CFTR and V‐ATPase trafficking and function in the intestinal brush border membrane

Abstract

The multi‐subunit proton pump, vacuolar ATPase (V‐ATPase) and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are both regulated by cAMP‐dependent traffic from endosomes to the plasma membrane in ion transporting epithelia. We hypothesized that V‐ATPase traffic and function are linked to CFTR in the intestine. The distribution of V‐ATPase Voa1, Voa2 and V1E subunits and CFTR were examined in wild‐type (WT), CFTR−/− mouse intestine and polarized intestinal CaCo‐2 BBe cells following cAMP stimulation. Functional dependence of V‐ATPase on CFTR was determined in CaCo‐2 BBe cells using the pH sensitive dye BCECF. Co‐immunoprecipitation confirmed Voa1, Voa2 and V1E interaction with CFTR. V‐ATPase Voa1 and Voa2 subunits were localized to the apical and basolateral domains in CaCo‐2 BBe cells. V‐ATPase Voa1 and Voa2 subunits were localized to distinct domains in untreated and treated enterocytes from WT and CFTR−/−intestine. Following cAMP activation, both transporters co‐localized in the apical membrane. Inhibition of V‐ATPase or CFTR significantly decreased proton efflux in CaCo2 BBe cells. These studies suggest functional synergy between both transporters in maintaining intracellular pH; and localization in the intestinal brush border membrane. NIH Grants: R01‐DK‐077065 to N. Ameen and P30‐DK‐34989 to the Yale Liver Center.