Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase alpha-subunit.
Na,K-ATPase in right-side-out oriented vesicles was stabilized in different conformations, and the location of intramembrane Cys residues of the alpha-subunit was assessed with membrane-permeable and membrane-impermeable Cys-directed reagents. In the presence of Mg2+ and Pi, 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 (), 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 alpha-subunit.
Lutsenko
S
S
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
Daoud
S
S
Kaplan
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D004285:Dogs; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism
D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine