title: Liddle-Mutation of the β-Subunit, but not the γ-Subunit, Attenuates Protein Kinase C-Mediated Inhibition of Human Epithelial Sodium Channels (hENaC).
abstract: Mammalian distal nephron and distal colon, prime sites for Na(+) homeostasis, contain amiloride-sensitive epithelial sodium channels (ENaC). Protein kinase C (PKC) inhibits ENaC by phosphorylating serine and threonine residues within COOH termini of the β- and/or γ-subunits. Although some of these phosphorylation sites are close to the PY motifs, it is unclear whether they remain susceptible to PKC in Liddle-mutated ENaC β- and/or γ-subunits, where PY motifs are truncated, resulting in increased apical ENaC expression. We therefore studied the effects of PKC in wild-type and Liddle-mutated human epithelial Na(+) channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. PKC activation using 500 nmol/l phorbol 12-myristate 13-acetate (PMA) decreased amiloride-sensitive Na(+) currents by 80 % in oocytes expressing wild-type hENaC, an effect largely prevented by co-exposure to 50 µmol/l calphostin C (a specific inhibitor of PKC), whereas 500 nmol/l phorbol didecanoate (PDD), an inactive phorbol ester which does not stimulate PKC, had no effect. In oocytes expressing hENaC containing the Liddle-mutated β-subunit, PMA elicited a 54 % decrease in amiloride-sensitive Na(+) currents, significantly (P < 0.0025) less than that in oocytes expressing wild-type hENaC. By contrast, in oocytes expressing hENaC containing the Liddle-mutated γ-subunit, PMA elicited a 68 % decrease in amiloride-sensitive Na(+) current, similar (P = 0.10) to that in oocytes expressing wild-type hENaC. We conclude that hENaC incorporating the Liddle-mutated β-subunit lacks one or more PKC phosphorylation sites, thereby significantly reducing the inhibitory effect of PKC on Na(+) channel activity, whereas hENaC incorporating Liddle-mutated γ-subunits remains as susceptible to PKC as wild-type hENaC.
authors.affiliation: Leeds Institute of Biomedical and Clinical Sciences, St James's University Hospital, Level 7, Clinical Sciences Building, Beckett Street, Leeds, LS9 7TF, UK.
authors.forename: Gerard G
authors.initials: GG
authors.lastname: Robins
authors.affiliation: Leeds Institute of Biomedical and Clinical Sciences, St James's University Hospital, Level 7, Clinical Sciences Building, Beckett Street, Leeds, LS9 7TF, UK. g.i.sandle@leeds.ac.uk.
authors.forename: Geoffrey I
authors.initials: GI
authors.lastname: Sandle
mesh_terms: D000818:Animals; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D009154:Mutation; D009281:Naphthalenes / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010703:Phorbol Esters / Q000494:pharmacology; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D053503:Epithelial Sodium Channels; D009281:Naphthalenes; D010703:Phorbol Esters; D021122:Protein Subunits; C119222:phorbol-12-myristate; D011493:Protein Kinase C; C058819:calphostin C
keywords: ENaC; Liddle mutations; Oocytes; Protein kinase C; Sodium channels