NA-K-2CL COTRANSPORTER (NKCC) OF INTESTINAL EPITHELIAL-CELLS - SURFACE EXPRESSION IN RESPONSE TO CAMP

During intestinal chloride secretion, epithelial uptake of salts is ac complished largely by a bumetanide-sensitive Na:K:2Cl cotransporter de signated here as NKCC, Using monoclonal antibodies directed against NK CC from the human crypt epithelial cell line, T84, we define its surfa ce localization as a function of cotransporter activation, Immunoelect ron microscopy, confocal localization, and selective surface biotinyla tion studies revealed that the 195-kDa NKCC protein is polarized to th e basolateral domain. Following immunoprecipitation, several polypepti des coprecipitated with the 195-kDa cotransporter including two promin ent proteins of molecular mass 160 and 130 kDa, Immunoblotting with th ree distinct anti NKCC monoclonal antibodies in conjunction with degly cosylation experiments suggested that the 160- and 130-kDa bands repre sented novel proteins unrelated to the cotransporter. Stimulation of T 84 monolayers with cAMP agonists, a condition which elicits chloride s ecretion and leads to microfilament-dependent NKCC activation, did not significantly increase the number of bumetanide-binding sites and onl y marginally increased surface expression of the 195-kDa cotransporter available for surface biotinylation, In contrast, cAMP agonist stimul ation increased the surface expression of the coprecipitating 160- and 130-kDa proteins similar to 6-fold, The increase in surface 160- and 130-kDa proteins was attenuated by phalloidin preloading the cells, a condition which also prevents activation of NKCC without influencing t he activity of other membrane transporters participating in chloride s ecretion, These studies define the polarized distribution of the NKCC protein on intestinal epithelia, indicate that NKCC may be associated with two other previously unidentified membrane proteins and such asso ciation is influenced by the F-actin cytoskeleton.