transfected with vector alone (pRBG4, sham transfected) and was
always studied in parallel with HEK-293 cells transfected with the
NaPi-3 expression construct (pRBG4-NaPi3) at the same time using the
same reagents. We have found that the
32PO4
influx in wild-type cells was 0.5-0.7 times that in the
sham-transfected cells (data not shown). Thus the transfection
procedure itself had an effect on transport. This demonstrates the
importance of using sham-transfected cells, rather than untransfected
wild-type cells, for controls, as we have done in this study. The
influx of
32PO4
was measured at 24, 48, 72, and 96 h posttransfection; the peak of
expressed NaPi-3 activity was at 48-72 h posttransfection, and
activity rapidly dropped by 96 h posttransfection (Fig.
3). All subsequent experiments were
performed at 48 ± 6 h. The background influx of
32PO4
measured in Na-free medium was not different among untransfected wild-type cells, sham-transfected cells, and NaPi-3-transfected cells,
indicating that no other Na-independent phosphate entry pathway was
induced by the NaPi-3 transfection. The influx values in Na-free media
were 3.6 ± 5.0 and 4.5 ± 6.2 nmol · g
protein1 · min
1
in sham- (n = 20) and
NaPi-3-transfected cells (n = 21),
respectively. These values were very low compared with the flux in
media containing 143 mM Na, so they represent <10% of the
Na-activated
32PO4
influx into sham-transfected cells and <1% of the influx into NaPi-3-transfected cells. Consequently, we have not subtracted the
background influx in Na-free media.
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Alkali cation activation of phosphate influx. The background (in sham-transfected cells using the vector only) and NaPi-3-specific 32PO4 influxes are strongly activated only by extracellular Na ions. The other alkali cation metals caused slight activations over isosmotic NMDG as the principal cation (Fig. 4). Li consistently caused activation that was 10-30 times the flux in the NMDG control solutions but only 1/25th-1/50th of that caused by Na ions. By flame emission spectroscopy the NMDG solution contained 1 µM Na, whereas the Li, K, Rb, and Cs stock solutions contained 20-30 µM Na. Thus Na and Li activate 32PO4 influx, but the maximum flux is 25-50 times greater in Na than in Li media.
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Na activation. The Na activation of 32PO4 influx has Michaelis-Menten kinetics with the same K Na1/2 values in sham- and NaPi-3-transfected cells (KNa1/2 = 128 mM) but a greater maximal reaction velocity (Vmax) in the NaPi-3-transfected cells (Fig. 5). The specific influx induced by NaPi-3 expression is the difference between these two curves, and thus it also has the same K Na1/2 value as the background transport system shown by the sham-transfected cells.
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Phosphate activation. The influx of 32PO4 in the presence of 143 mM Na (~1.1 × K Na1/2) is activated by extracellular Pi. At pH 7.65, 15% of the phosphate is monovalent H2PO4 and 85% is divalent HPO4. There is no formation of the ion pair NaHPO4. The phosphate activation was fit to a Michaelis-Menten hyperbola with the best K PO41/2 equal to 84 µM total phosphate for the cells transfected with the vector alone and a similar value for the cells transfected with NaPi-3 (Fig. 6). Thus the background Na-activated PO4 transporter and the expressed NaPi-3 cotransport have the same apparent Michaelis coefficient.
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Arsenate inhibition. Similar to the human erythrocyte Na-Pi cotransporter that is inhibited with an inhibition constant (Ki) of 2.6 mM arsenate (unpublished observations), renal Na-Pi cotransporters in several species are partially inhibited by arsenate (8). We therefore tested whether the background and NaPi-3-expressed Na-Pi cotransporters were arsenate inhibitable. The sham- and NaPi-3-transfected cells have an arsenate-inhibitable component of their Na-dependent 32PO4 influx. The difference between the fluxes in NaPi-3- and sham-transfected cells is the NaPi-3-mediated transport shown in Fig. 7. The Ki for KH2AsO4 in 0.1 mM total phosphate and 143 mM Na was 0.4 ± 0.1 mM.
