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)P(2)) levels on the activities of two electrogenic Na‐Pi cotransporters, NaPi‐IIa and NaPi‐IIb. To deplete plasma membrane PI(4,5)P(2) in Xenopus oocytes, we utilized Ciona intestinalis voltage‐sensing phosphatase (Ci‐VSP), which dephosphorylates PI(4,5)P(2) 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)P(2) and PI(4)P from the plasma membrane of cultured Neuro 2a cells. Depletion of PI(4,5)P(2) 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)P(2) and PI(4)P. These results indicate that NaPi‐IIb activity is regulated by PI(4,5)P(2), whereas NaPi‐IIa is not sensitive to either PI(4,5)P(2) 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.