Vesicle associated membrane protein (VAMP) 3 but not VAMP2 is involved in NKCC2 apical targeting and processing in thick ascending limbs (TALs)

Abstract

NKCC2 undergoes apical targeting to the cell surface in TALs in the absence of hormonal stimulation. VAMP2 and VAMP3 colocalize with NKCC2 in the apical membrane and subapical space of TALs and may differentially regulate protein processing, sorting and apical targeting of NKCC2. We hypothesized that both VAMP2 and VAMP3 regulate constitutive NKCC2 surface expression. We measured the effect of a) siRNAs against VAMP2 and VAMP3 and b) tetanus toxin; on surface and total NKCC2 expression by biotinylation of rat TAL suspensions and Western blot. VAMP2 siRNA decreased VAMP2 (85% decrease) without affecting VAMP3 and VAMP3 siRNA decreased VAMP3 (95% decrease) without affecting VAMP2 in control experiments. VAMP2 siRNA did not affect surface NKCC2 (81 ± 7 % of control) or total NKCC2 expression (93 ± 9 % of control). In contrast, VAMP3 siRNA decreased surface NKCC2 by 53 ± 13% (p<0.05) and total NKCC2 expression by 59 ± 8 % (p<0.01), suggesting that VAMP3 mediates both processing and apical targeting. We next used a low concentration of tetanus toxin, which cleaves and inactivates VAMP2 and VAMP3, to block apical targeting, but not processing or sorting. Fifty min treatment with 30 nM tetanus toxin decreased surface NKCC2 by 33 ± 5 % (p<0.05) without affecting total NKCC2 expression. Our data suggest that VAMP3 is involved in both NKCC2 processing and apical targeting in TALs whereas VAMP2 is not likely involved in these processes.NIH