Binding of the third Na⁺ ion to the cytoplasmic side of the Na,K‐ATPase is electrogenic

Abstract

A new experimental setup was constructed to allow parallel measurements of total internal reflection fluorescence and of capacitance changes in Na,K-ATPase-containing membranes. Effects correlated with cytoplasmic sodium binding to Na,K-ATPase were investigated. Ion binding-induced fluorescence changes of the electrochromic dye RH421 in membrane fragments adsorbed on a transparent capacitative electrode corresponded perfectly to capacitance increases detected by a lock-in technique. From these electric measurements it was possible to estimate a dielectric coefficient of about 0.25 for the electrogenic binding of the third Na + ion. Binding of K + to cytoplasmic sites was electroneutral.z 1999 Federation of European Biochemical Societies.

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

“…Since only Na + ions bind electrogenically in the E 1 conformation of the ion pump [12,30], all ions other than Na + should have only a marginal effect on the fluorescence intensity (⌬F max /F 0 < 3%). In the following it will be shown that this is true for all other monovalent cations, however, some di-and trivalent cations are able to affect the fluorescence intensity of the styryl dye.…”

“…7B. The third binding site is placed inside the protein dielectric (about 25%), is uncharged, exclusively selective for Na + , and binding to this site promotes a conformational relaxation that is propagated, possibly by another movement of the L6/7 loop [32,37], to the nucleotide binding site where Asp372 becomes competent to be phosphorylated by ATP [12,30,31].…”

“…This reaction can be monitored by fluorescent probes such as RH421 and FITC [30] or by direct electric measurements of changes of the membrane capacitance, and it has been shown that all three methods detect identical behavior of the protein as a function of the Na + concentration [12]. Since the experiments with RH421 as well as the changes of the membrane capacitance are related to electrogenic processes, it could be proven that binding of the third Na + ion is the only electrogenic event on the cytoplasmic side of the Na,K-ATPase.…”

“…The observation that exclusively Na + is able to bind as a third ion indicates that this site is extremely selective for Na + ; no other ions have been found to introduce a positive charge into the protein dielectric. From recent studies is was shown that the Na + ion has to cross about 25% of membrane dielectric (or membrane potential) to reach its site [12,25]. The special role of the third Na + -binding site can be seen also from the titrations with Na + only.…”

“…However, the properties of the cytoplasmic ion-binding sites, sodium binding and the competition of various cations at these sites as well as the nature and structure of these sites are only poorly understood so far. Recently, the electrogenicity of cytoplasmic Na + binding (in contrast to all other ions) was proven [12,26,32] and it was shown that the electrogenic binding of the third Na + ion to its (uncharged) site triggers a conformational rearrangement at the ATP-binding site which makes the enzyme competent to be phosphorylated, the next step in the transport cycle [30]. In forward direction three Na + ions are necessary for the phosphorylation of the enzyme, and no other ions may replace Na + .…”