Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels.
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.
Zimniak
L
L
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock, Arkansas, USA.
Winters
C J
CJ
Reeves
W B
WB
Andreoli
T E
TE
D000818:Animals; D015153:Blotting, Western; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics*; D007106:Immune Sera; D007679:Kidney Medulla / Q000378:metabolism*; D008051:Lipid Bilayers; D008138:Loop of Henle / Q000378:metabolism*; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits
D018118:Chloride Channels; D018076:DNA, Complementary; D007106:Immune Sera; D008051:Lipid Bilayers; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments