Modulation of renal type IIa Na+/Pi cotransporter kinetics by the arginine modifier phenylglyoxal.

The effects of the arginine-modifying reagent phenylglyoxal on the kinetics of the type IIa Na + /Pi cotransporter expressed in Xenopus, oocytes were studied by means of 32Pi uptake and electrophysiology. Phenylglyoxal incubation induced up to 60% loss of cotransport function but only marginally altered the Na+-leak. Substrate activation and pH dependency remained essentially unaltered, whereas the voltage dependency of Pi-induced change in electrogenic response was significantly reduced. Presteady-state charge movements were suppressed and the equilibrium charge distribution was shifted slightly towards hyperpolarizing potentials. Charge movements in the absence of external Na+ were also suppressed, which indicated that the empty-carrier kinetics were modified. These effects were incorporated into an ordered alternating access model for NaPi-IIa, whereby the arginine modification by phenylglyoxal was modeled as altered apparent electrical distances moved by mobile charges, together with a slower rate of translocation of the electroneutral, fully loaded carrier.

Cysteine mutagenesis reveals novel structure-function features within the predicted third extracellular loop of the type IIa Na(+)/P(i) cotransporter.

The transport function of the rat type IIa Na(+)/P(i) cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a "structure-function" profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an alpha-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated alpha-helical structure.

Molecular determinants of pH sensitivity of the type IIa Na/P(i) cotransporter.

Type II Na/P(i) cotransporters play key roles in epithelial P(i) transport and thereby contribute to overall P(i) homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/P(i) cotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/P(i) cotransporter.

PiUS (Pi uptake stimulator) is an inositol hexakisphosphate kinase.

A cDNA cloned from its ability to stimulate inorganic phosphate uptake in Xenopus oocytes (phosphate uptake stimulator (PiUS)) shows significant similarity with inositol 1,4,5-trisphosphate 3-kinase. However, the expressed PiUS protein showed no detectable activity against inositol 1,4,5-trisphosphate, nor the 1,3,4,5- or 3,4,5, 6-isomers of inositol tetrakisphosphate, whereas it was very active in converting inositol hexakisphosphate (InsP(6)) to inositol heptakisphosphate (InsP(7)). PiUS is a member of a family of enzymes found in many eukaryotes and we discuss the implications of this for the functions of InsP(7) and for the evolution of inositol phosphate kinases.

Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

Effect of two tyrosine mutations on the activity and regulation of the renal type II Na/Pi-cotransporter expressed in oocytes.

The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY402FAM matching the consensus sequence GYXXZ, and Y509RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated 32Pi-uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/Pi-cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter.

Dietary sulfate regulates the expression of the renal brush border Na/Si cotransporter NaSi-1.

Dietary inorganic sulfate (Si) intake is an important factor in the regulation of renal proximal tubular sodium-dependent Si transport (Na/Si cotransport). The purpose of the present study was to determine whether modulation of Na/Si cotransport activity by dietary Si is mediated through regulation of the renal expression of the recently cloned NaSi-1 protein located in the apical brush border membrane (BBM) of the proximal tubule. It was found that rats fed a high Si diet had a marked increase in the renal excretion of Si and a concomitant decrease in BBM Na/Si cotransport activity when compared with rats on a control Si diet. The 43% decrease in BBM Na/Si cotransport activity was associated with a 33% decrease in BBM NaSi-1 protein abundance, as determined by Western blotting, and a 2.7-fold decrease in cortical NaSi-1 mRNA abundance, as determined by Northern blotting. Furthermore, cortical mRNA from rats fed a high Si diet when injected into Xenopus laevis oocytes led to a 2.2-fold decrease in Na/Si cotransport activity compared with mRNA isolated from control Si diet rats. This study indicates that adaptation to a high Si diet is accompanied by a decrease in renal cortical NaSi-1 mRNA abundance, which results in reduced expression of the NaSi-1 protein at the level of the proximal tubular BBM.

The voltage dependence of a cloned mammalian renal type II Na+/Pi cotransporter (NaPi-2).

