SEARCH

SEARCH BY CITATION

Keywords:

  • cDNA cloning;
  • Phosphate transport;
  • Structurally related family;
  • Neuron

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

Abstract: We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain-specific Na+-dependent inorganic phosphate (Na+/Pi) cotransporter (BNPI), which is localized exclusively to neuron-rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na+-dependent Pi transport, indicating that DNPI is a novel Na+/Pi cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia-rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na+-dependent Pi transport in the neuron-rich regions of the brain, may form a new class within the Na+/Pi cotransporter family.

Inorganic phosphate (Pi) is essential for various cellular metabolic functions and signal transduction, and its homeostasis in the body is maintained primarily by the kidney (Murer and Biber, 1996). The cells in the proximal tubules of the kidney reabsorb Pi in the glomerular filtrate through complex Na+-dependent Pi (Na+/Pi) cotransport systems that are driven by the transmembrane electrochemical gradient for Na+. Several cDNAs encoding distinct Na+/Pi cotransporters, which are classified into type 1 (NaPi-1-related), type 2 (NaPi-2-related), and type 3 (viral receptor-related) on the basis of molecular structure, have been identified in kidney and some other tissues (Murer and Biber, 1996; Werner et al., 1998). Amino acid comparison of the proteins shows weak overall homology (∼20% identify) between these types.

A distinct type of the brain-specific Na+/Pi cotransporter (BNPI), which has ∼30% amino acid identity to the type 1 proteins, has been described (Ni et al., 1994, 1996). Northern blot analysis revealed that BNPI mRNA is expressed predominantly in brain, and in situ hybridization analysis revealed high levels of mRNA expression in certain neuron-rich regions of amygdala, cerebral cortex, and hippocampus. On the other hand, this mRNA was detected at low levels in substantia nigra, subthalamic nuclei, and thalamus, and no hybridization signals were detected in caudate nucleus and hypothalamus, suggesting that additional related proteins may be present in these regions to complement Pi transport in brain.

Rat pancreatic AR42J cells (Rosewicz et al., 1992) share the feature of pluripotency of the common precursor cells of the pancreas. Treatment of the cells with activin A converts them to neuron-like cells with accompanying neuron-specific morphological changes (Ohnishi et al., 1995); a combination of activin A and betacellulin converts them further into insulin-secreting neuroendocrine cells (Mashima et al., 1996). In the course of identifying genes expressed during the differentiation of AR42J cells by the method of mRNA differential display (Mashima et al., 1999), we have identified a cDNA encoding a novel brain-type Na+/Pi cotransporter (DNPI, for differentiation-associated Na+/Pi cotransporter). The overall high homology between DNPI and BNPI and their restricted mRNA expressions in brain indicate that they form a distinct class within the Na+/Pi cotransporter family.

Isolation of human DNPI cDNA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

An unrecognized cDNA fragment, AB20, the expression of which was up-regulated in the process of differentiation of AR42J cells into neuroendocrine cells (Mashima et al., 1999), was first used to screen a rat pancreatic islet cDNA library for a longer fragment that contains the 5′-upstream coding region. Partial sequencing of the isolated fragment and a database search revealed that the amino acid sequence encoded had ∼80% identity to that of rat BNPI (Ni et al., 1994). A human whole-brain cDNA library was subsequently screened for the human sequence using the 700-bp fragment of the rat cDNA as a probe. A human thalamus cDNA library was also screened for the entire cDNA sequence. Both cDNA strands were sequenced using sequence-specific primers and an ABI PRISM dRhodamine Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA, U.S.A.). The sequencing reactions were analyzed by an Applied Biosystems DNA sequencer model 377.

Expression of DNPI mRNA in Xenopus laevis oocytes

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

To express DNPI in Xenopus oocytes, DNPI poly(A)+RNA was synthesized from the human cDNA template using mCAP RNA Capping Kit (Stratagene, La Jolla, CA, U.S.A.) according to the manufacturer’s instructions. The capped RNAs were suspended in water at 1 μg/μl. Fifty nanoliters of the RNA solution or water was injected into oocytes of X. laevis. The follicular layers of oocytes were removed by incubation in sterile normal amphibian Ringer’s solution (112 mM NaCl, 2 mM KCl, 2 mM CaCl2, and 10 mM Tris-HCl, pH 7.4) containing 0.5 mg/ml collagenase for 15-30 min. The oocytes were then maintained at 18°C for 3 days in sterile modified Barth’s solution [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 7.5 mM Tris-HCl (pH 7.2), 100 units/ml penicillin, and 0.1 mg/ml gentamicin]. To measure Pi uptake, oocytes (five to 10 per individual condition) were washed in Na+-free uptake medium (100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES-Tris, pH 7.5). Half of the oocytes were then placed in Na+-containing uptake medium [100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-Tris (pH 7.5), 0.1 mM phosphate (KH2PO4), and 30 mCi of 32Pi (2 mCi/ml)] for 1 h at room temperature. The other half was used for Na+-independent uptake measurement in Na+-free uptake medium containing 32Pi. After incubation, the oocytes were washed four times with cold Na+-free uptake medium. Each oocyte was then transferred to a scintillation vial, dissolved in 0.2 ml of 10% sodium dodecyl sulfate, and counted. Statistical analysis was done by Student’s t test.

