Rab22 is involved in regulation of the epithelial sodium channel (ENaC)

Abstract

We previously demonstrated a role for small GTPases in the regulation of the epithelial sodium channel (ENaC) in epithelial cells of the distal kidney nephron. To further map the trafficking itinerary of ENaC in mouse cortical collecting duct (mCCD) cells, we investigated a number of other GTPases for their ability to alter ENaC function. Overexpression of wt‐Rab22 in mCCD cells resulted in a 45 +/−5% increase in ENaC‐mediated sodium transport over control transfect cells. A Rab22 dominant‐negative mutant, S19N reduced ENaC activity, while a GTPase deficient mutation Q64L enhanced ENaC activity 72 +/− 7% over control levels. To determine the location of a Rab22‐positive compartment we carried out indirect immunofluorescent labeling of both Rab11 and Rab22 in mCCD cells. Both Rabs localized to puncta (vesicles) near the membrane, and ENaC could be localized to both compartments, however there was a clear and non‐overlapping localization for each Rab. Finally, we tested the impact of the Rab22 mutants on cAMP activation of ENaC. There was a significantly greater response to cAMP stimulation in cells overexpressing the Q64L mutation compared to control or wt‐Rab22 transfected cells. These data introduce a new protein regulator in the trafficking itinerary of ENaC at a location distinct from the previously reported GTPases. The role of Rab22 and mechanism of ENaC regulation is still under investigation.