EXPRESSION OF VAMP2-LIKE PROTEIN IN KIDNEY COLLECTING DUCT INTRACELLULAR VESICLES - COLOCALIZATION WITH AQUAPORIN-2 WATER CHANNELS

Body water balance is controlled by vasopressin, which regulates Aquap orin-2 (AQP2) water channels in kidney collecting duct cells by vesicu lar trafficking between intracellular vesicles and the plasma membrane , To examine the molecular apparatus involved in vesicle trafficking a nd vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesic le-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed i n kidney collecting duct, Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla, Controls using preadsorbed antibody or preimmune serum were negative, Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicl es, VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue, Similarly, tetanus toxin cle aved VAMP2 in synaptic vesicles, In kidney inner medulla, VAMP2 was pr edominantly expressed in the membrane fraction enriched for intracellu lar vesicles, with little or no VAMP2 in the plasma membrane enriched fraction, This was confirmed by immunocytochemistry using semithin cry osections, which showed mainly vesicular labeling in collecting duct p rincipal cells, with no labeling of intercalated cells, VAMP2 immunola beling colocalized with AQP2 labeling in intracellular vesicles, as de termined by immunoelectron microscopy after double immunolabeling of i solated vesicles. Quantitative analysis of 1,310 vesicles revealed a h ighly significant association of both AQP2 and VAMP2 in the same vesic les (P < 0.0001), Furthermore, the presence of AQP2 in vesicles immuno isolated with anti-VAMP2 antibodies was confirmed by im munoblotting, In conclusion, VAMP2, a component of the neuronal SNARE; complex, is e xpressed in vesicles carrying AQP2, suggesting a role in vasopressin-r egulated vesicle trafficking of AQP2 water channels.