Modulation of pump function by mutations in the first transmembrane
region of the na+,k+-atpase [alpha]1 subunit.
Yamamoto, S., G. R. Askew, J. Heiny, H. Masaki, and A. Yatani.
Departments of Pharmacology and Cell Biophysics and Molecular
Genetics, Biochemistry and Microbiology, Molecular and Cellular
Physiology, University of Cincinnati College of Medicine, Cincinnati,
Ohio 45267
APStracts 2:0313C, 1995.
The Cys in the first transmembrane region of the Na+,K+-ATPase a1
subunit has been shown to be a critical determinant of cardiac
glycoside binding. To study the role of this Cys on ion transport
activity, we measured pump currents in HeLa cells expressing wild
-type or mutant a1 cDNAs. The endogenous ouabain-sensitive Na+,K+
-ATPase was selectively inhibited by growing the cells in 0.1 [mu]M
ouabain. A Cys to Tyr substituted mutant exhibited decreased
sensitivity to digitoxin but not digoxin, compared to wild-type. The
decreased affinity for digitoxin was due to a faster dissociation
rate. In contrast, the Cys to Ala substitution did not significantly
alter the sensitivity to digitoxin or digoxin. Both wild-type and
mutant cells displayed marked external K+-dependent pump currents;
however, the affinity for K+ was reduced by the mutations. The
decrease in K+ affinity was due to a slower association rate. The
results show that the Cys that interacts with cardiac glycosides also
participates in the sensitivity of the pump to external K+.
Received 17 March 1995; accepted in final form 9 August 1995.
APS Manuscript Number C148-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 September 1995.