Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase α-subunit

Svetlana Lutsenko, Sylvia Daoud, Jack H. Kaplan

Research output: Contribution to journalArticle

Abstract

Na,K-ATPase in right-side-out oriented vesicles was stabilized in different conformations, and the location of intramembrane Cys residues of the α-subunit was assessed with membrane-permeable and membrane-impermeable Cys-directed reagents. In the presence of Mg2+ and P(i), Cys964 was the most accessible for both membrane-impermeable 4-acetamido-4'- maleimidylstilbene-2,2'disulfonic acid (or stilbene disulfonate maleimide, SDSM) and membrane-permeable 7-diethylamino-3-(4'-maleimidyl)-4- methylcoumarin (CPM). In the presence of K+, Cys964 was modified only by hydrophobic CPM, indicating that the environment around Cys964 was different in these two conformations. Cys964 seems to mark the extracellular border of transmembrane segment M9. Cys911 in transmembrane segment M8 showed similar behavior; however, it was not so readily modified. Complete modification of Cys964 and Cys911 causes only partial (about 50%) inactivation of both ATPase activity and Rb+ (or K+) occlusion, indicating that the effect on cation occlusion is indirect and not within the occlusion cavity. The ATP binding capacity remains unaltered by the modifications. Treatment of the K+-stabilized post-tryptic preparation of purified Na,K-ATPase revealed labeling of several cysteines by CPM, none of which were labeled with SDSM. Removal of K+ ions from the preparation, which we have previously shown is accompanied by release of the M5M6 hairpin to the supernatant (1), causes changes in the organization of the C-terminal 21-kDa fragment. In particular Cys983 in M10 became labeled by both CPM and SDSM, pointing to a tight association between the C terminus and the M5M6 hairpin of the α-subunit.

Original languageEnglish (US)
Pages (from-to)5249-5255
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number8
DOIs
StatePublished - Feb 21 1997
Externally publishedYes

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Stilbenes
Cysteine
Adenosine Triphosphatases
Membranes
Conformations
Labeling
Cations
Adenosine Triphosphate
Association reactions
Ions
sodium-translocating ATPase
maleimide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase α-subunit. / Lutsenko, Svetlana; Daoud, Sylvia; Kaplan, Jack H.

In: Journal of Biological Chemistry, Vol. 272, No. 8, 21.02.1997, p. 5249-5255.

Research output: Contribution to journalArticle

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abstract = "Na,K-ATPase in right-side-out oriented vesicles was stabilized in different conformations, and the location of intramembrane Cys residues of the α-subunit was assessed with membrane-permeable and membrane-impermeable Cys-directed reagents. In the presence of Mg2+ and P(i), Cys964 was the most accessible for both membrane-impermeable 4-acetamido-4'- maleimidylstilbene-2,2'disulfonic acid (or stilbene disulfonate maleimide, SDSM) and membrane-permeable 7-diethylamino-3-(4'-maleimidyl)-4- methylcoumarin (CPM). In the presence of K+, Cys964 was modified only by hydrophobic CPM, indicating that the environment around Cys964 was different in these two conformations. Cys964 seems to mark the extracellular border of transmembrane segment M9. Cys911 in transmembrane segment M8 showed similar behavior; however, it was not so readily modified. Complete modification of Cys964 and Cys911 causes only partial (about 50{\%}) inactivation of both ATPase activity and Rb+ (or K+) occlusion, indicating that the effect on cation occlusion is indirect and not within the occlusion cavity. The ATP binding capacity remains unaltered by the modifications. Treatment of the K+-stabilized post-tryptic preparation of purified Na,K-ATPase revealed labeling of several cysteines by CPM, none of which were labeled with SDSM. Removal of K+ ions from the preparation, which we have previously shown is accompanied by release of the M5M6 hairpin to the supernatant (1), causes changes in the organization of the C-terminal 21-kDa fragment. In particular Cys983 in M10 became labeled by both CPM and SDSM, pointing to a tight association between the C terminus and the M5M6 hairpin of the α-subunit.",
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