Zimniak L, Winters C J, Reeves W B, Andreoli T E
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock, USA.
Kidney Int. 1995 Dec;48(6):1828-36. doi: 10.1038/ki.1995.481.
These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.
这些实验旨在利用基于同源性的克隆策略，鉴定可能编码髓袢升支粗段基底外侧膜氯离子通道的候选cDNA。我们使用来自兔外髓的1.8至3.2 kb mRNA片段制备了一个cDNA文库，该片段可在非洲爪蟾卵母细胞的细胞膜中诱导氯离子电导。用两个与其他氯离子通道中高度保守序列相对应的32P - 寡核苷酸探针筛选该cDNA文库。我们分离出了两个cDNA：rbClC - Ka和rbClC - Kb。从这两个cDNA推导的蛋白质序列具有99%的同源性。利用逆转录聚合酶链反应（RT - PCR）技术，发现培养的小鼠髓袢升支粗段细胞含有与这两个cDNA相对应的mRNA。与这两个cDNA相对应的mRNA的表达具有肾脏特异性，且在兔肾髓质中比兔肾皮质中更高。最后，通过将RT - PCR技术与显微切割的肾小球或肾小管节段相结合，我们在肾小球、近端曲管、髓袢升支粗段和皮质集合管中发现了rbClC - Ka的mRNA。
