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  • 1
    Electronic Resource
    Electronic Resource
    Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face (1991)
    Springer
    The journal of membrane biology 121 (1991), S. 163-176 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; ion pumps ; electrogenic transport ; potentiometric dyes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870530
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  • 2
    Electronic Resource
    Electronic Resource
    Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents (1987)
    Springer
    The journal of membrane biology 97 (1987), S. 179-191 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; ion pumps ; electrogenic transport ; Albers-Post cycle ; partial reactions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869221
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  • 3
    Electronic Resource
    Electronic Resource
    Fast charge translocations associated with partial reactions of the Na,K-pump: I. Current and voltage transients after photochemical release of ATP (1987)
    Springer
    The journal of membrane biology 97 (1987), S. 161-178 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; ion pumps ; electrogenic transport ; concentration jump ; “caged” ATP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Nonstationary electric currents are described which are generated by the Na,K-pump. Flat membrane sheets 0.2–1 μm in diameter containing a high density of oriented N,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 currentI p (t) can be evaluated from the recorded current signal, using estimated values of the circuit parameters of the compound membrane system.I p (t) exhibits a biphasic behavior with a fast rising period, followed by a slower decline towards a small quasistationary current. The time constant of the rising phase ofI p (t) is found to depend on the rate of photochemical ATP release. Further information on the microscopic orgin 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869220
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  • 4
    Electronic Resource
    Electronic Resource
    Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase (1985)
    Springer
    The journal of membrane biology 85 (1985), S. 49-63 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; reconstitution ; potential sensitive dye ; ion fluxes ; transport kinetics ; activation energy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01872005
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  • 5
    Electronic Resource
    Electronic Resource
    Conformational transitions and charge translocation by the Na,K pump: Comparison of optical and electrical transients elicited by ATP-concentration jumps (1989)
    Springer
    The journal of membrane biology 110 (1989), S. 67-86 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; active transport ; electrogenic pumps ; fluorescence probes ; conformational transitions ; caged ATP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary 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 (∼8000 μm−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-μsec 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870994
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  • 6
    Electronic Resource
    Electronic Resource
    A microscopic model for the current-voltage behaviour of the Na,K-pump (1986)
    Springer
    European biophysics journal 13 (1986), S. 309-321 
    Details
    ISSN: 1432-1017
    Keywords: Na, K-pump ; active transport ; electrogenic transport ; current-voltage behaviour ; Post-Albers scheme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract The current voltage characteristic of the Na, K pump is described on the basis of a modified Post-Albers cycle. The voltage dependence of the rate constants is derived from the elementary chargetranslocations associated with the single reaction steps. Charge displacements result from movements of the sodium- or potassium-loaded binding sites, as well as from motions of polar groups in the pump molecule. If part of the transmembrane voltage drops between the alkali-ion binding sites and the aqueous solution, the binding constants become voltage-dependent. Depending on the values of the microscopic parameters, the current-voltage characteristic may assume a variety of different shapes. Saturating behaviour results when one or more voltage-independent reaction steps become rate limiting. Non-monotonic current-voltage curves exhibiting regions of negative pump conductance are predicted when, at least in one of the transitions, charge is moved against the direction of overall charge-translocation. The theoretical predictions are compared with recent experimental studies of voltage-dependent pump currents.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00254213
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