CL- CHANNELS IN BASOLATERAL RENAL MEDULLARY VESICLES .6. RBCLC-K-ALPHA CDNA ENCODES BASOLATERAL MTAL CL- CHANNELS

The present experiments examined whether rbClC-Ka, a CIC family Cl- ch annel cDNA from rabbit outer medulla, encodes a basolateral membrane C l- channel mediating net medullary thick ascending limb (MTAL) Cl- abs orption. MTAL cells contain a Cl- channel having certain properties th at make it a plausible candidate for the basolateral Cl- channel in th at segment. Especially pertinent among properties is the fact that cyt osolic 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 wit h no complementarity to rbClC-Ka. The abundance of Cl- channels was as sessed 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 treat ed with the random oligonucleotide, Cl- channels were incorporated int o bilayers in 17% and 16% of trials, respectively. However, when vesic les were prepared from cells pretreated with antisense oligonucleotide , there was a virtual abolition of Cl- channel incorporation into bila yers but no effect on the frequency of K+ channel incorporation. In pa rallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced similar to 50% on Western blots. Final ly, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiser um resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorpti on in the MTAL is encoded by rbClC-Ka.