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 similar to 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. 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 [S-35]methionine autoradiography gave major bands at 29 and 32 kDa for the short clone. in situ hybridization of rat kidney using a S-35-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.