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Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels
L Zimniak1, C J Winters, W B Reeves
1Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock, Arkansas, USA.
Abstract:
The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from 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 rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-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 approximately 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.
Insights
Rabbit outer medulla chloride channel Ka (rbClC-Ka) encodes a basolateral membrane chloride channel crucial for net medullary thick ascending limb chloride absorption. Antisense inhibition of rbClC-Ka significantly reduced chloride channel activity, confirming its role.
Area of Science:
- Nephrology
- Molecular Biology
- Ion Transport
Background:
- The thick ascending limb (TAL) plays a vital role in renal salt reabsorption.
- A specific basolateral chloride channel is hypothesized to mediate chloride absorption in the medullary TAL (MTAL).
- The rabbit CIC family chloride channel, rbClC-Ka, is a potential candidate for this basolateral channel.
Purpose of the Study:
- To investigate whether rbClC-Ka encodes the basolateral chloride channel responsible for net chloride absorption in the MTAL.
- To determine the functional role of rbClC-Ka in MTAL chloride transport.
Main Methods:
- Cultured mouse MTAL cells were treated with rbClC-Ka-specific antisense oligonucleotides.
- Chloride channel activity was assessed by incorporating membrane vesicles into lipid bilayers.
- Protein abundance was analyzed using Western blots with rbClC-Ka antiserum.
Main Results:
- Antisense treatment significantly reduced chloride channel incorporation into lipid bilayers (from 17% to near zero).
- Western blots showed approximately 50% reduction in rbClC-Ka protein levels after antisense treatment.
- rbClC-Ka antiserum blocked chloride channel activity in lipid bilayers.
Conclusions:
- The results strongly support rbClC-Ka as the basolateral chloride channel mediating net chloride absorption in the MTAL.
- rbClC-Ka is essential for chloride transport in this segment of the nephron.