Tissue distribution of human DNPI and BNPI mRNAs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

Northern blot analysis was carried out using human DNPI and BNPI cDNA probes and human multiple tissue northern (MTN) blots I, II, III, and fetal II (Clontech, Palo Alto, CA, U.S.A.). The MTN blot I contains 2 μg of poly(A)+ RNAs from eight human tissues, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The MTN Blots II and III contain 2 μg of poly(A)+ RNAs from a human whole brain and 15 different brain sections, including cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe, putamen, amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus, and thalamus. The Fetal II Blot also contains 2 μg of poly(A)+ RNAs from human fetal brain, lung, liver, and kidney tissues. As the nucleotide sequences for protein-coding regions are highly homologous between the DNPI and BNPI cDNAs, their 3′-untranslated region was used as a probe to avoid cross-hybridization.

Isolation of human DNPI and BNPI cDNAs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

An adult human whole-brain cDNA library was first screened with a rat partial cDNA probe. Of 1 × 106 phages screened, eight positive clones were identified. Nucleotide sequencing revealed two of them to encode a human counterpart of rat DNPI and the others to encode human BNPI. The nucleotide sequence of the BNPI cDNA obtained (accession no. AB032436) was identical to that reported, except for the 3′-end. As both of the two DNPI cDNA sequences obtained lacked the 3′ half of the coding regions, a human thalamus cDNA library was subsequently screened for the entire sequence because the highest levels of DNPI mRNA expression were seen in thalamus by RNA blotting. Sixty-nine positive clones were identified in this process, and one of these, λhNPT2-25, which had the longest insert (3,946 bp), was sequenced (accession no. AB032435). The open reading frame of the sequence encoded a protein of 582 amino acids (Mr = 64,400) with 82% amino acid identity and 92% similarity to human BNPI (Ni et al., 1996) (Fig. 1A). The protein also has 48% identity and 66% similarity to Caenorhabditis elegans EAT-4, a homologue of a mammalian Na+/Pi cotransporter (Lee et al., 1999). A hydropathy analysis of the deduced amino acid sequence suggests that DNPI is a membrane protein exhibiting eight possible transmembrane segments (Fig. 1B), a feature similar in BNPI and EAT-4 (Ni et al., 1996; Lee et al., 1999). Construction of a phylogenetic tree based on comparison of the amino acid sequences indicates that DNPI, BNPI, and EAT-4 are closely related in one branch and form a distinct group (type 4) of members of the Na+/Pi cotransporter family (Fig. 2).

image

Figure 1. A: Comparison of amino acid sequences of human DNPI and BNPI. Gaps are introduced to generate the alignment. Identical residues are indicated by hyphens. B: Hydrophilicity plot of the putative Na/Pi cotransporter. Eight putative transmembrane segments predicted by the method of Kyte and Doolittle (1982) are numbered.

Download figure to PowerPoint

image

Figure 2. Graphical representation of the sequence relationship among members of the Na+/Pi cotransporter family. The phylogenetic tree was generated using amino acid sequences from each subtype (Werner et al., 1991, 1994; Johann et al., 1992; Magagnin et al., 1993; Sorribas et al., 1994; Hartmann et al., 1995; Verri et al., 1995; Ni et al., 1996; Lee et al., 1999).

Download figure to PowerPoint

Na+/Pi cotransport activity of DNPI in Xenopus oocytes

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

To determine whether DNPI is a novel Na+/Pi cotransporter, we expressed human DNPI in Xenopus oocytes and measured 32Pi uptake into the cells. When the mRNA was injected into oocytes, Na+-dependent 32Pi uptake was significantly increased above that of intrinsic activity (p < 0.0001; Fig. 3). Replacement of NaCl with choline chloride decreased Pi transport to basal levels. The experiment was repeated three times, giving the same results.

image

Figure 3. Expression of DNPI mRNA in X. laevis oocytes and measurement of uptake of 32Pi. When human DNPI mRNA was expressed in the oocytes, 32Pi uptake was increased 10-fold above that of Na+ (-) controls. The Na+/Pi cotransport was significantly higher than that of the intrinsic activity (p < 0.0001). The experiment was repeated three times, from which one representative result is shown.

