Uchida S, Sasaki S, Furukawa T, Hiraoka M, Imai T, Hirata Y, Marumo F
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
J Biol Chem. 1993 Feb 25;268(6):3821-4.
Complementary DNA encoding a rat kidney chloride channel (CIC-K1) was isolated by a polymerase chain reaction (PCR) cloning strategy. We designed degenerate primers, based on the regions where previously cloned chloride channels (CIC-0, -1, and -2) possess significant amino acid identity, and performed reverse transcription PCR with whole kidney mRNA. The 686-amino acid protein encoded by CIC-K1 is about 40% identical to the previously cloned chloride channels and has a similar hydropathy profile. Expression of CIC-K1 in Xenopus oocytes induced Cl- currents that activate instantaneously upon hyperpolarization and depolarization, and displayed a slightly outwardly rectifying current-voltage relationship. The message for CIC-K1 was 2.4 kilobases and was found predominantly in kidney, especially in the inner medulla. Reverse transcription PCR technique using micro-dissected nephron segments revealed that the main site of expression in kidney was the thin ascending limb of Henle's loop, which has the highest Cl- permeability among the nephron segments and is thought to be involved in a counter-current system for urine concentration in the inner medulla. The abundance of CIC-K1 mRNA in kidney increased about 4-fold as rats became dehydrated by deprivation of water for 5 days. The site of expression and the regulation by dehydration suggest that CIC-K1 function may be important in urinary concentrating mechanisms.
通过聚合酶链反应（PCR）克隆策略分离出编码大鼠肾氯通道（CIC-K1）的互补DNA。我们根据先前克隆的氯通道（CIC-0、-1和-2）具有显著氨基酸同一性的区域设计了简并引物，并用全肾mRNA进行逆转录PCR。CIC-K1编码的686个氨基酸的蛋白质与先前克隆的氯通道约有40%的同一性，并且具有相似的亲水性图谱。CIC-K1在非洲爪蟾卵母细胞中的表达诱导了Cl-电流，该电流在超极化和去极化时瞬间激活，并显示出轻微外向整流的电流-电压关系。CIC-K1的信使RNA为2.4千碱基，主要在肾脏中发现，尤其是在内髓质中。使用显微切割的肾单位节段进行的逆转录PCR技术表明，肾脏中的主要表达部位是亨氏袢细升支，它在肾单位节段中具有最高的Cl-通透性，并且被认为参与了内髓质尿液浓缩的逆流系统。随着大鼠因缺水5天而脱水，肾脏中CIC-K1 mRNA的丰度增加了约4倍。表达部位和脱水调节表明CIC-K1功能在尿液浓缩机制中可能很重要。
