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.
S Uchida, S Sasaki, K Nitta, K Uchida, S Horita, H Nihei, F Marumo