Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel.
The cloning and expression of an apical membrane water channel from rat kidney collecting duct (WCH-CD) homologous to a 28-kDa integral membrane protein (CHIP28) was reported recently (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). We obtained an approximately 1.8-kilobase clone from a rat kidney lambda gt10 cDNA library by a polymerase chain reaction cloning method; whereas the coding sequence (814 base pairs, predicted protein size 29 kDa) was identical to that reported, we identified an in-frame ATG codon at base pair -123 predicting a protein size of 33 kDa. On Northern blots probed by cDNAs corresponding to the WCH-CD coding sequence (base pairs +1 to +814) or 5'-untranslated sequence (-403 to -16), a single band at 1.9 kilobases was observed in kidney medulla greater than in cortex but not in other tissues; mRNA expression was increased strongly by dehydration. Translation and oocyte expression studies were performed to identify the translation start site. The short (base pairs +1 to +814) and long (base pairs -123 to +814) cDNAs were subcloned in vector pSP64 containing the 5'-untranslated Xenopus globin sequence upstream to the ATGs; a 30-base pair c-myc sequence was engineered at the COOH- terminal for antibody recognition.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Medicine, University of California, San Francisco 94143.
Hasegawa
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Skach
W R
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Frigeri
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Verkman
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AS
D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001483:Base Sequence; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D017403:In Situ Hybridization; D007473:Ion Channels / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D017385:Sequence Homology; D014867:Water / Q000378:metabolism*; D014981:Xenopus
C496022:Aqp1 protein, rat; D020346:Aquaporins; D018076:DNA, Complementary; D007473:Ion Channels; D008565:Membrane Proteins; D015335:Molecular Probes; D014867:Water; D051398:Aquaporin 1