SYNTAXIN-4 IS LOCALIZED TO THE APICAL PLASMA-MEMBRANE OF RAT RENAL COLLECTING DUCT CELLS - POSSIBLE ROLE IN AQUAPORIN-2 TRAFFICKING

To evaluate the possible role of a putative vesicle-targeting protein, syntaxin-4, in vasopressin-regulated trafficking of aquaporin-2 water channel vesicles to the apical plasma membrane of renal collecting du ct cells, we have carried out immunoblotting, immunocytochemistry, and reverse transcription (RT)-PCR experiments in rat kidney. Immunochemi cal studies used an affinity-purified, peptide-directed polyclonal ant ibody to rat syntaxin-4. Immunoblots using membrane fractions from inn er medullary collecting duct (IMCD) cell suspensions revealed a solita ry protein of 36 kD, the expected molecular mass of syntaxin-4. This p rotein was enriched in a plasma membrane-enriched membrane fraction fr om IMCD cells, Immunoperoxidase immunocytochemistry in 0.85-mu m cryos ections from rat inner medulla revealed discrete labeling of the apica l plasma membrane of IMCD cells. RT-PCR demonstrated the presence of s yntaxin-4 mRNA in microdissected IMCD segments, confirmed by direct se quencing of the PCR product. In addition, RT-PCR experiments demonstra ted syntaxin-4 mRNA in glomeruli, vasa recta, connecting tubules, and thin descending limbs of Henle's loops. The demonstrated localization of syntaxin-4 in the apical plasma membrane of collecting duct princip al cells, coupled with previous demonstration of syntaxin-4's putative cognate receptor VAMP2 in aquaporin-2-containing vesicles, supports t he view that these proteins could play a role of aquaporin-2 vesicle t argeting to the apical plasma membrane.