Electrogenic K + transport by the Na + –K + pump in rat cardiac ventricular myocytes

The method of voltage clamp fluorometry combined with sitedirected 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 ␣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 (Ϸ5%) 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 E1P-E2P 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 E1 and E2 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. P -type ATPases form a major class of primary active membrane transport proteins, so called because they become transiently phosphorylated on ATP hydrolysis. The most prominent member is the ubiquitously occurring Na ϩ ͞K ϩ -ATPase, which exports three Na ϩ ions and imports two K ϩ ions in each transport cycle and thereby maintains the electrochemical gradients of Na ϩ and K ϩ across the plasma membrane of most animal cells.The reaction cycle of the Na ϩ ͞K ϩ -ATPase is described in terms of the Albers-Post scheme (see Fig. 1A) (1, 2). The transduction of primary energy from ATP hydrolysis to active ion transport is brought about by conformational changes that occur for both the ␣ and ␤ subunit of the Na ϩ ͞K ϩ -ATPase (3-6). Several approaches were undertaken to reveal Na ϩ ͞K ϩ -ATPase conformational changes by using fluorescence labeling of the native enzyme with fluorescein-5Ј-isothiocyanate, N-(p-(2-benzimidazolyl)phenyl)-maleimide and styrylpyridinium dyes like RH421 (7-12), which were limited for several reasons. Purely biochemical assays to investigate conformationdependent proteolysis patterns cannot provide time-resolved data, in the case of fluorescence labeling with styryl dyes the site of...

Section: Voltage-dependent Charge Movement and Fluorescence Changementioning

confidence: 89%

Section: Voltage-dependent Charge Movement and Fluorescence Changementioning

confidence: 99%

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

Geibel

Kaplan

Bamberg

et al. 2003

“…Two consequences of the unequal transport stoichiometry of Na þ and K þ are that steady pumping produces an outwardly directed current (1), proportional in magnitude to the turnover rate (2), which can be monitored electrically (3)(4)(5)(6)(7)(8), and that at least one step in the transport cycle must move charge through the membrane field (9). The latter implies that, under favorable conditions, charge relaxations following voltage jumps can be used to learn details about specific steps during the transport cycle (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

“…The occlusion/deocclusion transition of the first Na þ to be released from the pump involves large enthalpic and entropic changes (Net ΔH ¼ 19.6 kcal∕mol and net ΔS ¼ 62.4 cal∕mol K). From the occlusion/deocclusion net enthalpy, it is expected that this transition would have a Q 10 value of 3.3 (18,22), characteristic of transitions involving protein conformational changes. Of note is the fact that it is smaller than the value obtained for the rate-limiting step (v f ) when Na þ ∕K þ pumps are exchanging Na þ for K þ (Fig.…”

mentioning

confidence: 99%

Energy landscape of the reactions governing the Na ⁺ deeply occluded state of the Na ⁺ /K ⁺ -ATPase in the giant axon of the Humboldt squid

Castillo

Giorgis

Basilio

et al. 2011

The Na ∕K pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na from the cell and import 2K per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na ∕K pump that represent the kinetics of extracellular Na binding/release and Na occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid sidechain rearrangements at the site.P-type ATPases | pump currents | thermodynamics D uring each normal transport cycle of the Na þ ∕K þ -ATPase pump, three Na þ are exported from the cell in exchange for two K þ imported, a process driven by hydrolysis of one molecule of ATP. By establishing the Na þ and K þ gradients across cell membranes, the Na þ ∕K þ pump enables action potentials, synaptic signaling, and most solute transport in and out of cells. Two consequences of the unequal transport stoichiometry of Na þ and K þ are that steady pumping produces an outwardly directed current (1), proportional in magnitude to the turnover rate (2), which can be monitored electrically (3)(4)(5)(6)(7)(8), and that at least one step in the transport cycle must move charge through the membrane field (9). The latter implies that, under favorable conditions, charge relaxations following voltage jumps can be used to learn details about specific steps during the transport cycle (10-28).In the absence of internal and external K þ , the nominal absence of internal ADP and presence of an ATP regenerating system, the Na þ ∕K þ pump allows exchange of 3Na þ between their binding sites in a deeply occluded conformation and the external milieu (21, 29) ( Fig. 1A; encircled by dotted lines). Under these conditions there is no steady pump current observed at any voltage. Nevertheless, sudden voltage steps produce pre-steadystate currents that can be recorded (12, 13, 16-18, 21, 23, 26).These currents allow direct measurements of the rates of partial reactions of the pump cycle. Using giant axons from the squid Loligo pealeii, we have characterized th...

“…The more voltage-sensitive partial reactions are those reactions involving ion binding and release through a channel-like structure accessible from the extracellular space ( Fig. 1) (7)(8)(9)(10). Because the Na ϩ -transport branch is more voltage-sensitive (8, 11) than the K ϩ -transport branch (7,12), under physiological conditions with high Na o ϩ , negative transmembrane voltages inhibit the pump by forcing Na ϩ ions back into the access channel producing a steep positive slope in the steady-state Na/K pump current (I P ) versus V curve.…”

mentioning

confidence: 99%

Altered Na ⁺ transport after an intracellular α-subunit deletion reveals strict external sequential release of Na ⁺ from the Na/K pump

Yaragatupalli

Olivera

Gatto

et al. 2009

The Na/K pump actively exports 3 Na ؉ in exchange for 2 K ؉ across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the ␣-subunit diminishes internal Na ؉ (Na duced an inward flow of Na ؉ through Na/K pumps at negative potentials. Guanidinium ؉ can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidinium o ؉ can also traverse normal Na/K pumps in the absence of both Na o ؉ and K o ؉ and can also inhibit Na/K pump currents in a Na ؉ -like voltagedependent manner, we conclude that the normal pathway transited by the first externally released Na ؉ is large enough to accommodate guanidinium ؉ .C-terminal truncation ͉ ion access channel ͉ ion binding ͉ Na,K-ATPase ͉ voltage dependence