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Dependence on extracellular pH. The background Na-activated 32PO4 influx (0.1 mM total phosphate) declined 5.5-fold as pH increased from 5.97 to 8.32 in the extracellular medium (Fig. 8). However, the Na-activated 32PO4 influx in NaPi-3-transfected cells was independent of pH. Because this influx included background and NaPi-3-specific fluxes, the difference in the fluxes, that due to the NaPi-3 expression, must have increased as pH increased from 5.97 to 8.32. Because all the cells were equivalently treated before the initiation of the influx, the increase in specific NaPi-3-mediated flux at high pH values must be due to increased turnover of the cotransporter or activation of resident transporters and not to any differences in the number of transporters in the cell membranes at the different pH values. The 5.5-fold decline in the flux in sham cells over 2 units of change in pH is much less than the 10-fold/pH unit expected for titration of a single carrier site or for the decrease in H2PO4 concentration if it were the sole substrate. Because the NaPi-3-specific flux increases only twofold, the same argument excludes any simple single-site mechanism for the pH dependence of NaPi-3 function in these cells.
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Temperature dependence. The temperature dependence of the 32PO4 influx was assessed in cells treated with the standard wash and preincubation at 37°C, but only then placed on the water-thermostated platform for 5 min before initiation of the flux measurement. The radioactive influx medium was prewarmed or cooled in the same water bath, and the temperature was measured with a probe placed in one of the 24 wells. Thus, although the cells at different temperatures began with the same contents, one would expect that their steady-state Na and phosphate contents at the end of the 30-min flux period would differ.
The sham- and NaPi-3-transfected (Fig. 9) fluxes measured in the absence of Na were nearly the same and had negative activation energies (EA):
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Pharmacology of inhibition.
In addition to arsenate, NaPi-3-mediated
32PO4
influx was inhibited by
p-chloromercuriphenylsulfonate
(pCMBS), vanadate, phloretin, and one amiloride analog, but not by
amiloride itself (Fig. 10). The inhibition by the sulfhydryl reagent
pCMBS (0.52 mM caused 95% inhibition of the NaPi-3-induced flux)
suggests that one or more of the native cysteine residues (13 total) on
the protein is important for transport function. The stilbene
disulfonates 4,4'-diisothiocyanostilbene-2,2'-disulfonic
acid (DIDS; not shown) and
4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) did not
inhibit flux. Phosphonoformate was a moderate inhibitor (0.09 mM caused 75% inhibition). In contrast, the
Ki in opossum
kidney (OK) cells is 6 mM. In Fig. 11 the
inhibition by amiloride analogs (0.09 mM), but not amiloride itself
[methylisopropyl amiloride (MIA) phenamil
5-(N,N-hexamethylene)-amiloride (HMA)
benzamil > ethylisopropylamiloride (EIPA)
5-(N,N-demethyl)-amiloride (DMA)
amiloride], suggests that the Na-binding site in the NaPi-3
protein has a specific conformation that is distinct from the
Na-binding site of the aldosterone-stimulated, amiloride-sensitive Na
channel of the renal distal tubule and cortical collecting duct and
different from the Na-binding site of the amiloride-sensitive Na/H
exchanger (11).
