Conformational changes in the alpha-subunit, and cation transport by Na+, K+-ATPase

The relationship has been studied of structural changes in the alpha-subunit (Mr approximately equal to 100 000) of Na+,K+-ATPase to the binding and translocation of Rb+ and Na+. Two conformations, E1 and E2, are distinguished by controlled proteolysis of the alpha-subunit and fluorescence techniques. The de-phospho forms, E1Na and E2K, are stabilized by binding of Na+ and K+ or Rb+ to cytoplasmic sites on pure Na+,K+-ATPase in membrane fragments. In phospholipid vesicles reconstituted with pure Na+,K+-ATPase, the transitions between E1Na and E2K are coupled to translocation of cation in ouabain- or vanadate-sensitive passive fluxes along gradients for K+ and Na+. The direction of these fluxes is opposite to that of the active Na+-K+ transport. Coupling of transitions between the phospho forms, E1P and E2P, and the cation translocation was studied after selective proteolysis of the alpha-subunit. Cleavage with trypsin at the carboxyl-terminal side (bond 1) of the aspartyl phosphate residue or with chymotrypsin at the amino-terminal side (bond 3) blocks Na+, K+-ATPase and Na+-K+ transport, but the two splits have widely different effects on partial reactions. Cleavage of bond 3 blocks transition from E1P to E2P and abolishes both (ADP + ATP)-Na+/Na+ exchange and (ATP + Pi)-Rb+/Rb+ exchange reactions. Cleavage of bond 1 interferes neither with the transitions nor with the exchange reactions. Thus, both the cation-induced transitions between E1Na and E2K and the transitions between the phospho forms, E1P and E2P, of the alpha-subunit are coupled to flipping of cation sites between the inside-exposed state (E1) and the outside-exposed state (E2).