2 ISOFORMS OF A CHLORIDE CHANNEL PREDOMINANTLY EXPRESSED IN THICK ASCENDING LIMB OF HENLE LOOP AND COLLECTING DUCTS OF RAT-KIDNEY

Complementary DNAs encoding rat kidney chloride channels (ClC-K2L and ClC-K2S) were isolated by a polymerase chain reaction cloning strategy . Degenerate primers were designed based on the significant amino acid identity of the previously cloned chloride channels (ClC-0, -1, -2, a nd -K1). The 687-amino acid protein encoded by ClC-K2L is about 80% id entical to rat ClC-K1 and about 40% identical to ClC-0, -1, and -2. Cl C-K2S encodes a 632-amino acid protein in which 55 amino acids contain ing the putative second membrane-spanning domain of ClC-K2L are delete d. Chloride currents induced by both clones were very similar in terms of inhibitor sensitivity and anion selectivity (Br- > I- > Cl- >> cyc lamate(-)). Northern blot with total ClC-K2L as a probe under high str ingency revealed its message predominantly in kidney, especially in th e outer and inner medulla. Reverse transcription polymerase chain reac tion technique using microdissected nephron segments revealed that the main site of expression of both clones in kidney was the thick ascend ing limb of Henle's loop and collecting ducts, where the existence of a variety of chloride channels and their importance for maintaining bo dy fluid homeostasis have been demonstrated. These results suggest tha t ClC-K2L and -K2S are chloride channels in the thick ascending limb a nd collecting ducts and may be important routes for transcellular chlo ride transport like ClC-K1.