The voltage dependence of the rat renal type II Na+/Pi cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (Pi) induced inward currents (Ip) in the presence of 96 mM Na+ over the potential range -140

Chloride conductance and Pi transport are separate functions induced by the expression of NaPi-1 in Xenopus oocytes.

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl- conductance (GCl), organic anion transport and Na+-dependent Pi-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and Pi-induced currents and transport. NaPi-1 expression induced Pi-transport, which was not different at 1-20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1-2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1-20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. Pi-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of Pi (>/= 1 mm Pi). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl- channel blocker NPPB, partially Na+-dependent and completely abolished in Cl- free solution. In contrast, Pi-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl- substitution. Endogenous Pi-uptake in water-injected oocytes amounted in all experiments to 30-50% of the Na+-dependent Pi-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous Pi-uptake system (Km for Pi > 1 mM; partial Na+- and Cl--dependence; lack of NPPB block) were similar to the NaPi-1 induced Pi-uptake, but no Ip could be recorded at Pi-concentrations

Abnormal sulfate metabolism in vitamin D-deficient rats.

To explore the possibility that vitamin D status regulates sulfate homeostasis, plasma sulfate levels, renal sulfate excretion, and the expression of the renal Na-SO4 cotransporter were evaluated in vitamin D-deficient (D-D-) rats and in D-D- rats rendered normocalcemic by either vitamin D or calcium/lactose supplementation. D-D- rats had significantly lower plasma sulfate levels than control animals (0.93+/-0.01 and 1.15+/-0.05 mM, respectively, P < 0.05), and fractional sulfate renal excretion was approximately threefold higher comparing D-D- and control rats. A decrease in renal cortical brush border membrane Na-SO4 cotransport activity, associated with a parallel decrease in both renal Na-SO4 cotransport protein and mRNA content (78+/-3 and 73+/-3% decreases, respectively, compared with control values), was also observed in D-D- rats. Vitamin D supplementation resulted in a return to normal of plasma sulfate, fractional sulfate excretion, and both renal Na-SO4 cotransport mRNA and protein. In contrast, renal sulfate excretion and renal Na-SO4 cotransport activity, protein abundance, and mRNA remained decreased in vitamin D-depleted rats fed a diet supplemented with lactose and calcium, despite that these rats were normocalcemic, and had significantly lower levels of parathyroid hormone and 25(OH)- and 1,25(OH)2-vitamin D levels than the vitamin D-supplemented groups. These results demonstrate that vitamin D modulates renal Na-SO4 sulfate cotransport and sulfate homeostasis. The ability of vitamin D status to regulate Na-SO4 cotransport appears to be a direct effect, and is not mediated by the effects of vitamin D on plasma calcium or parathyroid hormone levels. Because sulfate is required for synthesis of essential matrix components, abnormal sulfate metabolism in vitamin D-deficient animals may contribute to producing some of the abnormalities observed in rickets and osteomalacia.

Renal endosomal phosphate (Pi) transport in normal and diabetic rats and response to chronic Pi deprivation.

Chronic renal adaptation to dietary deprivation of Pi is accompanied by increased Na+/Pi co-transport across the brush border membrane of the renal proximal tubule. The increased activity of this co-transport system depends on de novo protein synthesis and insulin. The present study used normal and diabetic rats to determine if the endosomal pool of Na+/Pi co-transporters was altered by Pi deprivation and the possible role of insulin. In response to 5 days of dietary Pi deprivation there was a significant increase in endosomal Na+/Pi co-transport in control rats but there was no change in diabetic rats. The increase in endosomal Pi uptake was restored in diabetic rats treated with exogenous insulin. Na(+)-independent Pi uptake and proline uptake remained unchanged in all groups. The changes in endosomal Na+/Pi co-transport correlated with the abundance of the specific Na+/Pi co-transporter protein, as determined by Western blots. The pattern of endosomal changes paralleled that observed in brush border membranes. One possibility consistent with these findings is that the endosomal fraction contains newly synthesized Na+/Pi co-transporters targeted for delivery to the apical brush border membrane. Increased synthesis and delivery is required to maintain the adaptation to chronic Pi deprivation.

