Ligand-sensitive interactions among the transmembrane helices of Na+/K+-ATPase

Abstract

An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to hind Na+, K+, and ouabain, consists of four fragments of the α- subunit that contain all 10 transmembrane a domains, and the β-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 α-fragment containing the H1-H2 helices and a 22-kDa α-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 a-fragments and that fraction of β 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 β 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 a 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 β with the interacting H1-H2 and H7-H10 helices of a is altered by detergent solubilization and by structural changes in the extracellular domain of β, and (c) the three-dimensional packing of the interacting transmembrane helices of a and β are regulated by the specific ligands of the enzyme.