Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes |
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Authors: | Xu Hua Inouye Michael Missey Timothy Collins James F Ghishan Fayez K |
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Institution: | Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Avenue, Tucson, AZ 85274, USA. |
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Abstract: | 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. |
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