Cl- channels in basolateral renal medullary vesicles xi. rbclc-ka
cdna encodes basolateral mtal cl-channels.
Zimniak, Ludwika, Christopher J. Winters, W. Brian Reeves, Thomas E.
Andreoli.
Division of Nephrology, Department of Internal Medicine, University
of Arkansas College of Medicine and John L. McClellan Veterans
Administration Hospital, Little Rock, Arkansas
APStracts 3:0033F, 1996.
The present experiments examined whether rbClC-Ka, a ClC family Cl-
channel cDNA from rabbit outer medulla, encodes a basolateral
membrane Cl- channel mediating net mTAL Cl- absorption. mTAL cells
contain a Cl- channel having certain properties which make it a
plausible candidate for the basolateral Cl- channel in that segment.
Especially pertinent among these 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 C-terminus of the
rbClC-Ka protein. Using 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
about 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.
Received 18 December 1995; accepted in final form 9 February
1996.
APS Manuscript Number F418-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 24 February 96