Regulation of renal atp-sensitive k+ channel by membrane-bound protein phosphatases in rat principal tubule cell. Kubokawa, Manabu, Carmel M. McNicholas, Maria A. Higgins,wenhui Wang, and Gerhard Giebisch*. Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, Cellular Physiology Research Unit, Physiology Department, University College, Cork, Ireland , Department of Pharmacology, New York Medical College, Valhalla, New York 10595
APStracts 2:0061F, 1995.
The role of membrane-bound protein serine/threonine phosphatases (PP) in modulating the renal ATP-sensitive K+ (KATP) channel was examined using the patch-clamp technique in principal cells of rat cortical collecting duct. In the absence of ATP, channel activity rapidly (11.2 sec) declines (channel "run-down") upon excision of the membrane patches into control bath solutions (1 mM Mg2+, Ca2+-free). Both orthovanadate (5 mM), a broad spectrum inhibitor of phosphatases except for Ca2+-dependent PP (PP-2B), and okadaic acid (OA, 1 [mu]M), a potent inhibitor of PP types 1 and 2A (PP-1 and PP-2A), significantly slowed channel run-down. Removal of Mg2+ from the bath also slowed the run-down process. Incubation of cells with OA in the absence of Mg2+, or with orthovanadate in ATP-free solution, maintained channel activity at levels of 70% of control values for 3 min after membrane excision. In contrast, Ca2+ (0.1 mM) and calmodulin (1 [mu]M) in the presence of 1 mM Mg2+, a condition in which PP-2B is stimulated, had no significant effect on the channel activity that persisted in the presence of OA and orthovanadate. Application of exogenous PP-2A (1 u/ml) to the cytosolic side of membrane in inside-out patches significantly inhibited channel activity to 35.0% of control, but the inhibitory effects of PP-1 (1 u/ml) and PP-2B (20 [mu]g/ml) were minor. These results suggest that run-down of the renal KATP channel after membrane excision results mainly from dephosphorylation of the channel or an associated protein by membrane-bound phosphatases. Our data suggests that at least two types of phosphatases, OA-sensitive PP-2A and a Mg2+-dependent phosphatase, possibly PP-2C, are involved in this dephosphorylation process. 

Received 1 February 1995; accepted in final form 7 April 1995.
APS Manuscript Number F30-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 25 April 1995.