Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates epithelial sodium channel activity in A6 cells

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane lipid found in all eukaryotic cells, which regulates many important cellular processes, including ion channel activity. In this study, we used inside-out patch clamp technique, immunoprecipitation, and Western blot analysis to investigate the effect Of PIP2 on epithelial sodium channel activity in A6 cells. A6 cells were cultured in media supplemented with 1.5 mum aldosterone. Single sodium channel activity in excised, inside-out patches was increased by perfusion of the bath solution with 30 mum PIP2 Plus 100 mum GTP (NPo = 1.34 +/- 0.14) compared with the paired control (NPo = 0.09 +/- 0.02). However, neither 30 mum PIP2 (NPo = 0.11 +/- 0.02) nor 100 mum GTP (NPo = 0.10 +/- 0.02) alone stimulated the sodium channels. The PIP2-stimulated channel activity was abolished by application of 10 nm G protein betagamma subunits (NPo = 0.14 +/- 0.05). However, 10 nm Galpha(i-3) + 30 mum PIP2 increased both NP. and P.. The stimulating effect of 10 nm Galpha(i-3) + 30 muM PIP2 is similar to that of 30 muM PIP2 plus 100 muM GTP. Immunoprecipitation and Western blot analysis show that both Gi(alpha-3) and PIP2 bind beta and gamma epithelial Na+ channels (ENaC), but not a ENaC. These results indicate that PIP2 increases ENaC activity by direct interaction with beta or gamma xENaC in the presence of Galpha(i-3).