Selectivity of PKC isoforms in the regulation of Na+‐K+‐2Cl− cotransporter (NKCC1) in secretory epithelia

Abstract

NKCC1 drives basolateral Cl− entry in secretory epithelia. Activation of PKC by phorbol 12‐myristate 13‐acetate (PMA) reduces colonic epithelial Cl− secretory response to cAMP and surface NKCC1. The study was to define selectivity of PKCs in regulating epithelial NKCC1.T84 monolayers uninfected or infected with adenoviruses expressing human PKC (α, δ, ε)‐specific shRNAs or non‐target LacZ‐shRNA were treated with PMA or vehicle, followed by measurement of Cl− secretion (short‐circuit current, Isc). NKCC1 signal in the cells was assessed by immunofluorescence. NKCC1 surface expression was examined by basolateral surface biotinylation assay.We found proteins of PKC (α, δ, ε) in shPKCs cells were decreased by ~85% after 72h infection. PMA reduced forskolin (FSK)‐stimulated Cl− secretion much less in shPKCδ or shPKCε cells than shLacZ or uninfected cells. There was no significant difference in PMA‐reduced Cl− secretion between shPKCα and shLacZ. In shLacZ and shPKCα cells, PMA induced a redistribution of NKCC1 signal to intracellular vesicles; the effect was not observed in shPKCδ or shPKCε cells. PMA caused a dramatic decrease in basolateral biotinylated NKCC1 in shLacZ or shPKCα, but not in shPKCδ or shPKCε cells.In conclusion, depletion of PKC δ or ε, but not PKCα, prevents PMA inhibition of T84 Cl− secretion via a mechanism involving stabilization of the basolateral membrane NKCC1.Received NIH grant DK48010.