Ras couples phosphoinositide 3-OH kinase to the epithelial Na+ channel.
Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells approximately 3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPbetaS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110alpha) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.
Staruschenko
Alexander
A
University of Texas Health Science Center at San Antonio, Department of Physiology-7756, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. staruschchenk@uthscsa.edu
Pochynyuk
Oleh M
OM
Tong
Qiusheng
Q
Stockand
James D
JD
D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D053503:Epithelial Sodium Channels; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D015222:Sodium Channels / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D000450:Aldosterone; D019869:Phosphatidylinositol 3-Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins