Vesicle-Associated membrane protein 2 (VAMP2) but not VAMP3 mediates cAMP-stimulated trafficking of the renal Na+-K+-2Cl - Co-transporter NKCC2 in thick ascending limbs

Abstract

In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl- co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinaseA(PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied.Wefound that severalmembers of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report thatNKCC2co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface.cAMPstimulation enhanced VAMP2exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co- immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated thatcAMPstimulation enhancesVAMP2exocytosis and promotes VAMP2 interaction with NKCC2. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.