Membrane disposition of the M5-M6 hairpin of Na⁺,K⁺-ATPase α subunit is ligand dependent

Extensive proteolytic digestion of Na⁺,K⁺-ATPase (EC 3.6.1.37) by trypsin produces a preparation where most of the extramembrane portions of the a subunit have been digested away and the β subunit remains essentially intact. The fragment Gln-737-Arg-829 of the Na⁺,K⁺-ATPase α subunit, which includes the putative transmembrane hairpin M5-M6, is readily, selectively, and irreversibly released from the posttryptic membrane preparation after incubation at 37°C for several minutes. Once released from the membrane, the fragment aggregates but remains water soluble. Occlusion of K⁺ or Rb⁺ specifically prevents release of the Gln-737-Arg-829 fragment into the supernatant. Labeling of the posttryptic membrane preparation with cysteine- directed reagents revealed that Cys-802 (which is thought to be located within the M6 segment) is protected against the modification by Rb⁺ while this fragment is in the membrane but ran be readily modified upon release. Cation occlusion apparently alters the folding and/or disposition of the MS- M6 fragment in the membrane in a way that does not occur when the fragment migrates to the aqueous phase. The ligand-dependent disposition of the M5-M6 hairpin in the membrane along with recent labeling studies suggest a key role for this segment in cation pumping by Na⁺,K⁺-ATPase.