Cl^- channels in basolateral renal medullary vesicles XL rbClC-Ka cDNA encodes basolateral MTAL Cl^- channels

The present experiments examined whether rbClC-Ka, a ClC family Cl^- channel cDNAfrom rabbit outer medulla, encodes a basolateral membrane Cl^- channel mediating net medullary thick ascending limb (MTAL) Cl^- absorption. MTAL cells contain a Cl^- channel having certain properties that make it a plausible candidate for the basolateral Cl^- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl^- increases in the range 2-25 mM activated these Cl^- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbClC-Ka or a random oligonucleotide with no complementarity to rbClC-Ka. The abundance of Cl^- channels was assessed by the frequency of incorporation of Cl^- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-Ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl^- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl^- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl^- channel incorporation, the abundance of rbClC-Ka protein was reduced -50% on Western blots. Finally, exposure of Cl^- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl^- channel mediating net Cl^- absorption in the MTAL is encoded by rbClC-Ka.