Download figure to PowerPoint

Tissue distribution of human DNPI and BNPI mRNAs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

Northern blot analysis indicates that human DNPI mRNA is expressed predominantly in brain, generating two bands of 4.1 and 4.4 kb (Fig. 4). As the upstream sequences were identical among the cDNA clones, the difference in size of the bands could be generated by different polyadenylylation. Analysis of sublocalization in the brain indicates that the highest levels of DNPI mRNA expression are detected in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia-rich region of corpus callosum. In other regions examined, both the DNPI and BNPI mRNAs are moderately or highly expressed. These results suggest that these two closely related proteins coordinate Na+/Pi transport in neuron-rich regions of the brain.

image

Figure 4. RNA blot analysis of DNPI and BNPI mRNAs in various adult human (h) tissues, sections of brain tissue, and fetal human tissues. The blots were exposed to x-ray film for overnight. A single band of 2.8 kb for BNPI mRNA and two bands of 4.1 and 4.4 kb for DNPI mRNA are evident only in brain (left). Various levels of mRNA expression in the brain segments are also shown (middle). DNPI mRNA is highly expressed only in brain in fetus (right), whereas no BNPI mRNA is detectable in fetal tissues (data not shown). The sizes of internal RNA standards (kb) are indicated on the left side.

Download figure to PowerPoint

Northern blot analysis of the mRNAs from human fetal tissues, including brain, lung, liver, and kidney, revealed high levels of DNPI mRNA expression in the fetal brain, suggesting the importance of DNPI in brain development.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

This study has identified a novel brain-type Na+/Pi cotransporter, DNPI, although its functional significance in the brain is not yet known. Because DNPI has on overall high homology to BNPI, however, the roles of DNPI in the brain may be somewhat similar.

The saturable Pi transport in neuronal cells is Na+-dependent (Glinn et al., 1995). Depletion of Na+ in a primary culture of rat fetal cortical neurons has been shown to result in lowered levels of ATP/ADP in the cells, suggesting the importance of Na+/Pi cotransport in the production of high-energy Pi-containing compounds in neurons. Accordingly, cells that do not express BNPI should alternatively express other related molecules to maintain high-energy production and metabolism.

A recent examination by immunohistochemistry using anti-BNPI antibody localized BNPI almost exclusively to a subset of nerve terminals (Bellocchio et al., 1998), a feature similar to that found for DNPI (authors’ unpublished data). Both electron microscopy analysis and biochemical fractionation have shown that BNPI is associated preferentially with the membranes of synaptic vesicles, suggesting specific presynaptic roles in neurotransmission (Bellocchio et al., 1998). As both BNPI and DNPI show high sequence similarity to EAT-4, a nematode protein that plays a presynaptic role in glutamatergic transmission (Lee et al., 1999), these proteins may also augment excitatory transmission by increasing the Pi concentration for efficient glutamate synthesis.

In this study, we identified DNPI cDNA from differentially expressed genes during differentiation of AR42J cells into neuron-like cells. Although no DNPI mRNA was detected before treatment of the cells, a remarkable increase in its expression was observed after treatment. As BNPI transcripts were not detected throughout the course of differentiation (data not shown), DNPI rather than BNPI may be involved in the initial step of neuronal or neuroendocrine differentiation. This seems consistent with the high signals and no signals for DNPI and BNPI mRNAs, respectively, in fetal brain by northern blotting (Fig. 4) and with the previous observation that BNPI mRNA is detectable only in restricted regions of rat brain on embryonic day 17 by in situ hybridization analysis (Ni et al., 1995). In contrast, as BNPI mRNA has been shown to be dramatically increased in neonatal rat brain, BNPI may play an important role in postnatal development of the brain.

The present study demonstrates that the structurally related BNPI and DNPI, expressed primarily in neuron-rich regions in the brain, form a distinct class within the Na+/Pi cotransporter family. Characterization of the functional properties of these proteins should provide new insight into the cell-specific mechanisms of energy production and metabolism, as well as the developmental regulation of neuronal cells in the brain.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References

