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.
The mechanisms involved in the regulation of the epithelial sodium channel (ENaC) via the cAMP pathway are not yet completely understood. The aim of the present study was to investigate cAMP-mediated ENaC regulation in Xenopus laevis oocytes heterologously expressing the three subunits (alphabetagamma) of rat ENaC and to determine the ENaC regions important for mediating the stimulatory effect of cAMP. In oocytes treated for about 24 h with 1 mm 3-isobutyl-1-methylxanthine (IBMX) and 1 microm forskolin (FSK) so as to increase intracellular cAMP, the amiloride-sensitive whole cell current (DeltaI(Ami)) was on average 10-fold larger than DeltaI(Ami) in matched control oocytes. This effect on DeltaI(Ami) was paralleled by an increase in ENaC surface expression caused by a reduced rate of ENaC retrieval. In addition, IBMX/FSK also enhanced ENaC open probability from about 0.2 to 0.5. The stimulatory effect of IBMX/FSK was dependent on the presence of intact PY motifs in the C termini of the channel. Mutagenesis of putative protein kinase A and CK-2 consensus motifs in the cytosolic domains of the channel did not reveal critical sites involved in mediating the stimulatory effect of IBMX/FSK. In contrast, site-directed mutagenesis of two putative ERK-consensus motifs (T613A in betaENaC and T623A in gammaENaC) largely reduced the stimulatory effect of IBMX/FSK. Phosphorylation of these ERK sites has previously been reported to enhance the interaction of ENaC and Nedd4 (Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R., and Garty, H. (2002) J. Biol. Chem. 277, 13539-13547). Using co-expression experiments we demonstrated that mutating the two ERK sites attenuates the inhibitory effect of Nedd4-2 on ENaC currents. We conclude that an increase in intracellular cAMP favors the dephosphorylation of the two ERK sites, which reduces channel retrieval and increases P(O) by modulating ENaC/Nedd4 interaction. This defines a novel regulatory pathway likely to be relevant for cAMP-induced stimulation of ENaC in vivo.
Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstrasse 6, 91054 Erlangen, Germany.
Li-Min
LM
Yang
Ralf
R
Rinke
Christoph
C
Korbmacher
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis
D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors; D053503:Epithelial Sodium Channels; D018075:RNA, Complementary; D015222:Sodium Channels; D029867:Xenopus Proteins; D005576:Colforsin; D000242:Cyclic AMP; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618484:Nedd4 protein, rat; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; D017868:Cyclic AMP-Dependent Protein Kinases; D015056:1-Methyl-3-isobutylxanthine