Takeuchi Y, Uchida S, Marumo F, Sasaki S
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Kidney Int. 1995 Nov;48(5):1497-503. doi: 10.1038/ki.1995.439.
Two kidney-specific chloride channels, ClC-K1 and ClC-K2, have been isolated from rat kidney. In the present study, we sought to isolate human homologue of rat ClC-K2 chloride channel that was present in the thick ascending limb of Henle's loop and collecting ducts. Human kidney cDNA library was screened with the whole rat ClC-K2 cDNA probe. Two highly homologous but not identical cDNAs were isolated and sequenced. Northern analysis showed that both clones were expressed only in kidney among various human tissues, demonstrating that kidney-specific ClC family members were also present in human kidney. Because both clones had almost the same nucleotide identity (approximately 80%) with rat ClC-K2, we could not determine by sequence alone which human clone corresponded to rat ClC-K2. Accordingly, we performed reverse transcription PCR using dissected human nephron segments and identified the site of expression of each clone in human nephron segments. One clone was only expressed in the thin limb of Henle's loop and the other was expressed in glomeruli, proximal tubules, and collecting ducts. We identified the latter clone as human ClC-K2 based on the localization of rat ClC-K1 and ClC-K2. Identification of human ClC-K2 clone will be of help in understanding the genetic involvement of chloride channel in disorders of chloride transport such as Bartter's syndrome.
两种肾脏特异性氯离子通道，即ClC-K1和ClC-K2，已从大鼠肾脏中分离出来。在本研究中，我们试图分离大鼠ClC-K2氯离子通道的人类同源物，该通道存在于亨氏袢升支粗段和集合管中。用完整的大鼠ClC-K2 cDNA探针筛选人肾脏cDNA文库。分离并测序了两个高度同源但不相同的cDNA。Northern分析表明，在各种人类组织中，这两个克隆仅在肾脏中表达，表明肾脏特异性ClC家族成员也存在于人类肾脏中。由于这两个克隆与大鼠ClC-K2的核苷酸同一性几乎相同（约80%），仅通过序列我们无法确定哪个人类克隆与大鼠ClC-K2相对应。因此，我们使用解剖的人类肾单位节段进行逆转录PCR，并确定每个克隆在人类肾单位节段中的表达位点。一个克隆仅在亨氏袢细段中表达，另一个克隆在肾小球、近端小管和集合管中表达。基于大鼠ClC-K1和ClC-K2的定位，我们将后一个克隆鉴定为人类ClC-K2。鉴定人类ClC-K2克隆将有助于理解氯离子通道在诸如巴特综合征等氯离子转运障碍中的遗传作用。