We thank Ms. Makiko Koide for her excellent help in the experiment of DNPI mRNA expression in Xenopus oocytes. This study was supported by Grants-in-Aid for Scientific Research and for Creative Basic Research from the Japanese Ministry of Science, Education, Sports and Culture and the Mitsubishi Foundation.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Isolation of human DNPI cDNA
  5. Expression of DNPI mRNA in Xenopus laevis oocytes
  6. Tissue distribution of human DNPI and BNPI mRNAs
  7. RESULTS
  8. Isolation of human DNPI and BNPI cDNAs
  9. Na+/Pi cotransport activity of DNPI in Xenopus oocytes
  10. Tissue distribution of human DNPI and BNPI mRNAs
  11. DISCUSSION
  12. Acknowledgements
  13. References
  • 1
    Bellocchio E.E., Hu H., Pohorille A., Chan J., Pickel V.M., Edwards R.H. (1998) The localization of the brain-specific inorganic phosphate transporter suggests a specific presynaptic role in glutamatergic transmission. J. Neurosci.18 86488659.
  • 2
    Glinn M., Ni B., Paul S.M. (1995) Characterization of Na+-dependent phosphate uptake in cultured fetal rat cortical neurons. J. Neurochem.65 23582365.
  • 3
    Hartmann C.M., Wagner C.A., Busch A.E., Markovich D., Biber J., Lang F., Murer H. (1995) Transport characteristics of a murine renal Na/Pi-cotransporter. Pflugers Arch.430 830836.
  • 4
    Johann S.V., Gibbons J.J., O'Hara B. (1992) GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus. J. Virol.66 16351640.
  • 5
    Kyte J. & Doolittle R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol.157 105132.
  • 6
    Lee R.Y.N., Sawin E.R., Chalfie M., Horvitz H.R., Avery L. (1999) EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in Caenorhabditis elegans. J. Neurosci.19 159167.
  • 7
    Magagnin S., Werner A., Markovich D., Sorribas V., Stange G., Biber J., Murer H. (1993) Expression cloning of human and rat renal cortex. Proc. Natl. Acad. Sci. USA 90 59795983.
  • 8
    Mashima H., Ohnishi H., Wakabayashi K., Mine T., Miyagawa J., Hanafusa T., Seno M., Yamada H., Kojima I. (1996) Beta-cellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells. J. Clin. Invest.97 16471654.
  • 9
    Mashima H., Yamada S., Tajima T., Seno M., Yamada H., Takeda J., Kojima I. (1999) Genes expressed during the differentiation of pancreatic AR42J cells into insulin-secreting cells. Diabetes 48 304309.
  • 10
    Murer H. & Biber J. (1996) Molecular mechanisms of renal apical Na/phosphate cotransporter. Annu. Rev. Physiol.58 607618.
  • 11
    Ni B., Rosteck P.R.J, Nadi N.S., Paul S.M. (1994) Cloning and expression of a cDNA encoding a brain-specific Na+-dependent inorganic phosphate cotransporter. Proc. Natl. Acad. Sci. USA 91 56075611.
  • 12
    Ni B., Wu X., Yan G., Wang J., Paul S.M. (1995) Regional expression and cellular localization of the Na+-dependent inorganic phosphate cotransporter of rat brain. J. Neurosci.15 57895799.
  • 13
    Ni B., Du Y., Wu X., DeHoff B.S., Rosteck P.R.J, Paul S.M. (1996) Molecular cloning, expression, and chromosomal localization of a human brain-specific Na+-dependent inorganic phosphate cotransporter. J. Neurochem.66 22272238.
  • 14
    Ohnishi H., Ohgushi N., Tanaka S., Mogami H., Nobusawa R., Mashima H., Furukawa M., Mine T., Shimada O., Ishikawa H., Kojima I. (1995) Conversion of amylase-secreting rat pancreatic AR42J cells to neuronlike cells by activin A. J. Clin. Invest.95 23042314.
  • 15
    Rosewicz S., Vogt D., Harth N., Grund C., Franke W.W., Ruppert S., Schweitzer E., Riecken E.O., Wiedenmann B. (1992) An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties. Eur. J. Cell Biol.59 8091.
  • 16
    Sorribas V., Markovich D., Hayes G., Stange G., Forgo J., Biber J., Murer H. (1994) Cloning of a Na/Pi-cotransporter from opossum kidney cells. J. Biol. Chem.269 66156621.
  • 17
    Verri T., Markovich D., Perego C., Norbis F., Stange G., Sorribas V., Biber J., Murer H. (1995) Cloning and regulation of a rabbit renal Na/Pi-cotransporter. Am. J. Physiol.268 F626F633.
  • 18
    Werner A., Moore M.L., Mantei N., Biber J., Semenza G., Murer H. (1991) Cloning and expression of cDNA for a Na/Pi cotransporter system of kidney cortex. Proc. Natl. Acad. Sci. USA 88 96089612.
  • 19
    Werner A., Murer H., Kinne R.K. (1994) Cloning and expression of a renal Na/Pi-cotransporter system from flounder. Am. J. Physiol.267 F311F317.
  • 20
    Werner A., Dehmelt L., Nalbant P. (1998) Na+-dependent phosphate cotransporters: the NaPi protein families. J. Exp. Biol.201 31353142.