Ras Activates the Epithelial Na+ Channel through Phosphoinositide 3-OH Kinase Signaling

Abstract

Aldosterone induces expression and activation of the GTP-dependent signaling switch K-Ras. This small monomeric G protein is both necessary and sufficient for activation of the epithelial Na ؉ channel (ENaC). The mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras activates human ENaC reconstituted in Chinese hamster ovary cells in a GTP-dependent manner. K-Ras influences ENaC activity most likely by affecting open probability. Inhibition of phosphoinositide 3-OH kinase (PI3K) abolished K-Ras actions on ENaC. In contrast, inhibition of other K-Ras effector cascades, including the MAPK and Ral/Rac/Rho cascades, did not affect KRas actions on ENaC. Activation of ENaC by K-Ras, moreover, was sensitive to co-expression of dominant negative p85 PI3K . The G12:C40 effector-specific double mutant of Ras, which preferentially activates PI3K, enhanced ENaC activity in a manner sensitive to inhibition of PI3K. Other effector-specific mutants preferentially activating MAPK and RalGDS signaling had no effect. Constitutively active PI3K activated ENaC independent of K-Ras with the effects of PI3K and K-Ras on ENaC not being additive. We conclude that K-Ras activates ENaC via the PI3K cascade.

Rho Small GTPases Activate the Epithelial Na+ Channel

“…8.). In these experiments, ␣-and ␤ENaC migrated as overlying ϳ85-kDa bands and ␥ENaC as an ϳ80-kDa band, which is similar to that observed previously (8,29). As summarized in Fig.…”

“…In previous work, we showed that Ras GTPases activate ENaC in a GTP-dependent manner (8). However, we also observed activation of ENaC by endogenous GTPases other than Ras.…”

“…Electrophysiology-Whole cell macroscopic current recordings of ENaC reconstituted in CHO cells were made under voltage clamp conditions using standard methods (7,8,22). Current through ENaC was the inward, amiloride-sensitive Na ϩ current with a bath solution containing (in mM) 160 NaCl, 1 CaCl 2 , 2 MgCl 2, and 10 HEPES (pH 7.4) and a pipette solution containing (in mM) 120 CsCl, 5 NaCl, 2 MgCl 2 , 5 EGTA, 10 HEPES (pH 7.4), 2.0 ATP, and 0.1 GTP.…”

“…We have demonstrated previously that small G proteins within the Ras family (e.g. K-RasA) augment ENaC activity by stimulating PI3K signaling (8) with the phospholipid products of this lipid kinase (PI(3,4)P 2 and PI(3,4,5)P 3 ) increasing channel open probability. Regulation of ENaC by other small G proteins has not been reported.…”

Endothelin-1 Inhibits the Epithelial Na+ Channel through βPix/14-3-3/Nedd4-2

“…20 Our previous studies showed that several small G proteins, including K-Ras, RhoA, and Rab11a, alter ENaC activity. [21][22][23][24][25][26] The activity of small G proteins is modulated by guanine nucleotide exchange factors (GEFs). p21-activated kinase (Pak)-interacting exchange factor ␤ (␤Pix) is a member of the DBL (diffuse B cell lymphoma) family of Rho-GEFs for Rac1 and Cdc42.…”