Faculty Collaboration Database

Molecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel. J Gen Physiol 2007 Oct;130(4):399-413

Date

09/26/2007

Pubmed ID

17893193

Pubmed Central ID

PMC2151653

DOI

10.1085/jgp.200709800

Scopus ID

2-s2.0-34748880880 (requires institutional sign-in at Scopus site)   68 Citations

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) 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)P(3) and PI(4,5)P(2) 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 beta- and gamma- but not alpha-ENaC as necessary for PI(3,4,5)P(2) but not PI(4,5)P(2) 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 beta- and gamma-ENaC are critical to PI(3,4,5)P(3) augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of beta- and gamma-ENaC were identified as being critical to down-regulation of ENaC activity and P(o) in response to depletion of membrane PI(4,5)P(2). These regions of the channel played no identifiable role in a PI(3,4,5)P(3) response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P(2) to increase open probability. We conclude that beta and gamma subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P(3) and PI(4,5)P(2). This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

Author List

Pochynyuk O, Tong Q, Medina J, Vandewalle A, Staruschenko A, Bugaj V, Stockand JD

MESH terms used to index this publication - Major topics in bold

Amino Acid Motifs
Amino Acid Substitution
Animals
Binding Sites
CHO Cells
Cell Line, Transformed
Cricetinae
Cricetulus
Epithelial Sodium Channel Agonists
Epithelial Sodium Channels
Ion Channel Gating
Mice
Microscopy, Fluorescence
Patch-Clamp Techniques
Phosphatidylinositol 4,5-Diphosphate
Phosphatidylinositol Phosphates
Photoaffinity Labels
Protein Subunits