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

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) 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 PIP(2) on epithelial sodium channel activity in A6 cells. A6 cells were cultured in media supplemented with 1.5 microm aldosterone. Single sodium channel activity in excised, inside-out patches was increased by perfusion of the bath solution with 30 microm PIP(2) plus 100 microm GTP (NP(o) = 1.34 +/- 0.14) compared with the paired control (NP(o) = 0.09 +/- 0.02). However, neither 30 microm PIP(2) (NP(o) = 0.11 +/- 0.02) nor 100 microm GTP (NP(o) = 0.10 +/- 0.02) alone stimulated the sodium channels. The PIP(2)-stimulated channel activity was abolished by application of 10 nm G protein betagamma subunits (NP(o) = 0.14 +/- 0.05). However, 10 nm Galpha(i-3) + 30 microm PIP(2) increased both NP(o) and P(o). The stimulating effect of 10 nm Galpha(i-3) + 30 microm PIP(2) is similar to that of 30 microm PIP(2) plus 100 microm GTP. Immunoprecipitation and Western blot analysis show that both Gi(alpha-3) and PIP(2) bind beta and gamma epithelial Na(+) channels (ENaC), but not alpha ENaC. These results indicate that PIP(2) increases ENaC activity by direct interaction with beta or gamma xENaC in the presence of Galpha(i-3).