Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: I. Sodium Binding is Associated with a Conformational Rearrangement

Abstract

Abstract. To investigate Na+ binding to the ion-binding sites presented on the cytoplasmic side of the Na,KATPase, equilibrium Na + -titration experiments were performed using two fluorescent dyes, RH421 1 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na + in the presence of various Mg 2+ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na + ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na + ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na + ions and followed by the conformational transition E 1 /E 2 of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na + ions are bound.

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.…”

“…Fluorescence measurements were carried out in a Perkin-Elmer LS 50B fluorescence spectrophotometer as described in the recently published part I of this paper [30]. The excitation wavelength was set to 580 nm and the emission wavelength to 650 nm (slit width 15 nm and 20 nm, respectively).…”

“…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.…”

“…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 + .…”

“…But it is also known that the presence of divalent or trivalent cations affects Na + binding in terms of modified apparent affinities of Na + binding [15,18,33]. A previously developed fluorescence method, using the electrochromic styryl dye RH421 [7], could be successfully applied to detect the electrogenic binding of the third Na + ion, the only reaction step which was found to produce a reliable response with respect to cytoplasmic ion binding or release [30]. Therefore, an experimental concept has been developed to utilize this single detectable reaction step as a monitor to investigate interaction between and competition of various ions at the cytoplasmic sites.…”