Identification of a cDNA/protein leading to an increased Pi-uptake in Xenopus laevis oocytes.

In a previous report we documented an increased Na(+)-dependent transport of inorganic phosphate (P(i)) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211-216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P(i)-uptake stimulator) lead to a 3-4-fold stimulation of Na(+)-dependent P(i)-uptake (10ng cRNA injected, 3-5 days of expression). Na(+)-independent uptake of P(i) was also affected but transport of sulphate and L-arginine (in the presence or absence of sodium) remained unchanged. The apparent K(m)-values for the induced Na(+)-dependent uptake were 0.26 +/- 0.04 mM for P(i) and 14.8 +/- 3.0 mM for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of approximately 2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P(i)-uptake into Xenopus laevis oocytes, but which is not a P(i)-transporter itself.

Protein kinase C consensus sites and the regulation of renal Na/Pi-cotransport (NaPi-2) expressed in XENOPUS laevis oocytes.

Renal brush border membrane sodium/phosphate (Na/Pi)-cotransport activity is inhibited by hormonal mechanisms involving activation of protein kinases A and C. The recently cloned rat renal Na/Pi-cotransporter (NaPi-2) contains several protein kinase C but no protein kinase A consensus sites [17, 20]. In the present study we have expressed wild type and polymutant (protein kinase C consensus sites removed) NaPi-2-transporters in Xenopus laevis oocytes. The expression of transport function as well as the basic transport properties were unaffected by the removal of the consensus sites. Pharmacological activation of protein kinase C with phorbol 12,13-didecanoate (PDD) led to a time-dependent inhibition of expressed wild type Na/Pi-cotransport function; simultaneous exposure to staurosporine (0.3) prevented the PDD induced (50 nM) inhibition. The kinase-C-mediated inhibition was not prevented by the removal of the protein kinase C consensus sites. Pharmacological activation of protein kinase A (dibutyryl adenosine 3':5':cyclic monophosphate (cAMP)/forskolin) had no effect on wild type NaPi-2-induced oocyte Na/Pi-cotransport. It is concluded that the protein-kinase-C-mediated regulation of expressed Na/Pi-cotransport does not involve the predicted consensus sites. The involvement of "cryptic" phosphorylation sites and/or of a phosphorylated "regulatory" protein is discussed.

Transport characteristics of a murine renal Na/Pi-cotransporter.

A complementary deoxyribonucleic acid (cDNA) corresponding to a murine renal cortical Na/phosphate-(Na/Pi-) cotransporter was isolated and its transport properties characterized by electrophysiological techniques after expression in Xenopus laevis oocytes. A Na-dependent inward movement of positive charges ("short-circuit current") was observed upon superfusion with Pi (and with arsenate). Increasing the Na concentration led to a sigmoidal elevation in Pi-induced short-circuit current; the apparent Michaelis constant, Km, (around 40 mM Na) was increased by lowering the pH of the superfusate but was not influenced by altering the Pi concentration. Increasing the Pi (and arsenate) concentration led to a hyperbolic elevation in Na-dependent short-circuit current (apparent Km for Pi at 100 mM Na was around 0.1 mM; apparent Km for arsenate was around 1 mM); lowering the Na concentration decreased the apparent affinity for Pi. The Pi-induced short-circuit current was lower at more acidic pH values (at pH 6.3 it was about 50% of the value at pH 7.8); this pH dependence was similar if the Pi concentration was calculated in total, or if distinction was made between its mono- and divalent forms. Thus, the pH dependence of Na-dependent Pi transport (total Pi) may not be related primarily to a pH-dependent alteration in the availability of divalent Pi, but includes also a competitive interaction of Na with protons. The effect of Pi and Na concentration on the apparent Km values for Na or Pi, respectively, provides evidence for an ordered interaction of "cosubstrate" (Na first) and "substrate" (Pi or arsenate second).

Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability.

Renal proximal tubular Na-dependent phosphate transport (Na/Pi cotransport) has been studied extensively in the opossum kidney (OK) cell line. Recently, we cloned a complementary deoxyribonucleic acid (cDNA) (NaPi-4) from OK cells encoding an apical NaPi cotransport system. OK cells exposed to a low-Pi medium, as compared to high-Pi media, responded with an increase in Na/Pi cotransport, which was followed by an increase in NaPi-4 messenger ribonucleic acid (mRNA) abundance; maximal stimulation of Na/Pi cotransport was reached in 2 h, with no further increase for up to 16 h. NAPi-4 mRNA abundance was unaltered for 2 h, then increased to a maximum after 6-16 h in cells treated with low Pi medium. NaPi-4 mRNA decay rate was lowered by low-Pi media when compared to high-Pi media, with no increase in the NaPi-4 mRNA transcription rate. These data suggest that the upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after 2 h) in the expression of Na/Pi cotransport, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4-6 h), resulting in an increase in NaPi-4 mRNA abundance, due to an increased stability.

Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate.

Previously, we isolated a cDNA (NaPi-1) related to a rabbit renal proximal tubular Na-Pi cotransporter (A. Werner, M.L. Moore, N. Mantei, J. Biber, G. Semenza, and H. Murer. Proc. Natl. Acad. Sci. USA 88:9608-9612, 1991.). In this study, we isolated an additional (rabbit renal) cDNA (NaPi-6), which induces Na-dependent Pi uptake in Xenopus laevis oocytes. Substrate specificity and kinetic properties corresponded to those known for rabbit renal brush-border membrane (BBM) Na-Pi cotransport. NaPi-6 was cloned by homology using NaPi-2 cDNA, a rat renal BBM Na-Pi cotransporter (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979-5983, 1993). NaPi-6 encodes a protein of 642 amino acids, exhibiting at least eight transmembrane domains. NaPi-6 mRNA and protein in kidneys of rabbits fed a low-Pi diet (LPD; 0.11% Pi) for 1 wk were increased by 1.5- and 4-fold, respectively, compared with those of rabbits fed a high-Pi diet (HPD; 1.20% Pi). This effect was correlated with an increase in Na-Pi cotransport of BBM vesicles isolated from animals adapted to LPD (2.5-fold with respect to HPD). In contrast, NaPi-1 mRNA and protein were not altered in response to LPD. Thus rabbit proximal tubular BBMs contain two different Na-Pi cotransport systems: NaPi-1 (type I) and NaPi-6 (type II). Only the type II transport system seems to be under regulatory control in response to low-Pi dietary intake.

Electrophysiological analysis of Na+/Pi cotransport mediated by a transporter cloned from rat kidney and expressed in Xenopus oocytes.

Phosphate (Pi) reabsorption in renal proximal tubules involves Na+/Pi cotransport across the brush border membrane; its transport rate is influenced by the Na(+)-coupled transport of other solutes as well as by pH. In the present study, we have expressed a cloned rat renal brush border membrane Na+/Pi cotransporter (NaPi-2) in Xenopus laevis oocytes and have analyzed its electrophysiologic properties in voltage- and current-clamp studies. Addition of Pi to Na(+)-containing superfusates resulted in a depolarization of the membrane potential and, in voltage-clamped oocytes, in an inward current (IP). An analysis of the Na+ and/or Pi concentration dependence of IP suggested a Na+/Pi stoichiometry of 3:1. IP was increased by increasing the pH of the superfusate; this phenomenon seems to be mainly related to a lowering of the affinity for Na+ interaction by increasing H+ concentration. The present data suggest that known properties of Pi handling at the tubular/membrane level are "directly" related to specific characteristics of the transport molecule (NaPi-2) involved.

Cloning of a Na/Pi cotransporter from opossum kidney cells.

