Na+-pump a subunit containing amino acids v115-i126 of gastric h,
k-atpase is inhibited by ouabain and sch28080.
Lyu(, Rong-Ming, and Robert A. Farley( (.
(Department of Physiology and Biophysics, and ( Department of
Biochemistry and Molecular Biology, University of Southern California
School of Medicine, Los Angeles, CA 90033
APStracts 3:0395C, 1996.
Na,K-ATPase is inhibited by cardiac glycosides, and is insensitive to
SCH28080, an inhibitor of gastric H,K-ATPase. Gastric H,K-ATPase is
not inhibited by cardiac glycosides. Both ouabain and SCH28080
binding are inhibited by potassium, and it has been suggested that
the inhibitors bind to corresponding regions on the a subunit of each
ion pump. In order to identify regions of each pump that interact
with the specific inhibitors, chimeric a subunits consisting of
selected regions from Na,K-ATPase and gastric H,K-ATPase have been
prepared. One chimera (gM( ) has been constructed from cDNA of the
sheep a1 subunit of Na,K-ATPase by replacing the last 12 amino acids
of the first predicted transmembrane region (I99-I110) with
corresponding amino acids from rat gastric H,K-ATPase. gM( was
expressed in yeast cells together with either the rat Na,K-ATPase b1
subunit (NKb1) or rat gastric H,K-ATPase b subunit (HKb). Western
blots show that the expression level of the chimeric a subunit was
comparable to the Na,K-ATPase a1. Ouabain binds with high affinity to
gM(+NKb1 (Kd = 9.5 nM), but not to gM(+HKb. The Kd for ouabain
binding to Na,K-ATPase was 7.8 nM. Na,K-ATPase activity of gM(+NKb1
was inhibited both by ouabain and by SCH28080. The IC50 for SCH28080
inhibition was 20-60 nM. 10 mM SCH28080 did not inhibit Mg2+- and Pi
-dependent ouabain binding to gM(+NKb1. 0.75 mM Ouabain inhibited
palytoxin-induced K+ efflux from yeast cells expressing either
gM(+NKb1 or gM(+HKb, and SCH28080 increased the palytoxin-induced K+
efflux from yeast cells expressing gM(+NKb1 or gM(+HKb. These results
implicate a small number of amino acids in the first transmembrane
part of gastric H,K-ATPase as partial determinants of the sensitivity
to SCH28080. The data also suggest that ouabain and SCH28080 do not
bind to the same site on the chimera.
Received 4 September 1996; accepted in final form 4 December
1996.
APS Manuscript Number C514-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996