LOCALIZATION AND FUNCTIONAL-CHARACTERIZATION OF RAT KIDNEY-SPECIFIC CHLORIDE CHANNEL, CIC-K1

To investigate the physiological role of a kidney-specific chloride ch annel (ClC-K1), we sought to determine its exact localization by immun ohistochemistry and its functional regulation using Xenopus oocyte exp ression 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 inne r medulla. In comparison with the immunostaining with anti-aquaporin-C HIP 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 (t AL) which has the highest chloride permeability among nephron segments , Immunoelectron microscopy confirmed that the staining of ClC-K1 in t AL was observed in the region of both apical and basolateral plasma me mbranes. Expressed chloride current in Xenopus oocytes by ClC-K1 cRNA was regulated by extracellular pH and extracellular calcium, Furosemid e inhibited the expressed current (K-i = 100 mu M), whereas N-ethyl-ma leimide stimulated the current, These functional characteristics were consistent with the in vitro perfusion studies of chloride transport i n tAL. The localization and the functional characteristics described h ere indicate that ClC-K1 is responsible for the transepithelial chlori de transport in tAL.