AJP - Renal Physiology, Vol 273, Issue 6 1030-F1038, Copyright © 1997 by American Physiological Society
Cl- channels in basolateral renal medullary membranes. XII. Anti-rbClC-Ka antibody blocks MTAL Cl- channels
C. J. Winters, L. Zimniak, W. B. Reeves and T. E. Andreoli
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock, USA.
Cl- channels in the medullary thick ascending limb (MTAL) studied by either
patch-clamp technique or reconstitution into lipid bilayers are activated
by increases in intracellular Cl- concentrations. rbClC-Ka, a ClC Cl-
channel, may represent this channel. We therefore evaluated the role of
rbClC-Ka in transcellular MTAL Cl- transport in two separate ways. First,
an antibody was raised against a fusion protein containing a 153-amino acid
fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the
antibody was localized to basolateral regions of MTAL and cortical thick
ascending limb (CTAL) segments and also to the cytoplasm of intercalated
cells in the cortical collecting duct. Second, Cl- uptake and efflux were
measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide
sensitive and was stimulated by treatment with a combination of vasopressin
+ forskolin + dibutyryl adenosine 3',5-cyclic monophosphate (DBcAMP). Cl-
efflux was also increased significantly by vasopressin + forskolin + DBcAMP
from 114 +/- 20 to 196 +/- 36 nmol.mg protein-1.45 s-1 (P = 0.003). Cl-
efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate
(154 +/- 26 vs. 70 +/- 21 nmol.mg protein-1.45 s-1, P = 0.003). An
anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in
lipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 28
vs. 53 +/- 14 nmol.mg protein-1.45 s-1, P = 0.02). These results support
the view that rbClC-Ka is the basolateral membrane Cl- channel that
mediates vasopressin-stimulated net Cl- transport in the MTAL segment.
