Cloning of a Na/Pi cotransporter from opossum kidney cells

J Biol Chem. 1994 Mar 4;269(9):6615-21.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Biological Transport / drug effects
  • Carrier Proteins / biosynthesis*
  • Carrier Proteins / metabolism
  • Cells, Cultured
  • Cloning, Molecular
  • DNA, Complementary / chemistry
  • DNA, Complementary / metabolism
  • Female
  • Gene Library
  • Humans
  • Hydrogen-Ion Concentration
  • Kidney / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides / pharmacology
  • Oocytes / physiology
  • Opossums
  • Phosphates / metabolism
  • Poly A / isolation & purification
  • Poly A / metabolism
  • Protein Biosynthesis
  • RNA / isolation & purification
  • RNA / metabolism
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / metabolism
  • Rats
  • Sequence Homology, Amino Acid
  • Sodium / metabolism
  • Sodium-Phosphate Cotransporter Proteins
  • Symporters*
  • Xenopus laevis

Substances

  • Carrier Proteins
  • DNA, Complementary
  • Oligodeoxyribonucleotides
  • Phosphates
  • RNA, Messenger
  • Sodium-Phosphate Cotransporter Proteins
  • Symporters
  • Poly A
  • RNA
  • Sodium

Associated data

  • GENBANK/L26308