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Interaction of NKCC2 With SNARE Proteins in the Thick Ascending Limb
Author(s) -
Caceres Paulo Sebastian,
Ortiz Pablo Alfredo
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1152.4
Subject(s) - syntaxin , endocytic cycle , immunoprecipitation , microbiology and biotechnology , chemistry , snare complex , membrane protein , lipid bilayer fusion , biology , biochemistry , membrane , endocytosis , cell , gene
NaCl absorption by the apical Na/K/2 Cl cotransporter (NKCC2) in thick ascending limbs (TAL) is important for blood pressure regulation and urinary concentration. NKCC2 levels in the apical membrane are maintained via exocytic insertion and endocytic retrieval. Inserted NKCC2 may derive from a newly synthesized pool or from those retrieved and then recycled. These processes are called trafficking, and involve several fusion events. Vesicle fusion is mediated by SNARE proteins from the VAMP, SNAP and syntaxin families. NKCC2 may directly interact with these proteins during trafficking into the apical membrane. We showed that VAMP2 and 3 are involved in NKCC2 trafficking in TALs. We hypothesized that NKCC2 directly interacts with VAMP 2, 3, and other SNAREs in TALs. We first studied SNARE expression and localization. We found that TALs express VAMP 2 and 3, SNAP 23 and syntaxins 3 and 4. VAMP 2 and 3, syntaxin 3 and SNAP 23 were localized to the apical membrane and subapical space similar to NKCC2. Syntaxin 4 was distributed throughout the cell. Next we performed co‐immunoprecipitation studies study protein interactions. VAMP 2 and 3 co‐immunoprecipitated with NKCC2 but the other SNAREs did not. Antibody specificity was tested with peptides and brain lysates. We conclude that NKCC2 interacts with VAMP 2 and 3 in TALs. SNAP23 and syntaxin 3 may be involved in NKCC2 trafficking without directly binding NKCC2. Funding: NIH, AHA