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.
Sorribas
V
V
Institute of Physiology, University of Zürich, Switzerland.
Markovich
D
D
Hayes
G
G
Stange
G
G
Forgo
J
J
Biber
J
J
Murer
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D005260:Female; D015723:Gene Library; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D009893:Opossums; D010710:Phosphates / Q000378:metabolism; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D012333:RNA, Messenger; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D011061:Poly A; D012313:RNA; D012964:Sodium