Dataset Information

Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.

ABSTRACT: The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na(+)K(+)-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep alpha(1)-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ( approximately 5%) on addition of extracellular K(+). When the enzyme was subjected to voltage jumps under Na(+)Na(+)-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the E(1)P-E(2)P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of Na(+) by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating K(+) concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing K(+) concentrations. As a result, the stationary distribution between E(1) and E(2) states and voltage-dependent relaxation times can be determined at any time and membrane potential under Na(+)Na(+) exchange as well as Na(+)K(+) turnover conditions.

SUBMITTER: Geibel S

PROVIDER: S-EPMC298709 | biostudies-literature | 2003 Feb

REPOSITORIES: biostudies-literature

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Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.

Geibel Sven S Kaplan Jack H JH Bamberg Ernst E Friedrich Thomas T

Proceedings of the National Academy of Sciences of the United States of America 20030127 3

The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na(+)K(+)-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep alpha(1)-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the cat ...[more]

PMID: 12552111

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Project description:Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E(1)P and E(2)P states and measured Rb(+) uptake under various ionic and pH conditions. The steady-state E(1)P/E(2)P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb(+) uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E(1)P/E(2)P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V(0.5), the voltage, at which the E(1)P/E(2)P ratio is 50?50, by -100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E(1)P?E(2)P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb(+) uptake yielded an activation energy of ?90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E(1)P?E(2)P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na(+) profoundly alters the voltage-dependent E(1)P/E(2)P distribution indicating that Na(+) ions can act as surrogates for protons regarding the E(2)P?E(1)P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ?0.5 and ?0.2, respectively.

Project description:Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na(+) and K(+) gradients across the plasmalemma. Cardiac isozymes contain one catalytic ?-subunit isoform (?1, ?2, or ?3) associated with an auxiliary ?-subunit isoform (?1 or ?2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different ?-? isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na(+)o and K(+)o. We demonstrate that although ?1 and ?3 pumps are functional at all physiologically relevant voltages, ?2?1 pumps and ?2?2 pumps are inhibited by ?75% and ?95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump's voltage dependence. Our observations provide a simple explanation for the physiological relevance of the ?2 subunit (?20% of total ? subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na(+) entry occurs. This strong voltage dependence of ?2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the ?2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca(2+), due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na(+) and Ca(2+) concentrations.

Dataset Information

Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.

Publications

Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.

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