Uchida S, Sasaki S, Nitta K, Uchida K, Horita S, Nihei H, Marumo F
Second Department of Internal Medicine, Tokyo Medical and Dental University, School of Medicine, Japan.
J Clin Invest. 1995 Jan;95(1):104-13. doi: 10.1172/JCI117626.
To investigate the physiological role of a kidney-specific chloride channel (ClC-K1), we sought to determine its exact localization by immunohistochemistry and its functional regulation using Xenopus oocyte expression system. The antiserum specifically recognized a 70-kD protein in SDS-PAGE of membrane protein from rat inner medulla and an in vitro translated ClC-K1 protein. Immunohistochemistry revealed that ClC-K1 was exclusively localized to the thin limb of Henle's loop in rat inner medulla. In comparison with the immunostaining with anti-aquaporin-CHIP antibody that only stains the descending thin limb of Henle's loop (tDL), ClC-K1 was found to be localized only in the ascending limb (tAL) which has the highest chloride permeability among nephron segments. Immunoelectron microscopy confirmed that the staining of ClC-K1 in tAL was observed in the region of both apical and basolateral plasma membranes. Expressed chloride current in Xenopus oocytes by ClC-K1 cRNA was regulated by extracellular pH and extracellular calcium. Furosemide inhibited the expressed current (Ki = 100 microM), whereas N-ethyl-maleimide stimulated the current. These functional characteristics were consistent with the in vitro perfusion studies of chloride transport in tAL. The localization and the functional characteristics described here indicate that ClC-K1 is responsible for the transepithelial chloride transport in tAL.
为了研究肾脏特异性氯离子通道（ClC-K1）的生理作用，我们试图通过免疫组织化学确定其确切定位，并利用非洲爪蟾卵母细胞表达系统研究其功能调节。该抗血清在大鼠髓质内层膜蛋白的SDS-PAGE中特异性识别一种70-kD的蛋白质以及体外翻译的ClC-K1蛋白。免疫组织化学显示，ClC-K1仅定位于大鼠髓质内层的髓袢细段。与仅对髓袢降支细段（tDL）进行染色的抗水通道蛋白-CHIP抗体免疫染色相比，发现ClC-K1仅定位于髓袢升支细段（tAL），而髓袢升支细段是肾单位各节段中氯离子通透性最高的部位。免疫电子显微镜证实，在tAL中ClC-K1的染色见于顶端和基底外侧质膜区域。ClC-K1编码RNA（cRNA）在非洲爪蟾卵母细胞中表达的氯离子电流受细胞外pH值和细胞外钙的调节。呋塞米抑制所表达的电流（抑制常数Ki = 100 microM），而N-乙基马来酰亚胺则刺激该电流。这些功能特性与tAL中氯离子转运的体外灌注研究结果一致。此处所描述的定位和功能特性表明，ClC-K1负责tAL中的跨上皮氯离子转运。
