EXPRESSION, FUNCTIONAL-ANALYSIS, AND IN-SITU HYBRIDIZATION OF A CLONED RAT-KIDNEY COLLECTING DUCT WATER CHANNEL
EXPRESSION, FUNCTIONAL-ANALYSIS, AND IN-SITU HYBRIDIZATION OF A CLONED RAT-KIDNEY COLLECTING DUCT WATER CHANNEL
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DOI：
10.1152/ajpcell.1994.266.1.c189
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发表时间：
1994-01-01
影响因子：
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通讯作者：
VERKMAN, AS
中科院分区：
文献类型：
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作者：
MA, TH;HASEGAWA, H;VERKMAN, AS
最近报道了与28 kda完整膜蛋白（CHIP28）同源的大鼠肾收集管（WCH-CD）的克隆和表达（K. Fushimi， S. Uchida， Y. Hara， Y. Hirata， F. Marumo，和S. Sasaki）。自然科学，1993年第1期：549-552。采用聚合酶链式反应克隆法，从大鼠肾lambda gt10 cDNA文库中获得了一个类似1.8千碱基的克隆；而编码序列（814个碱基对，预测蛋白质大小为29 kDa）与报道的相同，我们在-123碱基对上发现了一个帧内ATG密码子，预测蛋白质大小为33 kDa。用WCH-CD编码序列（碱基对+1 ~ +814）或5′-未翻译序列（碱基对-403 ~ -16）对应的cdna进行Northern blot检测，在肾髓质中观察到1.9千碱基的单条带多于皮质，而在其他组织中则没有；脱水后mRNA表达明显增加。通过翻译和卵母细胞表达研究来确定翻译起始位点。短cdna（碱基对为+1 ~ +814）和长cdna（碱基对为-123 ~ +814）在载体pSP64上进行亚克隆，该载体在ATGs上游含有5'-未翻译的非洲爪蟾珠蛋白序列；在COOH末端设计了一个30个碱基对的c-myc序列，用于抗体识别。经50 ng转录的cRNA注射后，爪蟾卵母细胞的透水性分别为（cm/s × 10(-3)） 20 +/- 3（短克隆）、1.3 +/- 0.2（长克隆）、11 +/- 3（无珠蛋白序列的短克隆）、0.7 +/- 0.1（注水对照）和20 +/- 4 (CHIP28k)；0.3 mM HgCl2对表达短克隆的卵母细胞增加的透水性有75%的抑制作用，而腺苷3′，5′-环单磷酸激动剂对其没有影响。在胰腺微粒体存在的情况下，短克隆的无细胞翻译在29 kDa处形成一条糖基化带（32 kDa）；长克隆的翻译效率要低得多。在卵母细胞中翻译，然后进行抗c-myc免疫沉淀和[S-35]蛋氨酸放射自显影，短克隆的主要条带在29和32 kDa。使用s -35标记的187碱基cRNA反义探针（碱基对+343至+529）原位杂交大鼠肾脏显示编码WCH-CD的mRNA仅定位于髓质和皮质集管。这些研究表明WCH-CD是一个收集管道水路，并提供了翻译和表达数据，表明第二个ATG密码子是主要的翻译起始位点。
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.