Inorganic phosphate (Pi) is an essential constituent for maintenance of cell activity. Na-Pi cotransporter (SLC34) is the main molecule for Pi homeostasis in kidney. NaPi-IIa (SLC34A1) is localized at apical brush border membrane in the renal proximal tubule and reabsorbs Pi from glomerular filtrate (Forster et al, 2002). One of the regulation mechanisms of this transporter activity in the tubule is to reduce the molecule number by vesicular transport system through endocytosis. However, other mechanisms, especially in situ modulation of the activity on plasma membrane, are not known. It is known that the activity of various ion channels and some transporters is regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) abundant in plasma membrane (Suh et al, 2008, Thornell et al, 2015). Because NaPi-IIa functions on plasma membrane, we hypothesized that the transporter activity may depend on PI(4,5)P2. To test this idea, we expressed mouse NaPi-IIa in Xenopus oocytes and evaluated its PI(4,5)P2 dependence by using Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) that has phosphatase activity for phosphoinositide depending on membrane potential. During NaPi-IIa current recording, depolarization pulse was applied to activate Ci-VSP to induce reduction of PI(4,5)P2 on plasma membrane. PI(4,5)P2 level was monitored by measuring rat KCNQ2/3 current coexpressed with mNaPi-IIa and Ci-VSP. Na-bicarbonate cotransporter (rb2NBC) was used as a positive control. We first measured PI(4,5)P2 dependence of rb2NBC. After depolarization, rb2NBC current was significantly decreased (pre 53.1 ± 7.1 nA, post 40.5 ± 6.6 nA, n = 8, paired t-test; p < 0.0001, rate of change 0.741 ± 0.036) where reduced PI(4,5)P2 level was observed. However, NaPi-IIa current did not change before and after depolarization (pre 105.5 ± 21.2 nA, post 108.2 ± 15.9 nA, n = 4, p = 0.733, rate of change 1.064 ± 0.077). These results suggest that mouse NaPi-IIa has no or extremely low PI(4,5)P2 dependence. Ci-VSP can dephosphorylate PI(4,5)P2 upon membrane depolarization, but produces PI(4)P. However, the magnitude of NaPi-IIa current may not be affected by Ci-VSP if the activity of mNaPi-IIa both depend on PI(4)P and PI(4,5)P2 . We are now examining PI(4)P dependence of mNaPi-IIa by using Pseudojanin, a chimera protein, which has both activities of 5-phosphatase and 4-phosphatase (Hammond et al, 2012).