Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements.

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.

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face.

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.

Kinetic properties of F0F1-ATPases. Theoretical predictions from alternating-site models.

We present an analysis of models based on current structural concepts of the F0F1 synthases, accounting for coupling between proton transport and ATP synthesis. It is assumed that each of the three alpha beta-subunits of the synthase can exist in three different conformational states E, Eo and E*. Proton translocation is coupled to cyclic interconversion of the conformations of the alpha beta-subunits. The conformational changes of these subunits are assumed to be coordinated so that all three interconvert simultaneously, in a rate-limiting transition. Binding and release of the ligands ATP, ADP, Pi, and protons are assumed to be equilibrium steps. In one family of models, interconversion of the alpha beta-subunits of F1 is coupled to the translocation event in F0 acting as a proton carrier. In a second family of models, protons combine with F0F1 and are translocated during the interconversion step in a chemiport. Kinetic tests involving the mutual effects of [ATP], [ADP], H+', and H+" are described, allowing us to make a distinction between the different models and submodels.

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy.

Na+/K+-ATPase can be isolated from the outer medulla of mammalian kidney in the form of flat membrane fragments containing the enzyme in a density of 10(3)-10(4) protein molecules per microm2 (Deguchi et al. (1977) J. Cell. Biol. 75, 619-634). In this paper we show that these membrane fragments can be bound to a germanium plate coated with a phospholipid bilayer. With this system infrared spectroscopic studies of the enzyme have been carried out using the technique of attenuated total reflection (ATR). At a coverage of the lipid surface corresponding to 30-40% of a monolayer of membrane fragments, characteristic infrared bands of the protein such as the amide I and II bands can be resolved. About 24% of the NH-groups of the peptide backbone are found to be resistant to proton/deuterium exchange within a time period of several days. Evidence for orientation of the protein with respect to the supporting lipid layer is obtained from experiments with polarized light, the largest polarization effects being associated with the -COO- band at 1400 cm-1. Experiments with aqueous media of different ionic composition indicate that the average orientation of transition moments changes when K+ in the medium is replaced by Tris+ or Na+.

Conformational transitions and change translocation by the Na,K pump: comparison of optical and electrical transients elicited by ATP-concentration jumps.

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.

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles.

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.

Voltage dependence of partial reactions of the Na+/K+ pump: predictions from microscopic models.

A theoretical treatment of the voltage dependence of electroneutral Na+-Na+ and K+-K+ exchange mediated by the Na+/K+ pump is given. The analysis is based on the Post-Albers reaction scheme in which the overall transport process is described as a sequence of conformational transitions and ion-binding and ion-release steps. The voltage dependence of the exchange rate is determined by a set of 'dielectric coefficients' reflecting the magnitude of charge translocations associated with individual reaction steps. Charge movement may result from conformational changes of the transport protein and/or from migration of ions in an access channel connecting the binding sites with the aqueous medium. It is shown that valuable mechanistic information may be obtained by studying the voltage dependence of transport rates at different (saturating and nonsaturating) ion concentrations.

Transient behaviour of the Na+/K+-pump: microscopic analysis of nonstationary ion-translocation.

In recent years fast perturbation techniques have been applied for investigating the mechanism of the Na+/K+-pump. Experiments in which nonstationary pump-currents and ion fluxes are measured after a voltage or ATP-concentration jump yield kinetic information which cannot be obtained from ordinary steady-state experiments. In this paper a theoretical treatment is described by which transient pump-currents and ion fluxes can be analyzed in a unified way. The method is based on the assumption that the operation of the pump involves a sequence of conformational transitions and ion-binding and -release steps. The charge displacements associated with the individual reaction steps are described by a set of dielectric coefficients. The nonstationary behaviour of the Na+/K+-pump is analyzed on the basis of the Albers-Post reaction cycle. It is shown that the different studies of transient pump-currents and ion fluxes carried out so far lead to internally consistent conclusions with respect to the nature of the electrogenic steps of the transport cycle.

Internal motions in proteins and gating kinetics of ionic channels.

Single-channel current recordings have revealed a complex kinetic behavior of ionic channels. Many channels exhibit closed-time distributions in which long waiting times occur with a much higher frequency than predicted by a simple exponential decay function. In this paper a model for opening-closing transitions that accounts for internal motions in the protein matrix is discussed. The model is based on the notion that the transition between a conductive and a nonconductive state of the channel represents a local process in the protein, such as the movement of a small segment of a peptide chain or the rotation of a single amino-acid residue. When the blocking group moves into the ion pathway, a structural defect is created consisting in a region of loose packing and/or poor hydrogen bonding. By rearrangements of neighboring groups, the defect may migrate within the protein matrix, carrying out a kind of random walk. Once the defect has moved away from the site where it was formed, a transition back to the open state of the channel is possible only when the defect has returned by chance to the original position. The kinetic properties of this model are analyzed by stochastic simulation of defect diffusion in a small domain of the protein. With a suitable choice of domain size and diffusion rate, the model is found to predict closed-time distributions that agree with experimental observations.

Kinetics of the Na+/alanine cotransporter in pancreatic acinar cells.

