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 duct cells, we have carried out immunoblotting, immunocytochemistry, and reverse transcription (RT)-PCR experiments in rat kidney. Immunochemical studies used an affinity-purified, peptide-directed polyclonal antibody to rat syntaxin-4. Immunoblots using membrane fractions from inner medullary collecting duct (IMCD) cell suspensions revealed a solitary protein of 36 kD, the expected molecular mass of syntaxin-4. This protein was enriched in a plasma membrane-enriched membrane fraction from IMCD cells. Immunoperoxidase immunocytochemistry in 0.85-microm cryosections from rat inner medulla revealed discrete labeling of the apical plasma membrane of IMCD cells. RT-PCR demonstrated the presence of syntaxin-4 mRNA in microdissected IMCD segments, confirmed by direct sequencing of the PCR product. In addition, RT-PCR experiments demonstrated 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 principal cells, coupled with previous demonstration of syntaxin-4's putative cognate receptor VAMP2 in aquaporin-2-containing vesicles, supports the view that these proteins could play a role of aquaporin-2 vesicle targeting to the apical plasma membrane.
Mandon
B
B
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1598, USA.
Chou
C L
CL
Nielsen
S
S
Knepper
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2; D051400:Aquaporin 3; D051403:Aquaporin 6; D020346:Aquaporins; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D007473:Ion Channels / Q000378:metabolism*; D007672:Kidney Cortex / Q000378:metabolism; D007679:Kidney Medulla / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D014018:Tissue Distribution
C496025:Aqp2 protein, rat; C496029:Aqp3 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D017931:DNA Primers; D007473:Ion Channels; D008565:Membrane Proteins; D010455:Peptides; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D012333:RNA, Messenger; D051400:Aquaporin 3