Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel

T. Ma, H. Hasegawa, William Skach, A. Frigeri, A. S. Verkman

Research output: Contribution to journalArticle

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Abstract

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 ~1.8-kilobase clone from a rat kidney λgt10 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. Water permeability in Xenopus oocytes injected with 50 ng of transcribed cRNA was (in cm/s x 10-3) 20 ± 3 (short clone), 1.3 ± 0.2 (long clone), 11 ± 3 (short clone with no globin sequence), 0.7 ± 0.1 (water-injected control), and 20 ± 4 (CHIP28k); the increased water permeability in oocytes expressing the short clone was inhibited by 75% by 0.3 mM HgCl2 but not affected by adenosine 3',5'-cyclic monophosphate agonists. Cell-free translation of the short clone gave a band at 29 kDa that became glycosylated (32 kDa) in the presence of pancreatic microsomes; translation of the long clone was much less efficient. Translation in oocytes followed by anti-c-myc immunoprecipitation and [35S]methionine autoradiography gave major bands at 29 and 32 kDa for the short clone. In situ hybridization of rat kidney using a 35S-labeled 187-base cRNA antisense probe (base pairs +343 to +529) showed localization of mRNA encoding WCH-CD only to medullary and cortical collecting ducts. These studies indicate that WCH-CD is a collecting duct water channel and provide translation and expression data indicating that the second ATG codon is the major translation initiation site.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume266
Issue number1 35-1
StatePublished - 1994
Externally publishedYes

Fingerprint

Collecting Kidney Tubules
Aquaporin 2
Functional analysis
Aquaporins
In Situ Hybridization
Rats
Base Pairing
Globins
Clone Cells
Cloning
Ducts
Water
Complementary DNA
Oocytes
Complementary RNA
Mercuric Chloride
Messenger RNA
Polymerase chain reaction
Dehydration
Gene Library

Keywords

  • protein translation
  • vasopressin
  • water channel-collecting duct channel
  • Xenopus oocyte

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

Cite this

Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel. / Ma, T.; Hasegawa, H.; Skach, William; Frigeri, A.; Verkman, A. S.

In: American Journal of Physiology - Cell Physiology, Vol. 266, No. 1 35-1, 1994.

Research output: Contribution to journalArticle

Ma, T. ; Hasegawa, H. ; Skach, William ; Frigeri, A. ; Verkman, A. S. / Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel. In: American Journal of Physiology - Cell Physiology. 1994 ; Vol. 266, No. 1 35-1.
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KW - protein translation

KW - vasopressin

KW - water channel-collecting duct channel

KW - Xenopus oocyte

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