A B S T R A C T Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kinetic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E l ~ E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na + ion at the cytoplasmic face is electrogenic. This Na + ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg 2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)EI-P ---, P-Ez(Na2), which is accompanied by the release of one Na + ion. 5-IAF does not respond to the release of the subsequent two Na + ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E,P ~ P-E2 is the rate limiting process of the Na + translocation. The corresponding rate constant was determined to be 22 s -1 at 20~ From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na § ions are released subsequently with a rate constant of at least 5,000 s -1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3)'EI-P --, P-E2"(Na2) is the major charge translocating step in the reaction sequence Na3'El ~ (Na3)'E1-P ~ P-E2"(Na~) ~ P-E 2. The subsequent release of 2 Na + ions contributed less than 25% to the total electric current transient. 198 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 104 9 1994 binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E~(Na2) and P-E~ (K~). (e) Most of our experiments can be described by a modified Post-Albers scheme. A set of the kinetic parameters in this scheme has been determined by the experiments presented or by data from literature. Numerical simulations using this set are consistent with the presented data.

In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na,K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na+ are electroneutral steps, and that release of Na+ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K+ indicate that binding and occlusion of K+ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na+ or K+ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel ("ion well"). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP+) or tetraphenylborate (TPB-), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP+ leads to a decrease of binding affinity for Na+ and K+, which is thought to result from the TPP(+)-induced change of electric field strength in the access channel.

Membrane fragments containing a high density of Na,K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na,K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.

The electrogenic properties of the Na,K-ATPase were studied by correlating transient electrical events in the pump molecule with conformational transitions elicited by an ATP-concentration jump. Flat membrane fragments containing a high density (approximately 8000 microm(-2)) of oriented Na,K-ATPase molecules were bound to a planar lipid bilayer acting as a capacitive electrode. ATP was released in the medium from a photolabile inactive ATP derivative ("caged" ATP) by a 40-microsec light flash. Electrical signals resulting from transient charge movements in the protein under single-turnover conditions were recorded in the external measuring circuit. In parallel experiments carried out under virtually identical conditions, the fluorescence of membrane fragments containing Na,K-ATPase with covalently-bound 5-iodoacetamido-fluorescein (5-IAF) was monitored after the ATP-concentration jump. When the medium contained Na+, but no K+, the fluorescence of the 5-IAF-labeled protein decreases monotonously after release of ATP. In the experiments with membrane fragments bound to a planar bilayer, a transient pump current was observed which exhibited virtually the same time behavior as the fluorescence decay. This indicates that optical and electrical transients are governed by the same rate-limiting reaction step. Experiments with chymotrypsin-modified Na,K-ATPase suggest that both the fluorescence change as well as the charge movement are associated with the deocclusion of Na+ and release to the extracellular side. In experiments with Na+-free K+ media, a large inverse fluorescence change is observed after the ATP-concentration jump, but no charge translocation can be detected. This indicates that deocclusion of K+ is an electrically silent process.

The addition ol' cardiuc @ycosidcs lo the phaaphoenryme leads to large Ruorcscrtrcc chunyct3 Fig. 3~). Sign, amplitude und time courw dcpcndcd on Nn' concentration. At low Nu" (cnzymo in atnle P-E& the fluonxtacc decoded, W&I-W nt high Nn" (~IWW~ in state (Na,)E,-P) a fluotwccnee incrcusc could bc ob= served, The liunl lluorcrscu~r was the su~nc in ;\I1 Nu' concentretions, Similnr results huvc been obtrrincd with other anulogucq (digkoxigcnin, ~tmphunthidin, dig