In vitro phosphorylation of COOH termini of the epithelial Na+ channel andits effects on channel activity in Xenopus oocytes

Recent findings have suggested the involvement of protein phosphorylation i n the regulation of the epithelial Na+ channel (ENaC). This study reports t he in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifi cally phosphorylated by kinase-enriched cytosolic fractions derived from ra t colon. The phosphorylation observed was not mediated by the serum- and gl ucocorticoid-regulated kinase sgk. For the gamma -subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immed iate vicinity of the PY motif (T630). The analogous residue on beta (S620) was phosphorylated as well. The possible role of gamma T630 and beta S620 i n channel function was studied in Xenopus laevis oocytes. Mutating these re sidues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na+ and had a high basal Na+ curr ent. Thus mutating gT630 or bS620 may limit the maximal channel activity ac hieved by a combination of sgk and low Na+.