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

Ludwika Zimniak, Christopher J. Winters, W. Brian Reeves, Thomas E. Andreoli

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)F1066-F1072
JournalAmerican Journal of Physiology
Volume270
Issue number6 PART 2
StatePublished - 1996
Externally publishedYes

Keywords

  • Channel cloning
  • Chloride ion absorption
  • Thick limb

ASJC Scopus subject areas

  • Physiology (medical)

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