Distribution and diversity of na-k-cl cotransport proteins: a study
with monoclonal antibodies.
Lytle, Christian, Jian-Chao Xu, Daniel Biemesderfer, and Bliss Forbush
Iii.
Departments of Cellular and Molecular Physiology and Internal
Medicine, Yale University School of Medicine, 333 Cedar St., New
Haven, CT 06510 and Division of Biomedical Sciences, University of
California, Riverside, Riverside, CA 92521
APStracts 2:0345C, 1995.
The Na-K-Cl cotransporter (NKCC) is present in most animal cells where
it functions in cell volume homeostasis and epithelial salt
transport. We developed six monoclonal antibodies (designated T4, T8,
T9, T10, T12, and T14) against a fusion protein fragment encompassing
the carboxy-terminal 310 amino acids of the human colonic NKCC. These
T-antibodies selectively recognized putative NKCC proteins in a
diverse variety of animal tissues. Western blot analysis of membranes
isolated from 23 types of cells identified single bands of
immunoreactive protein ranging in mass from 146 kDa to 205 kDa. The
amount of immunoreactive protein detected in these cells correlated
with loop diuretic binding site density. Proteins identified
previously as Na-K-Cl cotransporters by loop diuretic photoaffinity
labeling were mutually recognized by multiple T-antibodies. Most of
the T-antibodies effectively immunoprecipitated the denatured form of
the NKCC protein. Immunocytochemical studies on the rabbit parotid
gland demonstrated that NKCC is restricted to the basolateral margin
of the acinar cells and absent from the ducts, in accord with the
central role of Na-K-Cl cotransport in chloride secretion. In the
rabbit kidney, NKCC was localized to the apical membrane of thick
ascending limb cells, consistent with its role in chloride
reabsorption.
Received 1 September 1995; accepted in final form 26 September
1995
APS Manuscript Number C536-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 31 October 95