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RT-PCR using NPT1- and NaPi-3-specific primers. To determine and compare the isoforms of renal Na-phosphate cotransporters present in these cells, we carried out RT-PCR on total RNA isolated from HEK-293 cells transfected with vector (pRBG4) or vector containing NaPi-3 (pRBG4-NaPi3). We found that RT-PCR identified the presence of NaPi-3 mRNA in pRBG4- and pRBG4-NaPi3-transfected cells; however, we were not able to distinguish any significant difference in the amount of RT-PCR product (data not shown). The expected RT-PCR product for NaPi-3 was not detected if the RNA template was first treated with ribonuclease (data not shown). We did not detect any product if the RT-PCR was carried out using primers specific for NPT1, the other known human renal Na-phosphate cotransporter. All primers used for RT-PCR are specific for the intended targets and amplify the intended product as determined from control experiments using purified plasmids containing NaPi-1 (the rabbit homologue of NPT1) or NaPi-3 cDNAs. Thus we conclude that native HEK-293 cells transfected with pRBG4 already express an NaPi-3 isoform and that transfection of these cells with pRBG4-NaPi3 increases the amount of this isoform. The amount of new NaPi-3 resulting from transfection is increased at least two- to threefold as detected by functional assays on the plasma membrane; however, the RT-PCR method did not permitus to quantitate an increased level of mRNA for NaPi-3 in transfected cells compared with sham-transfected cells.
Expression of NaPi-3 protein in transfected and sham-transfected HEK-293 cells. To further assess the expression of NaPi-3 protein in pRBG4- and pRBG4-NaPi3-transfected cells, Western analysis was carried out using a new antibody specific for NaPi-3. A polyclonal antiserum specific for NaPi-3 was prepared against a peptide corresponding to the NH2-terminal residues 77-93 (K-L-A-L-E-E-E-Q-K-P-E-S-R-L-V-P-K) of NaPi-3. This sequence is unique to the type II facilitated Na-phosphate cotransporters (e.g., NaPi-2, NaPi-3, and NaPi-4) and is not found in the type I facilitated Na-phosphate cotransporter (e.g., NaPi-1). This antibody specifically detects a 125,000-mol wt protein in total cell lysates prepared from untransfected HEK-293 cells, sham-transfected (pRBG4) HEK-293 cells, and NaPi-3-transfected (pRBG4-NaPi3) HEK-293 cells (Fig. 12A, lanes A-C). The reaction of this antiserum was specifically blocked by coincubation with the immunizing peptide (Fig. 12A, lanes D-F). Sometimes an additional protein band at 61,000 mol wt was seen in transfected cells (pRBG4 or pRBG4-NaPi3); however, the amount of this band was not reduced by competition with the immunizing peptide. There are three potential glycosylation sites on the proposed large extracellular loop of the protein (16), and the nonglycosylated NaPi-3 is predicted to be 68,900 mol wt on the basis of the derived amino acid sequence. To assess whether the 125,000-mol wt protein was a glycosylated form of NaPi-3, we treated samples with endoglycosidase Hf to deglycosylate NH2-linked glycoproteins at the last N-acetylglucosamine residue linked to the protein (Fig. 12B). Although the samples were not completely deglycosylated, a fraction of the 125,000-mol wt protein in all cases (transfected and untransfected HEK-293 cells) was deglycosylated to a smaller form with an apparent molecular weight of 76,800. We have also determined the presence of NaPi-3 protein in human kidney samples (Fig. 12B, lanes G and H). We have found that our antibody recognizes a 125,000-mol wt protein that is present in the human renal medulla and cortex. Finally, we quantitated the amount of NaPi-3 protein expressed in pRBG4- and pRBG4-NaPi3-transfected cells (Fig. 12C). Duplicate samples were analyzed on separate gels using an amount of total protein that we previously determined yielded bands that were in the linear response range of the film used (data not shown). The amount of protein in each sample was then determined by densitometry and corrected for the background density. We found that the average net amount of NaPi-3 protein in pRBG4-transfected cells was 28 density units/µg protein, whereas the amount in pRBG4-NaPi3-transfected cells was 92.5 density units/µg protein. Thus transfection with pRBG4-NaPi3 increases the amount of NaPi-3 protein by 3.3-fold compared with sham-transfected cells. This increase in amount of protein corresponds to the average increase in functional activity after transfection (see Fig. 3 legend).
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DISCUSSION |
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Comparison of
32PO4 influx in
vector-only- and NaPi-3-transfected cells.
