Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes.
The recently cloned NaPi-IIb cotransporter is an apical membrane protein that is involved in the absorption of phosphate in the intestine. To expedite functional and structural studies, the human intestinal NaPi-IIb cotransporter was stably expressed in hamster fibroblast (PS120) cells. The hNaPi-IIb cDNA stably transfected cells exhibited a 1.8-fold higher sodium-dependent phosphate uptake than vector DNA transfected cells, and had a K(m) for Pi of approximately 106 microM and a K(m) for Na(+) of approximately 34 mM. The hNaPi-IIb cotransporter was also expressed in Xenopus oocytes and it exhibited a K(m) for Pi of approximately 113 microM and a K(m) for Na(+) of approximately 65 mM. The hNaPi-IIb cotransporter expressed in both PS120 cells and oocytes was inhibited by high external pH. Furthermore, the phosphate uptake mediated by the hNaPi-IIb cotransporter was inhibited by 5 mM phosphonoformic acid (PFA), 1 mM arsenate and 100 nM phorbol myristate acetate (PMA). These results demonstrate that the human intestinal NaPi-IIb cotransporter is functional when expressed in hamster fibroblasts, and that this model system may be useful in the future to identify NaPi-IIb cotransporter-specific inhibitors.
Xu
Hua
H
Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Avenue, Tucson, AZ 85274, USA.
Inouye
Michael
M
Missey
Timothy
T
Collins
James F
JF
Ghishan
Fayez K
FK
D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D017931:DNA Primers; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D013379:Substrate Specificity; D027981:Symporters / Q000378:metabolism / Q000502:physiology*; D014981:Xenopus
D017931:DNA Primers; D011994:Recombinant Proteins; C493557:SLC34A2 protein, human; D050608:Sodium-Phosphate Cotransporter Proteins; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters