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J Biol Chem
2008 May 09;28319:13225-32. doi: 10.1074/jbc.M704532200.
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Regulation of the epithelial Na+ channel by the protein kinase CK2.
Bachhuber T
,
Almaça J
,
Aldehni F
,
Mehta A
,
Amaral MD
,
Schreiber R
,
Kunzelmann K
.
???displayArticle.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
Vte 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
(Gamil) 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 αβS631AγT559A-ENaC
and effects of amiloride (10 μm) and TBB (10 μm).
C and D, summaries of the effects of amiloride and TBB on
Gamil generated by αβγ-ENaC and
αβS631AγT559A-ENaC. E,
comparison of Gamil produced by
wt-(αβγ)-, single mutants
(αβS631Aγ, αβγT559A)-,
and a doublemutant
(αβS631AγT559A)-ENaC.F,
summaries of Gamil produced by dimeric wt-(αβ,
αγ)- and mutant (αβS631A,
αγT559)-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 Gamil (A, C, E) and
membrane expression of αFlag-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 Gamil after 40 h. TBB inhibited membrane
expression of α-ENaC via single mutants
(αβS631Aγ,αβγT559A)
but not that of the double mutant
(αβS631AγT559A).
Gamil was largely reduced for all mutants, and
Gamil produced by the double mutant was no longer
inhibited by TBB. Dashed lines indicate membrane expression and
Gamil 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
βFlag-ENaC and γFlag-ENaC and
Gamil by 10 μm TBB. B and
C, TBB inhibited membrane expression of βFlag-ENaC
and γFlag-ENaC in single mutants
(αβS631Aγ, αβγT559A),
but not in double mutants
(αβS631AγT559A).
Gamil was largely reduced for all mutants, and
Gamil produced by the double mutants was no longer
inhibited by TBB. Dashed lines indicate membrane expression and
Gamil 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
αFlag-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
αβS631AγT559A-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.
Awayda,
Protein kinase regulation of a cloned epithelial Na+ channel.
1996, Pubmed,
Xenbase
Awayda,
Protein kinase regulation of a cloned epithelial Na+ channel.
1996,
Pubmed
,
Xenbase
Bachhuber,
Cl- interference with the epithelial Na+ channel ENaC.
2005,
Pubmed
,
Xenbase
Becchetti,
Phosphatase inhibitors increase the open probability of ENaC in A6 cells.
2002,
Pubmed
Bildl,
Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating.
2004,
Pubmed
,
Xenbase
Cai,
Calcium dependence of polycystin-2 channel activity is modulated by phosphorylation at Ser812.
2004,
Pubmed
Canessa,
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
1994,
Pubmed
,
Xenbase
Debonneville,
Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression.
2001,
Pubmed
,
Xenbase
Diakov,
A novel pathway of epithelial sodium channel activation involves a serum- and glucocorticoid-inducible kinase consensus motif in the C terminus of the channel's alpha-subunit.
2004,
Pubmed
,
Xenbase
Dinudom,
The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels.
2004,
Pubmed
Falin,
Acute downregulation of ENaC by EGF involves the PY motif and putative ERK phosphorylation site.
2007,
Pubmed
Feldman,
Deactivation of vasodilator responses by GRK2 overexpression: a mechanism or the mechanism for hypertension?
2002,
Pubmed
Firsov,
Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.
1996,
Pubmed
,
Xenbase
Hu,
Casein kinase II and calcineurin modulate TRPP function and ciliary localization.
2006,
Pubmed
Kimura,
Cloning and functional characterization of squid voltage-dependent Ca2+ channel beta subunits: involvement of N-terminal sequences in differential modulation of the current.
2003,
Pubmed
,
Xenbase
Kunzelmann,
Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator.
2000,
Pubmed
Meggio,
Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate.
1994,
Pubmed
Michlig,
Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.
2005,
Pubmed
,
Xenbase
Rajamanickam,
EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.
2007,
Pubmed
,
Xenbase
Schermer,
Phosphorylation by casein kinase 2 induces PACS-1 binding of nephrocystin and targeting to cilia.
2005,
Pubmed
Shi,
Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation.
2002,
Pubmed
,
Xenbase
Shi,
Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits.
2002,
Pubmed
,
Xenbase
Snyder,
Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel.
2002,
Pubmed
Snyder,
cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2.
2004,
Pubmed
Treharne,
Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site.
2007,
Pubmed
,
Xenbase
Yan,
Intracellular trafficking of a polymorphism in the COOH terminus of the alpha-subunit of the human epithelial sodium channel is modulated by casein kinase 1.
2007,
Pubmed
,
Xenbase
Yang,
Stimulation of the epithelial sodium channel (ENaC) by cAMP involves putative ERK phosphorylation sites in the C termini of the channel's beta- and gamma-subunit.
2006,
Pubmed
,
Xenbase