HEK-293 cells are developed from HEK cells that have been transformed
with fragments of adenovirus. All the resulting immortal cell lines are
not the same, and the characteristics of the cells we obtained from
American Type Culture Collection at passage
32 may be different from separately maintained lines or
from later passage numbers. The kinetic characteristics of the
Na-Pi cotransport in these cells
and/or in cells induced by transfection with the vector pRBG4
alone (sham transfected) are quite similar to the characteristics of
these cells transfected with NaPi-3 in pRBG4 and expressing increased
Na-dependent
32PO4
influx (Table 1). Although the flux is
increased, there is the same selectivity for cation activation of
32PO4
influx (Fig. 5, Na Li > K = Rb = Cs = NMDG), the same
K1/2 for external
Na (Fig. 5, 128-129 mM), the same
K PO41/2 for external phosphate (Fig. 6, 81-84 µM), the same negative
EA in the absence
of Na and positive
EA in the
presence of Na (Fig. 9), the same insensitivity to inhibition by DNDS,
phosphonoacetate, amiloride, EIPA, DMA, benzamil, and phenamil, and the
same inhibition by phloretin and pCMBS (Figs. 10 and 11). There are
some differences between
32PO4
fluxes in vector-only- and Na-Pi-3-transfected cells. There is little
or no pH dependence of the flux in the presence of Na in
NaPi-3-transfected cells, whereas in the vector-only-transfected cells
the flux in the presence of Na decreases 75% as pH increases. The
percent inhibition by phosphonoformate arsenate, vanadate, and
5-(N-methyl-N-isobutyl)-amiloride
is greater in the NaPi-3-transfected cells. We have no explanation for
these differences, since we believe that the same protein mediates the
Na-dependent
32PO4
flux in sham- and NaPi-3-transfected cells.
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Comparison of the NaPi-3 mRNA in vector-only- and NaPi-3-transfected cells. These kinetic similarities are confirmed by the presence of NaPi-3 mRNA in the vector-only- and the NaPi-3 transfected cells. Although there were no quantitative differences in the amount of RT-PCR product produced from equal amounts of total RNA, there was clearly an increased functional expression of NaPi-3 protein in the NaPi-3-transfected cells. We were also not able to determine whether the 1.4- to 2-fold increase in the Na-dependent phosphate influx in vector-only-transfected cells compared with native cells (transfected HEK-293 cells) was entirely due to increased NaPi-3 function, although most of the functional characteristics of the transport were similar to those of the NaPi-3-transfected cells. The simplest but unproven explanation is that the transfection procedure itself upregulated native NaPi-3 expression. However, because we always measure the vector-only-transfected cells in parallel with the NaPi-3-transfected cells, the difference in function between these two cell types must be a consequence of the transfected NaPi-3 cDNA sequence.
Comparison of the NaPi-3 protein in vector-only- and NaPi-3-transfected cells. The Western analysis shows an increase in the amount of NaPi-3 protein in NaPi-3-transfected cells. It also shows that this protein is expressed in the sham-transfected, as well as untransfected, cells. This native transporter is probably responsible for the Na-dependent transport in the sham-transfected cells and thus explains the similarities in the kinetics between sham- and NaPi-3-transfected cells. The "deglycosylated" molecular weight is slightly higher than the predicted molecular weight of the unglycosylated protein. There are many potential explanations for this result, including the possibility that the samples analyzed here are only partially deglycosylated or that the NaPi-3 protein is glycosylated at multiple sites, but some do not contain a chitobiose core and thus are not substrates for endoglycosidase Hf. It is known that the rat isoform corresponding to NaPi-3 is glycosylated at two sites on the protein (16). These results are consistent with the conclusion that the 125,000-mol wt NaPi-3 protein (the native form in HEK-293 cells or the recombinant form expressed in pRBG4-NaPi3-transfected cells) is the fully glycosylated form of the protein and that the protein is highly glycosylated, perhaps at multiple sites. The highly glycosylated form of NaPi-3 is not an artifact of cell culture or HEK-293 cells, since we have shown that it is the form expressed in vivo in human kidney. Interestingly, the amount of NaPi-3 per microgram of total cellular protein is ~2.5-fold higher in medulla than in cortex on the basis of densitometry of the Western blot shown in Fig. 12 (data not shown). Because Na-dependent phosphate reabsorption is primarily in the proximal tubule, one would predict that a greater proportion of NaPi-3 should reside in the renal cortex. It is possible that in humans this isoform of the cotransporter is predominantly in the nephrons of the juxtamedullary portion of the kidney.
