Regulatory phosphorylation of the secretory na-k-cl cotransporter:
modulation by cytoplasmic chloride.
Lytle, Christian, and Bliss Forbush Iii.
Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672,
Department of Cellular and Molecular Physiology, Yale University
School of Medicine, 333 Cedar St., New Haven, CT 06510, Division of
Biomedical Sciences, University of California, Riverside, CA,
92521
APStracts 2:0286C, 1995.
The effect of cytoplasmic chloride ([Cl-]i) on the activation state
([3H]benzmetanide binding rate) and phosphorylation state (32P
incorporation) of the Na-K-Cl cotransporter was evaluated in
secretory tubules isolated from the dogfish shark rectal gland.
Reduction of [Cl-]i at relatively constant cell volume (by removal of
extracellular Cl- or Na+, or by addition of bumetanide) increased
cotransporter activation and phosphorylation. Raising [K+]o from 4 mM
to 80 mM, a maneuver which elevated [Cl-]i above normal, reduced
basal cotransport activity and rendered it entirely refractory to
forskolin. High [K+]o also blocked activation and phosphorylation in
response to cell shrinkage, despite the fact that [Cl]i was already
greatly elevated as a consequence of osmotic water loss. The
phosphatase inhibitor calyculin-A also promoted activation, but not
in cells pre-exposed briefly to high [K+]o. In summary, maneuvers
which lower [Cl-]i activate the cotransporter, while those which
elevate [Cl-]i (or prevent it from decreasing) block activation in
response to secretory stimuli. Cell chloride appears to govern its
own rate of entry via Na-K-Cl cotransport by impeding regulatory
phosphorylation of the Na-K-Cl cotransport protein.
Received 5 May 1995; accepted in final form 14 July 1995.
APS Manuscript Number C246-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 10 August 1995.