Opossum kidney (OK) cells have been extensively used to study cellular mechanisms of renal proximal tubular Na/P(i) cotransport. We have cloned a cDNA (NaPi-4) most likely encoding an apical Na/P(i) cotransporter from OK cells. The cloning strategy was based on homology to the recently cloned human renal (NaPi-3) Na/P(i) cotransporter (Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5979-5983). Kinetic characterization (P(i) interaction, sodium interaction, and pH dependence) of NaPi-4-induced Na/P(i) uptake showed high similarity to apical Pi transport in OK cell monolayers. The NaPi-4 cDNA is 2548 base pairs long and encodes a protein of 70.5 kDa, containing at least 8 predicted transmembrane domains. Northern blot analysis with OK cell mRNA shows a NaPi-4-related signal (2.5 kilobases) in cells grown on impermeant and permeant supports. Hybrid depletion with NaPi-4 antisense oligonucleotides abolished the mRNA-induced Na/P(i) cotransport in oocytes. Similarly, NaPi-4 antisense oligonucleotides inhibited (up to 70%) Na/P(i) cotransport in OK cell monolayers. We presume that NaPi-4 is closely related to the OK cell apical Na/P(i) cotransporter.

Effect of low-phosphate diet on sodium/phosphate cotransport mRNA and protein content and on oocyte expression of phosphate transport.

Recently, we have isolated a complementary DNA most likely related to rabbit kidney cortex brush border membrane sodium/phosphate (Na/Pi) cotransport activity [NaPi-1 (1)]. To further elucidate the cellular mechanisms involved in dietary 'adaptation' of renal Na/Pi cotransport, we have exposed young rabbits for 2 weeks to either a low phosphate (Pi) diet (LPD) or a high Pi diet (HPD). Initial linear uptake of Na/Pi cotransport in isolated brush border membrane vesicles was increased in rabbits on a LPD compared with those on a HPD. Injection of equal amounts of total mRNA isolated from kidney cortex of LPD or HPD rabbits into Xenopus laevis oocytes resulted in a higher stimulation of Na-dependent oocyte Pi uptake in LPD than HPD preparations. No difference in the content of 'specific' mRNA (NaPi-1 cDNA probe, Northern blots) and of the content of the 'specific' brush border membrane protein (NaPi-1 antipeptide antibody, Western blots) between LPD and HPD preparations was observed. We conclude that 'chronic' dietary Pi deprivation leads to a protein synthesis-dependent alteration of Na/Pi cotransport activity which does not involve a change in the total amount of a protein related to the recently cloned NaPi-1 protein.

Expression cloning of a human renal cDNA that induces high affinity transport of L-cystine shared with dibasic amino acids in Xenopus oocytes.

A renal cDNA clone (rBAT) that induces system bo,+-like amino acid transport activity in Xenopus oocytes has recently been isolated (Bertran, J., Werner, A., Moore, M. L., Strange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacín, and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5601-5605). Here we show the isolation of a cDNA clone by screening a human kidney cortex cDNA library for expression of sodium-independent transport of L-[3H]arginine in Xenopus oocytes. The cRNA of this clone induces in oocytes, in addition to the uptake of L-arginine, that of L-[35S]cystine and L-[3H]leucine. Expressed uptake of these amino acids is mutually cis-inhibitable by the other 2 amino acids. Expressed uptake of L-cystine is saturable and shows an apparent Km in the micromolar range. All these characteristics resemble induction of system bo,+ related to rBAT in the oocytes. Human rBAT mRNA (approximately 2.5 kilobases) is found in kidney, small intestine (i.e., jejunum), pancreas, and liver. Human kidney poly(A)+ RNA (mRNA) induces sodium-independent uptake of L-cystine, L-arginine, and L-leucine in Xenopus oocytes. Hybrid depletion with an antisense oligonucleotide of the isolated clone greatly prevents (80-97%) human kidney mRNA-dependent induction of the uptake of these amino acids (i.e., L-cystine, L-arginine, and L-leucine). The isolated clone (2304 base pairs in length) contains a poly(A) tail and encodes a predicted 78.8-kDa protein which is 85 and 80% identical to the rabbit and rat rBAT, respectively. This predicted protein corresponds to a membrane glycoprotein, and contains six potential N-glycosylation sites which might be functional in the oocyte: [35S] methionine labeling of oocytes shows a specific band of 94 kDa in crude membranes of these human cRNA-injected oocytes; treatment of these oocytes with tunicamycin shifts the cRNA-specific translation product to approximately 72 kDa. We conclude that we have isolated a functional cDNA corresponding to human rBAT. The isolation of this human cDNA would lead to the study of the possible involvement of rBAT in human hyperaminoacidurias.