Alkali cation activation. The alkali cation activation of 32PO4 influx is only significant for Na and Li over Cs, Rb, K, chloine, or NMDG. In this study, Li is only 2% as great as Na. In rat renal brush-border vesicles the Li activation is 8.5% as great as Na (8). In contrast, in LLC-PK1 cells it is 30% as great as Na, and in human red blood cells and K562 cells it is 20% as great (unpublished observations). Finally, in rat small intestine, Li and Na are equally good activators of phosphate flux. Thus NaPi-3 is the most selective Na-phosphate cotransport system reported.
Comparison of the Km values of expressed NaPi-3 with renal tissues, cell lines, and other expression systems. The phosphate activation of the Na-dependent 32PO4 influx is always hyperbolic and follows Michaelis-Menten kinetics. The K PO41/2 for phosphate activation of 32PO4 influx here was 0.081 mM (Fig. 6). The K PO41/2 was twice this value when NaPi-3 was expressed in Xenopus laevis oocytes (i.e., 0.170 ± 0.023 mM) (16). The native transporter in OK cells is NaPi-4 and has K PO41/2 of 0.18 ± 0.03 mM in the cells and 0.1 ± 0.01 to 0.13 ± 0.04 mM when NaPi-4 is expressed in oocytes (24). In porcine renal brush-border membranes, K PO41/2 is 0.149 ± 0.011 mM. In the pig kidney cell line LLC-PK1, K PO41/2 is 0.026 mM. In the rat renal brush-border vesicles, K PO41/2 ranges from 0.07-0.08 mM (2) to 0.9-1.4 mM (17); in the cloned rat renal gene NaPi-2, expressed in oocytes, K PO41/2 is 0.130 ± 0.015 mM when determined by tracer fluxes (16) and 0.31 ± 0.03 mM when determined from the phosphate activation of inward current (3). Some of these differences arise from comparing different genes (animals), but the same gene in different expression environments gives different K PO41/2 values, even when the pH and temperatures are comparable.
The activation of the Na-dependent 32PO4 influx by extracellular Na is usually sigmoidal, with a Hill coefficient (n) of ~2, suggesting that >1 Na accompanies the phosphate. The expression of NaPi-3 in oocytes causes a new electrogenic transport with a net positive charge flow in the direction of the net phosphate flux (4). This is consistent with a stoichiometry ofTemperature dependence. The negative EA for 32PO4 influx in the absence of Na demonstrates the complexity of temperature dependence of biological systems and the failure of precise physical chemical analysis to be of much use. If there were a single rate-limiting step of the influx with an Eyring energy barrier, the EA would be positive. Here, however, the cotransporter protein is at a complex lipid-water interface, and there are probably several rate-limiting steps and several temperature-sensitive elements in the transporting ensemble. The large SD along the bottom lines in Fig. 9 is the result of the logarithmic transformation of the flux and SDs for small flux values.
