Abstract
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).
Highlights
Amiloride-sensitive epithelial Naϩ channels in the distal nephron play a critical role in regulation of sodium transport across renal epithelial cells and play a central role in maintenance of salt balance and normal blood pressure
These results indicate that PIP2 increases epithelial Na؉ channels (ENaC) activity by direct interaction with  or ␥ xENaC in the presence of Gi-3 protein (G␣i-3)
This idea is supported by the observation that stimulation of ENaC activity by PIP2 ϩ GTP was abolished by addition of G protein ␥ subunit protein in the bath solution (Table I)
Summary
Amiloride-sensitive epithelial Naϩ channels in the distal nephron play a critical role in regulation of sodium transport across renal epithelial cells and play a central role in maintenance of salt balance and normal blood pressure. The direct regulation of ENaC activity by membrane lipid has, to our knowledge, never before been demonstrated. Recent studies using short circuit measurements demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) is present in A6 cells and that this enzyme is required for regulation of ENaC by insulin [6], aldosterone, and vasopressin (9 –12). The products of PI 3-kinase include phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-triphosphate [13] These studies indicate that phosphoinositides are involved in the regulation of ENaC activity, and these membrane lipids may be common effector molecules for hormonal regulation. We examined the PIP2 regulation of single ENaC activity in A6 cells with the patch clamp technique and identified PIP2 binding to ENaC subunits by coimmunoprecipitation and Western blot analysis
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