Properties of the sodium-pump ion binding sites in state E1

Two classes of ion-binding sites can be distinguished in state E-1 of the Na,K-ATPase: The first class is able to bind up to two cations of different valence, the second is selective for one Na+. Binding to this site is electrogenic and the site can be occupied exclusively Na+. This process arms the enzyme for phosphorylation by ATP. The electrochromic styryl dye RH421 was used to investigate the competition between Na+ and a variety of other cations. Apparent inhibition strength against Na+ binding was determined for monovalent cations (Rb+> K+ > Cs+ > Li+), divalent cations (Ca2+ > Sr2+ > Mg2+ > Ba2+) and trivalent cations (Br2TITU). Inhibition of Na+ binding to native and truncated Na,K-ATPase by Mg2+ was compared. While mono- and trivalent ions seem to affect the protein only at the ion binding sites, Mg2+ binds also at a site independent of the ion-binding moiety; we assume that it is the nucleotide binding site. All results are compatible with a model in which that Na+ enters its specific site only if the two less specific land negatively charged) sites are already occupied by Na+. There is no indication that under any condition "mixed" binding occurs with a Na+ ion bound to its specific site and any other ions bound to the negatively-charged sites.