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 language | English (US) |
|---|---|
| Journal | American Journal of Physiology |
| Volume | 270 |
| Issue number | 6 PART 2 |
| State | Published - Dec 1 1996 |
| Externally published | Yes |
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Keywords
- Channel cloning
- Chloride ion absorption
- Thick limb
ASJC Scopus subject areas
- Physiology (medical)
Cite this
Cl- channels in basolateral renal medullary vesicles XL rbClC-Ka cDNA encodes basolateral MTAL Cl- channels. / Zimniak, Ludwika; Winters, Christopher J.; Reeves, William B; Andreoli, Thomas E.
In: American Journal of Physiology, Vol. 270, No. 6 PART 2, 01.12.1996.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Cl- channels in basolateral renal medullary vesicles XL rbClC-Ka cDNA encodes basolateral MTAL Cl- channels
AU - Zimniak, Ludwika
AU - Winters, Christopher J.
AU - Reeves, William B
AU - Andreoli, Thomas E.
PY - 1996/12/1
Y1 - 1996/12/1
N2 - 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.
AB - 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.
KW - Channel cloning
KW - Chloride ion absorption
KW - Thick limb
UR - http://www.scopus.com/inward/record.url?scp=0029820101&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029820101&partnerID=8YFLogxK
M3 - Article
C2 - 8764326
AN - SCOPUS:0029820101
VL - 270
JO - The American journal of physiology
JF - The American journal of physiology
SN - 0002-9513
IS - 6 PART 2
ER -