Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes

摘要

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 ～106μM and a K_m for Na~+ of ～34 mM. The hNaPi-IIb cotransporter was also expressed in Xenopus oocytes and it exhibited a K_m for Pi of ～113 μM and a K_m for Na~+ of ～65mM. The hNaPi-IIb cotransporter expressed in both PS120 cells and oocytes was inhibited by high external pH. Furthermore, 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. ～ ～ ～