| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, October 5, 1994; and in revised form, December 1, 1994) By combining the tools of site-directed mutagenesis and
[
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 2993-3000
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Isoform of
Na
,K-ATPase Stabilizes a
K-induced Conformational Change
H]ouabain binding, the functional role of
glutamic acid 327 in the fourth transmembrane domain of the sheep
1 isoform of Na
,K-ATPase was
examined with respect to its interactions with ouabain,
Na, K, Mg
, and
inorganic phosphate. Using site-directed mutagenesis, this glutamic
acid was substituted with alanine, aspartic acid, glutamine, and
leucine. The mutant proteins were constructed in a sheep 1 protein
background such that [H]ouabain binding could be
utilized as a highly specific probe of the exogenous protein expressed
in NIH 3T3 cells. Na competition of
[H]ouabain binding to the mutant forms of
Na,K-ATPase revealed only slight
alterations in their affinities for Na and in their
abilities to undergo Na-induced conformational changes
which inhibit ouabain binding. In contrast, K competition of [H]ouabain binding to all
four mutant forms of Na,K-ATPase
displayed severely altered interactions between these proteins and
K. Interestingly, [H]ouabain
binding to the mutant E327Q was not inhibited by the presence of
K. This mutant was previously reported to be
functionally able to support cation transport with a 5-fold reduced K
for K-dependent ATPase
activity (Jewell-Motz, E. A., and Lingrel, J. B.(1993) Biochemistry 32, 13523-13530; Vilsen, B.(1993) Biochemistry 32,
13340-13349). Thus, it appears that this glutamic acid in the
fourth transmembrane domain may be important for stabilizing a
K-induced conformation within the catalytic cycle of
Na,K-ATPase that is not rate-limiting
in the overall ATPase cycle but that displays a greatly reduced
affinity for ouabain.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. M. Paul, M. J. Palladino, and G. J. Beitel A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control Development, January 1, 2007; 134(1): 147 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Imagawa, T. Yamamoto, S. Kaya, K. Sakaguchi, and K. Taniguchi Thr-774 (Transmembrane Segment M5), Val-920 (M8), and Glu-954 (M9) Are Involved in Na+ Transport, and Gln-923 (M8) Is Essential for Na,K-ATPase Activity J. Biol. Chem., May 13, 2005; 280(19): 18736 - 18744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mense, L. A. Dunbar, R. Blostein, and M. J. Caplan Residues of the Fourth Transmembrane Segments of the Na,K-ATPase and the Gastric H,K-ATPase Contribute to Cation Selectivity J. Biol. Chem., January 21, 2000; 275(3): 1749 - 1756. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tepperman, L. A. Millette, C. L. Johnson, E. A. Jewell-Motz, J. B. Lingrel, and E. T. Wallick Mutational analysis of Glu-327 of Na+-K+-ATPase reveals stimulation of 86Rb+ uptake by external K+ Am J Physiol Cell Physiol, December 1, 1997; 273(6): C2065 - C2079. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Kuntzweiler, J. M. Arguello, and J. B Lingrel Asp804 and Asp808 in the Transmembrane Domain of the Na,K-ATPase alpha Subunit Are Cation Coordinating Residues J. Biol. Chem., November 22, 1996; 271(47): 29682 - 29687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Arguello, R. D. Peluffo, J. Feng, J. B Lingrel, and J. R. Berlin Substitution of Glutamic 779with Alanine in the Na,K-ATPase alpha Subunit Removes Voltage Dependence of Ion Transport J. Biol. Chem., October 4, 1996; 271(40): 24610 - 24616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ambesi, R. L. Pan, and C. W. Slayman Alanine-scanning Mutagenesis along Membrane Segment 4of the Yeast Plasma Membrane H+-ATPase. EFFECTS ON STRUCTURE AND FUNCTION J. Biol. Chem., September 20, 1996; 271(38): 22999 - 23005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Pedersen, J. H. Rasmussen, and P. L. Jørgensen Expression in High Yield of Pig alpha1beta1 Na,K-ATPase and Inactive Mutants D369N and D807N in Saccharomyces cerevisiae J. Biol. Chem., February 2, 1996; 271(5): 2514 - 2522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Asano, Y. Tega, K. Konishi, M. Fujioka, and N. Takeguchi Functional Expression of Gastric H[IMAGE],K[IMAGE]-ATPase and Site-directed Mutagenesis of the Putative Cation Binding Site and the Catalytic Center J. Biol. Chem., February 2, 1996; 271(5): 2740 - 2745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Argüello and J. B Lingrel Substitutions of Serine 775 in the alpha Subunit of the Na,K-ATPase Selectively Disrupt K[IMAGE] High Affinity Activation without Affecting Na[IMAGE] Interaction J. Biol. Chem., September 29, 1995; 270(39): 22764 - 22771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Kuntzweiler, E. T. Wallick, C. L. Johnson, and J. B Lingrel J. Biol. Chem., July 7, 1995; 270(27): 16206 - 16212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. V. Petrov, K. P. Padmanabha, R. K. Nakamoto, K. E. Allen, and C. W. Slayman Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase J. Biol. Chem., May 19, 2000; 275(21): 15709 - 15716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ambesi, M. Miranda, K. E. Allen, and C. W. Slayman Stalk Segment 4 of the Yeast Plasma Membrane H+-ATPase. MUTATIONAL EVIDENCE FOR A ROLE IN THE E1-E2 CONFORMATIONAL CHANGE J. Biol. Chem., June 30, 2000; 275(27): 20545 - 20550. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