Electric currents associated with Na+-coupled alanine transport in pancreatic acinar cells were investigated by the technique of tight-seal whole-cell recordings. In a previous study the observed concentration dependence of alanine-dependent currents was found to be consistent with a 'simultaneous' transport mechanism with 1:1 stoichiometry. In the present work the sidedness of the cotransporter was investigated by comparing inward (I") and outward currents (I') measured under mirror-symmetrical conditions. I' and I" were found to be nearly equal (within a factor of approx. 2) in a wide range of Na+ and alanine concentrations. The transport model was further tested by 'infinite-cis' experiments with fixed, saturating concentrations of Na+ and L-alanine on one side of the membrane and variable concentrations on the other. By measuring transmembrane currents as a function of Na+ and alanine concentrations, numerical values of the equilibrium dissociation constants of both substrates could be estimated.

Torque and rotation rate of the bacterial flagellar motor.

This paper describes an analysis of microscopic models for the coupling between ion flow and rotation of bacterial flagella. In model I it is assumed that intersecting half-channels exist on the rotor and the stator and that the driving ion is constrained to move together with the intersection site. Model II is based on the assumption that ion flow drives a cycle of conformational transitions in a channel-like stator subunit that are coupled to the motion of the rotor. Analysis of both mechanisms yields closed expressions relating the torque M generated by the flagellar motor to the rotation rate v. Model I (and also, under certain assumptions, model II) accounts for the experimentally observed linear relationship between M and v. The theoretical equations lead to predictions on the relationship between rotation rate and driving force which can be tested experimentally.

Fast charge translocations associated with partial reactions of the Na,K-pump: I. Current and voltage transients after photochemical release of ATP.

Nonstationary electric currents are described which are generated by the Na,K-pump. Flat membrane sheets 0.2-1 micron in diameter containing a high density of oriented Na,K-ATPase molecules are bound to a planar lipid bilayer acting as a capacitive electrode. In the aqueous phase adjacent to the bound membrane sheets, ATP is released within milliseconds from an inactive, photolabile precursor ("caged" ATP) by an intense flash of light. After the ATP-concentration jump, transient current and voltage signals can be recorded in the external circuit corresponding to a translocation of positive charge across the pump protein from the cytoplasmic to the extracellular side. These electrical signals which can be suppressed by inhibitors of the Na,K-ATPase require the presence of Na+ but not of K+ in the aqueous medium. The intrinsic pump current Ip(t) can be evaluated from the recorded current signal, using estimated values of the circuit parameters of the compound membrane system. Ip(t) exhibits a biphasic behavior with a fast rising period, followed by a slower decline towards a small quasi-stationary current. The time constant of the rising phase of Ip(t) is found to depend on the rate of photochemical ATP release. Further information on the microscopic origin of the current transient can be obtained by double-flash experiments and by chymotrypsin modification of the protein. These and other experiments indicate that the observed charge-translocation is associated with early events in the normal transport cycle. After activation by ATP, the pump goes through the first steps of the cycle and then enters a long-lived state from which return to the initial state is slow.

Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents.

Nonstationary pump currents which have been observed in K+-free Na+ media after activation of the Na,K-ATPase by an ATP-concentration jump (see the preceding paper) are analyzed on the basis of microscopic reaction models. It is shown that the behavior of the current signal at short times is governed by electrically silent reactions preceding phosphorylation of the protein; accordingly, the main information on charge-translocating processes is contained in the declining phase of the pump current. The experimental results support the Albers-Post reaction scheme of the Na,K-pump, in which the translocation of Na+ precedes translocation of K+. The transient pump current is represented as the sum of contributions of the individual transitions in the reaction cycle. Each term in the sum is the product of a net transition rate times a "dielectric coefficient" describing the amount of charge translocated in a given reaction step. Charge translocation may result from the motion of ion-binding sites in the course of conformational changes, as well as from movement of ions in access channels connecting the binding sites to the aqueous media. A likely interpretation of the observed nonstationary currents consists in the assumption that the principal electrogenic step is the E1-P/P-E2 conformational transition of the protein, followed by a release of Na+ to the extracellular side. This conclusion is supported by kinetic data from the literature, as well as on the finding that chymotrypsin treatment which is known to block the E1-P/P-E2 transition abolishes the current transient. By numerical simulation of the Albers-Post reaction cycle, the proposed mechanism of charge translocation has been shown to reproduce the experimentally observed time behavior of pump currents.

Voltage dependence of sodium-calcium exchange: predictions from kinetic models.

Voltage effects on the Na-Ca exchange system are analyzed on the basis of two kinetic models, a "consecutive" and a "simultaneous" reaction scheme. The voltage dependence of a given rate constant is directly related to the amount of charge which is translocated in the corresponding reaction step. Charge translocation may result from movement of an ion along the transport pathway, from displacement of charged ligand groups of the ion-binding site, or from reorientation of polar residues of the protein in the course of a conformational transition. The voltage dependence of ion fluxes is described by a set of coefficients reflecting the dielectric distances over which charge is translocated in the individual reaction steps. Depending on the charge of the ligand system and on the values of the dielectric coefficients, the flux-voltage curve can assume a variety of different shapes. When part of the transmembrane voltage drops between aqueous solution and binding site, the equilibrium constant of ion binding becomes a function of membrane potential. By studying the voltage dependence of ion fluxes in a wide range of sodium and calcium concentrations, detailed information on the microscopic properties of the transport system may be obtained.