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.

Figure 1

(A) Albers–Post scheme for the Na⁺/K⁺-ATPase reaction cycle. The enzyme can assume two distinct conformations: E₁ with ion binding sites facing the cytoplasm, and E₂ with ion binding sites open to the extracellular space. The main electrogenic event was assigned to Na⁺ transport steps that are kinetically coupled to the E₁P–E₂P transition (underlaid in gray). (B) Na⁺/K⁺-ATPase α subunit modeled into the 1EUL structure of the SERCA Ca²⁺-ATPase (25) by using SWISSMODEL (courtesy of Jan B. Koenderink). Helix M5 and residue N790 (circle) are marked in red. Mutant N790C allowed for site-specific labeling by TMRM and yielded strong fluorescence changes in response to extracellular K⁺ or voltage pulses. Amino acids contributing to cation binding are colored as follows: E327, blue (helix M4); D776 and E779, red (helix M5); D804 and D808, green (helix M6). Two Ca²⁺ ions (yellow) from the 1EUL structure are also shown.

Figure 2

(A) Amino acids of the extracellular M5–M6 loop of the Na⁺/K⁺-ATPase. Amino acids of helices M5 and M6 are shown in black boxes. (B) Stationary pump currents of Na⁺/K⁺-ATPase single cysteine mutants at 0 mV in response to 10 mM K⁺. The dotted line indicates the stationary current level of the NaK_WT and NaK_ØCys constructs. Data originated from three to five oocytes; values are means ± SE. (C) Fluorescence images of TMRM-labeled oocytes. (Upper) Uninjected control. (Lower) Oocyte injected with NaK_ØCys(N790C) α and β subunit cRNA. Labeling and illumination conditions were identical. (D) Parallel recording of pump current (Lower) and fluorescence change (Upper) from an oocyte expressing NaK_ØCys(N790C) in response to 10 mM K⁺ and 5 mM ouabain (see perfusion protocol) at 0 mV.

Figure 3

(A) Fluorescence change signals in the absence of K⁺ on voltage pulses from 0 mV to values as stated. (B) Voltage jump-induced transient currents, obtained as ouabain-sensitive difference currents (see Materials and Methods), recorded in parallel to traces in A. (C) Rate constants (reciprocal of time constants) for fluorescence changes (□) and transient currents (●) as depicted in A and B. Data are means ± SE from five oocytes. (D) Voltage dependence of fluorescence saturation values (□) and translocated charge (●) from experiments as shown in A and B. Data are means ± SE from five oocytes. Fits of a Boltzmann function are superimposed, yielding the following parameters: ΔF-V curve (dashed): V_0.5 = −117 mV ± 1.4mV, z_q = 0.76 ± 0.2; Q–V curve (solid): V_0.5 = −110 mV ± 9 mV, z_q = 0.85 ± 0.12.

Figure 4

(A–E) Voltage pulse-induced fluorescence responses from a continuous recording of an oocyte expressing NaK_ØCys(N790C) at different K⁺ concentrations. Fluorescence increase is indicated by the black arrow. (F) Control measurement after removal of extracellular K⁺. (G) Inhibition by 5 mM ouabain in the presence of 30 mM K⁺. (Inset) The applied voltage protocol.

Figure 5

(A) Voltage dependence of fluorescence saturation values at different K⁺ concentrations: 0 mM (□), 0.3 mM (○),1 mM (▵), 3 mM (▿),10 mM (⋄). (B) [K⁺] dependence of apparent rate constants of fluorescence signals on off voltage jumps to −80 mV. Data are means ± SE from three oocytes.