The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels

Abstract

Epithelial Na(+) channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel beta subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na(+) channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.

Fig. 1.

ENaC are regulated by phosphorylation. (A) Amiloride-sensitive currents during dialysis with 0-Na solution or 0-Na⁺ solution plus 10 mM 2-deoxy-d-glucose/5 μM oligomycin plus either 5 mM AMP-PCP or 5 mM ATP. (B) Mean steady-state current-voltage relations of the amiloride-sensitive current under the conditions in A. (C) Amiloride-sensitive conductance during dialysis with 0-Na solution or 0-Na⁺ solution plus 2-deoxy-d-glucose/oligomycin plus 5 mM AMP-PNP, AMP-PCP, ATP, AMP-PNP plus 10 μM OA, or AMP-PNP plus 1,000 units/ml PPI2. (D) Amiloride-sensitive conductance during dialysis with 0-Na⁺ solution plus 200 μg/ml heparin ± OA or 2 μg/ml anti-Nedd4 antibody. Amiloride-sensitive current was calculated by subtracting the current after addition to the bath of 100 μM amiloride from that measured before the addition of amiloride. Data are given as mean ± SEM. Statistical significance was assessed by using Student's unpaired t test.

Fig. 2.

Grk2 regulates ENaC. (A) Amiloride-sensitive conductance during dialysis with 0-Na⁺ solution plus βC10 peptide ± the phosphatase inhibitors OA (10 μM) or PPI2 (1,000 units/ml). (B) Amiloride-sensitive conductance during dialysis with 0-Na⁺ solution plus the CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (100 μM) or LY294002 (50 μM). (C) Amiloride-sensitive conductance during dialysis with 0-Na⁺ solution ± antibodies (2 μg/ml) against Grk2 or Grk3. Antibody was inactivated (inact) at 60°C for 45 min. (D) In vitro incorporation of radioactive phosphate by Grk2 into various phosphopeptides derived from the terminal pentadecapeptide of mβ-ENaC (CQPLDTMESDSEVEAI). WT is unphosphorylated CQPLDTMESDSEVEAI, S631P phosphorylated at S631 [CQPLDTMES(P)DSEVEAI], S633P at S633 [CQPLDTMESDS(P)EVEAI], and S631PS633P at both serines [CQPLDTMES(P)DS(P)EVEAI].

Fig. 3.

Phosphorylation of ENaC regulates their sensitivity to ubiquitin protein ligases. (A) The effect of OA (10 μM), PPI2 (1,000 units/ml), or recombinant Grk2 (1 μg/ml) on the inhibition of the amiloride-sensitive conductance produced by dialyzing the cells with 70 mM Na⁺ solution. (B) The effect on the amiloride-sensitive conductance of dialyzing the cells with 0-Na⁺ solution containing recombinant Δ1–362mNedd4-2 (Δ(WW1,WW2)Nedd4-2, 100 μg/ml) ± the 100 μg/ml K48R mutant of ubiquitin, 10 μM OA, or 1 μg/ml recombinant Grk2.

Fig. 4.

Model summarizing the present findings. These findings indicate that the ENaC exists in the following two states: a phosphorylated state that has high activity and a dephosphorylated state that has low activity. The equilibrium between the phosphorylated and the dephosphorylated states is maintained by the kinase Grk2 and PP1. Inhibition of the kinase by the inclusion in the pipette solution of nonhydrolyzable analogues of ATP, such as AMP-PNP and AMP-PCP, or the Grk2 inhibitor, heparin, or of an excess of its substrate the terminal decapeptide of the β subunit of the channel (βC10), leads to decreased channel activity. Conversely, inhibition of the phosphatase with OA or PPI2, leads to an increase in the activity of the channel. Only the dephosphorylated form of the channel is sensitive to inhibition by Nedd4/Nedd4-2 ubiquitin protein ligases. Hence, the inhibition of the channel by increased intracellular Na⁺, which is mediated by these ubiquitin protein ligases, can be overcome by maintaining the channel in the phosphorylated state.