Regulation of the epithelial Na+ channel by the protein kinase CK2

Abstract

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.

FIGURE 1.

CK2 activates ENaC in native epithelia and in epithelial cells. Original Ussing chamber recordings of the transepithelial voltages V_te detected in mouse trachea (A), mouse colon (C), and M1 cells (E). Effects of amiloride (A, 10 μm) and the CK2 inhibitor TBB (10 μm). Concentration-dependence of the effects of TBB on amiloride-sensitive transport in trachea (B), colon (D), and M1 cells (F). The asterisk (^*) indicates significant effects of TBB (paired t-tests, number of experiments: 9-13 for each series).

FIGURE 2.

CK2 activates ENaC in Xenopus oocytes. A, current recording from a Xenopus oocyte expressing αβγ-ENaC and effects of amiloride (10 μm) and TBB (10 μm). Oocytes were voltage-clamped from -90 mV to +10 mV in steps of 10 mV, and the resulting currents were recorded. B, summary of the effects of amiloride and TBB on whole cell conductance in ENaC-expressing oocytes. C, summary of the change of amiloride-sensitive conductance (G_amil) after injection of water, the CK2 inhibitor poly(E:Y) (50 μm) and CK2 activator poly(K) (50 μm), respectively. D, current recording of an ENaC-expressing oocyte and the effects of amiloride (A, 10 μm) and okadaic acid (100 nm). E, summary of the effect of okadaic acid on the amiloride-sensitive whole cell conductance measured in oocytes. The asterisk (^*) indicates significant effects (paired t-test). The number sign (#) indicates a significant difference for the effects of amiloride before and after incubation with TBB (paired t-test, 6-25 experiments for each series).

FIGURE 3.

Elimination of CK2 phosphorylation sites on ENaC inhibits channel activity. A, current recording from a Xenopus oocyte expressing αβγ-ENaC and effects of amiloride (10μm) and TBB (10μm). Oocytes were voltage-clamped from -90 mV to +10 mV in steps of 10 mV, and the resulting currents were recorded. B, current recording from a Xenopus oocyte expressing αβ_S631Aγ_T559A-ENaC and effects of amiloride (10 μm) and TBB (10 μm). C and D, summaries of the effects of amiloride and TBB on G_amil generated by αβγ-ENaC and αβ_S631Aγ_T559A-ENaC. E, comparison of G_amil produced by wt-(αβγ)-, single mutants (αβ_S631Aγ, αβγ_T559A)-, and a doublemutant (αβ_S631Aγ_T559A)-ENaC.F, summaries of G_amil produced by dimeric wt-(αβ, αγ)- and mutant (αβ_S631A, αγ_T559)-ENaC channels. The asterisk (^*) and number sign (#) indicate a significant difference (paired t-test, 13-25 experiments for each series).

FIGURE 4.

CK2 controls membrane expression of ENaC in Xenopus oocytes. Time course for G_amil (A, C, E) and membrane expression of α_Flag-ENaC (B, D, F). Ooctyes were kept in ND97 or in ND97 containing TBB (10 μm), or were injected with heparin (10 μm), poly(E:Y) (50 μm), or equal amounts (30 nl) of water. The asterisk (^*) indicates significant differences when compared with ND96 or water (unpaired t-tests, 6-13 experiments for each series).

FIGURE 5.

CK2 is essential for ENaC activity and antagonizes the inhibitory effect of Nedd4-2 on ENaC. A, summary of α-ENaC membrane expression and G_amil after 40 h. TBB inhibited membrane expression of α-ENaC via single mutants (αβ_S631Aγ,αβγ_T559A) but not that of the double mutant (αβ_S631Aγ_T559A). G_amil was largely reduced for all mutants, and G_amil produced by the double mutant was no longer inhibited by TBB. Dashed lines indicate membrane expression and G_amil of wt-ENaC. B, whole cell conductances relative to wt-ENaC. A mutation in the PY motif (Y618A) of β-ENaC increased Na⁺ conductance, and S631A no longer inhibited ENaC conductance. The Grk2 mutant S633A inhibited ENaC, but not as a double mutant S631A/S633A. C, inhibition of xNedd4-2 expression by siRNA-xNedd4-2 but not scrambled siRNA. The abundant poly(A)-binding protein indicates equal loading. Summary of ENaC whole cell conductances measured in the absence or presence of siRNA-xNedd4-2 or scrambled siRNA (see “Materials and Methods”). D, summary of the amiloride-sensitive short-circuit current and effects of TBB (10 μm) in control M1 cells and M1 cell treated with scrambled RNAi, mNedd4-2-RNAi, and mCK2-RNAi (see “Materials and Methods”). The asterisk (^*) indicates significant effects of TBB (paired t-tests). The number sign (#) indicates a significant difference compared with control (unpaired t-test, 6-24 experiments for each series).

FIGURE 6.

CK2 is not essential for membrane expression of β-ENaC and γ-ENaC. A, inhibition of membrane expression of β_Flag-ENaC and γ_Flag-ENaC and G_amil by 10 μm TBB. B and C, TBB inhibited membrane expression of β_Flag-ENaC and γ_Flag-ENaC in single mutants (αβ_S631Aγ, αβγ_T559A), but not in double mutants (αβ_S631Aγ_T559A). G_amil was largely reduced for all mutants, and G_amil produced by the double mutants was no longer inhibited by TBB. Dashed lines indicate membrane expression and G_amil of wt-ENaC. The asterisk (^*) indicates a significant effect of TBB (paired t-tests, 6-12 experiments for each series).

FIGURE 7.

wt-ENaC translocates CK2 to the cell membrane. A, DIC image of the oocyte membrane (left panels) and immunostaining of α_Flag-ENaC in an ENaC-expressing (right upper panel) and a non-injected (right lower panel) oocyte. B, immunostaining of the three ENaC subunits (green) and CK2 (red) in wt-ENaC (left panel) and αβ_S631Aγ_T559A-ENaC (right panel)-expressing oocytes. Bars indicate 10 μm. Experiments were performed in at least triplicates.

FIGURE 8.

Model for CK2 action on ENaC. Binding of the ubiquitin ligase Nedd4-2 leads to ubiquitination of ENaC and subsequent degradation of the channel and/or channel inactivation. Phosphorylation of ENaC at Ser-631 reduces affinity of ENaC for Nedd4-2, thereby maintaining membrane localization and ENaC activity.