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 a domains, and the beta-subunit, a fraction of which is cleaved at Arg(142)-Gly(143). 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 H-1-H-2 helices and a 22-kDa alpha-fragment containing the H-7-H-10 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 Arg(142)-Gly(143). 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 H-1-H-2 transmembrane helices of alpha are adjacent to some of its H-7-H-10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta with the interacting H-1-H-2 and H-7-H-10 helices of a 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.