The apparent activation of the Na-dependent flux is the difference in the EA values in the presence and absence of Na: 40 and 35 kcal/mol for sham- and NaPi-3 transfected cells, respectively. Both values are large and consistent with specific membrane protein-mediated transport processes and not consistent with free diffusion. The Arrhenius plot of the temperature dependence of Na-PO4 cotransport in rat renal cortex brush-border vesicles (2) shows a break point at 18-23°C, with EA only 2.8 kcal/mol above that point and 26 kcal/mol below that point. Although these rates were measured by single points at 10 s, they may not be initial rates and thus underestimate the flux and consequently the EA at the higher temperatures. Alternatively, the brush-border preparation may be so rich in transporters that at elevated temperatures the transport becomes limited by some near-diffusional off-rate rather than the transporter rate. The report of a low temperature dependence above 23°C in this preparation casts doubt on the analysis and thus the proposed existence of two parallel mediated pathways at 37°C (1).pH dependence. Understanding the pH dependence of NaPi-3 may be important for integrating this proximal tubule apical membrane transporter into its role in tubule reabsorption of phosphate. This is because the luminal pH changes as bicarbonate is reabsorbed and the internal pH is more alkaline at the apical than at the basolateral membrane, where the mitochondria are apposed. In addition, an outwardly directed pH gradient across the apical membrane may contribute to phosphate uptake. The pH dependence of transport measured here may include several consequences of titration reactions besides those on the NaPi-3 cotransporter. For example, the transport is enhanced in hyposmotic media in OK cells (14) and in transfected HEK-293 cells (data not shown). Although the solutions were isosmotic in the pH experiments, the cells would be expected to swell as pH decreased because of titration of intracellular proteins and the entrance of accompanying anion. Thus inhibition of transport by protonation of the transporter is expected to be opposed by isosmotic swelling in low-pH solutions.
NaPi-3 expressed in oocytes was activated fourfold by increasing the pH from 6.0 to 7.5 or 8.0 with no pH dependence of the flux in water-injected controls (16). Here, in HEK-293 cells the NaPi-3-specific flux increased 2.1-fold over the same range of pH values but mostly due to a decline in the flux in sham-transfected cells. In rat renal brush-border vesicles the initial phosphate influx is always increased by increasing pH between 6.0 and 8.5 (1, 2). At room temperature the activation is between 6- and 20-fold (1). This agrees with the pH dependence of fluxes at room temperature mediated by rat NaPi-2 expressed in MDCK cells (22). However, at 35°C (2) or 37°C (1), where the rates of transport may be underestimated (see Temperature dependence), the activation is increased 4-fold and monotonic at 0.1 mM phosphate and only 1.2-fold and apparently biphasic at 3.0 mM phosphate with a peak flux at pH 7-7.4. The basis for these pH dependencies is unknown. Some data suggest that low pH reduces the affinity of the transport system for Na and thus reduces the flux; other data refute that conclusion (1). The changes in monovalent and divalent substrate concentrations as well as possible direct titration of the transporter make conclusions difficult, especially if there may be two transporters (NaPi-1 and NaPi-3) operating in the vesicles. The conclusion that divalent phosphate is the substrate is attractive but not necessarily true, even though the apparent K PO41/2 is constant as a function of pH if divalent phosphate is assumed to be the substrate. In human erythrocytes, where it is known that only monovalent phosphate interacts with the external transport site of band 3 (5), the Na-phosphate cotransporter Vmax increases threefold from pH 6.9 to 7.75 with an approximately constant K PO41/2 (80 µM) if monovalent phosphate is assumed to be the substrate (23).Inhibition of NaPi-3-mediated transport. We found no inhibition of NaPi-3 expressed in HEK-293 cells by the reversible stilbene DNDS, whereas in rat and porcine brush-border membrane vesicles, related stilbenes, H2-DIDS (4,4'-diisothiocyanodihydro-stilbene-2,2'-disulfonate) and DIDS, are reported to irreversibly inhibit what should be transport mediated by NaPi-3 homologues in those species. On the other hand, the phosphonocarboxylic acids (phosphonoformate arsenate and phosphonoacetate) gave comparable inhibitions in brush-border membrane vesicles and in this report, although bovine renal brush-border membrane vesicles are much less sensitive to these agents.
Amiloride and its analogs had very little inhibitory effect. Only MIA at 90 µM caused significant (65%) inhibition of the NaPi-3-mediated 32PO4 influx. Although amiloride has a low 50% inhibitory concentration (IC50, 0.34 µM) for the epithelial Na channel and an intermediate IC50 (84 µM) for the Na/H antiporter, it does have a high IC50 (1,100 µM) for the Na/Ca exchanger (11), similar to that for the Na site on NaPi-3 (IC50 for amiloride > 1,000 µM). However, the relative effects of amiloride analogs on the Na/Ca exchanger and on NaPi-3 are quite different. EIPA, HMA, benzamil, and phenamil, which have potencies relative to amiloride of 8.5, 11, 11, and 5.5 on Na/Ca exchange, are without effect on NaPi-3-mediated phosphate fluxes. MIA, which has a potency 8.5 times that of amiloride on the Na/Ca exchanger, has a relative potency >10 on NaPi-3-mediated phosphate flux. We conclude that the Na site on NaPi-3 has a low affinity for amiloride and a structure/environment different from that of other Na transporters, including Na/H exchanger, Na/Ca exchanger, Na-K-ATPase, Na-coupled glucose transporter, and the tetrodotoxin-sensitive Na channel (for review see Ref. 11). In summary, in HEK-293 cells, human NaPi-3-mediated Na-PO4 cotransport can be increased 2.5- to 5-fold by transfection with NaPi-3 in the expression vector pRBG4. The kinetics of the native transporters and those expressed after transfection are similar with respect to Na activation, phosphate activation, temperature dependence, and pharmacology. Together with the functional expression, there is a commensurate increase in NaPi-3 protein but not a detectable increase in mRNA by RT-PCR. ![]() |
ACKNOWLEDGEMENTS |
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We thank P. M. Smith for maintaining and transfecting the HEK-293 cells, Y. Yang and J. D. Hill for preparing the plasmid cDNA for transfection, B. Stockman for performing SDS-polyacrylamide gel electrophoresis and Western analysis, J. M. Sands for providing the human kidney sample, and B. M. Medley for preparing the manuscript.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute National Research Service Award HL-08989 (R. T. Timmer) and Grant R37-HL-28674-15 (R. B. Gunn).
1 The flux values calculated in this study are the result of the linear regressions of five to six data points over 30 min. Each flux condition was performed in duplicate. We believe that this method is superior to one-point flux measurements for two reasons. First, a spurious point due to contamination of radioactivity or altered optical density of protein reading can often be excluded when it deviates from the line formed by the other five points. Second, we have observed on occasion high intercepts on the ordinate due to nonspecific phosphate binding (1). These fluxes, although often the data values lay along a straight line, have been excluded, whereas there is no way to exclude or even be aware of this problem in a one-point flux assay. The variability in the flux from duplicate conditions on the same plate reflects uncontrolled variables such as the level of expression in each well or the protein per well that may not perfectly correlate with the number of cells, much less the number of functioning cotransporters on the cell plasma membranes. It is for this reason that the graphs of duplicate flux measurements may each give excellent straight lines with small standard deviations of the slope, yet the ranges of the two values do not overlap. The average percent difference between duplicates in Figs. 6, 7, and 9-12 is 15%. In the absence of Na (low flux values), the average percent difference was 31%. In the presence of inhibitors, the average percent difference was 20%. In the absence of inhibition and in the presence of Na (higher flux values), the average difference was 12%. We have seen small "discontinuities" in the fluxes in a series of experiments that occur when data in one plate are graphed adjacent to data from another plate, although the flux measurements were contemporaneous. This may reflect the differences in temperature and plating density/expression as well as unintended differences in influx media as the intended variable is changed.
Address reprint requests to R. B. Gunn.
Received 9 January 1997; accepted in final form 8 December 1997.
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