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 amin o and carboxyl termini. Several hormones and proteins regulate channel acti vity, but the molecular nature of this regulation is unknown. We conducted experiments to determine a possible new site within the carboxyl terminus o f the a-subunit involved in enhanced channel activity through endogenous ki nases. When an alpha -subunit that was truncated to remove a PY motif was e xpressed in Xenopus oocytes with wild type human beta- and gamma -ENaC subu nits, channel activity was greatly enhanced. The removal of the entire intr acellular carboxyl terminus of the alpha -subunit eliminated this enhanced basal activity. Using several point mutations, we localized this site to tw o amino acid residues (Pro(595)-Gly(596)) near the second membrane-spanning domain. The nonspecific kinase inhibitor staurosporine inhibits basal chan nel activity of wild type ENaC but was ineffective in inhibiting channels m utated 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 channel s on the cell surface. This region is potentially important in effecting ki nase-mediated increases in ENaC activity.