Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na+ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P3 and PI(4,5)P2 to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P2 but not PI(4,5)P2 modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P3 augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and Po in response to depletion of membrane PI(4,5)P2. These regions of the channel played no identifiable role in a PI(3,4,5)P3 response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P2 to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P3 and PI(4,5)P2. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

Insulin activates epithelial sodium channel (ENaC) via phosphoinositide 3-kinase in mammalian taste receptor cells

“…Recent studies indicate that ENaC activity can be increased directly by phosphatidylinositides (35,37). In the present study, we revealed the importance of PI4-kinases in the taste system.…”

Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

“…In addition to ROMK1, ENaC is also regulated by cholesterol 34,35 and PI(4,5)P 2 . [36][37][38] However, unlike ROMK1 channels, ENaC appears to be localized in microvilli. 39 Therefore, investigating how cholesterol and PI(4,5)P 2 regulate ENaC in microvilli would serve as interesting topics for our future studies.…”

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

“…Recently, however, PI(3,4,5)P 3 was shown to regulate TRPC6 by disrupting its association with calmodulin (11), and PI(4,5)P 2 has been reported to be important for ENaC channel gating and PI(3,4,5)P 3 for ENaC channel open probability and number (9,10). These results suggest that different phosphoinositides in a cell can regulate different functional aspects of the same protein.…”