Regulation of epithelial sodium channel activity through a region of the carboxyl terminus of the alpha -subunit. Evidence for intracellular kinase-mediated reactions.
The epithelial sodium channel (ENaC) is a heteromultimer composed of three subunits, each having two membrane-spanning domains with intracellular amino and carboxyl termini. Several hormones and proteins regulate channel activity, but the molecular nature of this regulation is unknown. We conducted experiments to determine a possible new site within the carboxyl terminus of the alpha-subunit involved in enhanced channel activity through endogenous kinases. When an alpha-subunit that was truncated to remove a PY motif was expressed in Xenopus oocytes with wild type human beta- and gamma-ENaC subunits, channel activity was greatly enhanced. The removal of the entire intracellular carboxyl terminus of the alpha-subunit eliminated this enhanced basal activity. Using several point mutations, we localized this site to two amino acid residues (Pro(595)-Gly(596)) near the second membrane-spanning domain. The nonspecific kinase inhibitor staurosporine inhibits basal channel activity of wild type ENaC but was ineffective in inhibiting channels mutated at this site. The major effect of these mutations was not on channel kinetics but was largely, if not entirely, on the number of active channels on the cell surface. This region is potentially important in effecting kinase-mediated increases in ENaC activity.
Volk
K A
KA
Department of Internal Medicine, University of Iowa College of Medicine and the Veterans Affairs Medical Center, Iowa City, Iowa 52246, USA.
Snyder
P M
PM
Stokes
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014981:Xenopus
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D053503:Epithelial Sodium Channels; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D015222:Sodium Channels; D011494:Protein Kinases