Distinct functional properties of two electrogenic isoforms of the SLC34 Na-Pi cotransporter

Inorganic phosphate (P_i ) is crucial for proper cellular function in all organisms. In mammals, type II Na-Pi cotransporters encoded by members of the Slc34 gene family play major roles in the maintenance of P_i homeostasis. However, the molecular mechanisms regulating Na-Pi cotransporter activity within the plasma membrane are largely unknown. In the present study, we used two approaches to examine the effect of changing plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂ ) levels on the activities of two electrogenic Na-Pi cotransporters, NaPi-IIa and NaPi-IIb. To deplete plasma membrane PI(4,5)P₂ in Xenopus oocytes, we utilized Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which dephosphorylates PI(4,5)P₂ to phosphatidylinositol 4-phosphate (PI(4)P). Upon activation of Ci-VSP, NaPi-IIb currents were significantly decreased, whereas NaPi-IIa currents were unaffected. We also used the rapamycin-inducible Pseudojanin (PJ) system to deplete both PI(4,5)P₂ and PI(4)P from the plasma membrane of cultured Neuro 2a cells. Depletion of PI(4,5)P₂ and PI(4)P using PJ significantly reduced NaPi-IIb activity, but NaPi-IIa activity was unaffected, which excluded the possibility that NaPi-IIa is equally sensitive to PI(4,5)P₂ and PI(4)P. These results indicate that NaPi-IIb activity is regulated by PI(4,5)P₂ , whereas NaPi-IIa is not sensitive to either PI(4,5)P₂ or PI(4)P. In addition, patch clamp recording of NaPi-IIa and NaPi-IIb currents in cultured mammalian cells enabled kinetic analysis with higher temporal resolution, revealing their distinct kinetic properties.