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J. Biol. Chem., Vol. 283, Issue 19, 13225-13232, May 9, 2008

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Regulation of the Epithelial Na⁺ Channel by the Protein Kinase CK2^*

Tanja Bachhuber¹, Joana Almaça¹², Fadi Aldehni, Anil Mehta^¶, Margarida D. Amaral^¶||, Rainer Schreiber, and Karl Kunzelmann³

From the Institut für Physiologie, Universität Regensburg, Universitätsstraβe 31, D-93053 Regensburg, Germany, the ^¶Department of Maternal and Child Health Sciences, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom, the Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal, and the ^||Centre of Human Genetics, National Institute of Health, 1649-016 Lisboa, Portugal

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 β- and -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 (β_S631AT599A) 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 β_S631AT599A-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of β-ENaC. Channels lacking the Nedd4-2 binding motif in β-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 β-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.

Received for publication, June 1, 2007 , and in revised form, February 7, 2008.

^* This work was supported in part by Deutsche Forschungsgemeinschaft SFB699 A6, the Wellcome Trust (to A. M.), and Fundacao para a Ciencia e a Tecnologia grants (Portugal) (to M. D. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Both authors contributed equally to the present work.

² Recipient of the SFRH/BD/29134/2006 PhD fellowship from FCT (Portugal).

³ To whom correspondence should be addressed. Tel.: 49-0-941-943-4302; Fax: 49-0-941-943-4315; E-mail: uqkkunze{at}mailbox.uq.edu.au.

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