Ligand-sensitive interactions among the transmembrane helices of Na+/K+-ATPase.
An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the alpha-subunit that contain all 10 transmembrane alpha domains, and the beta-subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa alpha-fragment containing the H1-H2 helices and a 22-kDa alpha-fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two alpha-fragments and that fraction of beta whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of beta without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of alpha are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta with the interacting H1-H2 and H7-H10 helices of alpha is altered by detergent solubilization and by structural changes in the extracellular domain of beta, and (c) the three-dimensional packing of the interacting transmembrane helices of alpha and beta are regulated by the specific ligands of the enzyme.
Sarvazyan
N A
NA
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008, USA.
Ivanov
A
A
Modyanov
N N
NN
Askari
A
A
D000595:Amino Acid Sequence; D000818:Animals; D004285:Dogs; D007679:Kidney Medulla / Q000201:enzymology; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D000254:Sodium-Potassium-Exchanging ATPase