Dempski RE et al. (2005), The beta subunit of the Na+/K+-ATPase follows t...

XB-ART-1989

J Gen Physiol 2005 May 01;1255:505-20. doi: 10.1085/jgp.200409186.

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The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme.

Dempski RE , Friedrich T , Bamberg E .

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The Na+/K+-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of Na+ and K+. It is comprised of at least two subunits, a large catalytic alpha subunit that mediates ATP hydrolysis and ion transport, and an ancillary beta subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the alpha subunit have been extensively studied, little is known about the participation of the beta subunit in conformational changes of the enzyme. To elucidate the role of the beta subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep beta1 subunit were individually replaced for cysteines. This enabled sulfhydryl-specific labeling with the environmentally sensitive fluorescent dye tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes. Investigation by voltage-clamp fluorometry identified three reporter positions on the beta1 subunit that responded with fluorescence changes to alterations in ionic conditions and/or membrane potential. These experiments for the first time show real-time detection of conformational rearrangements of the Na+/K+-ATPase through a fluorophore-labeled beta subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the beta subunit to certain reaction steps of the Na+/K+-ATPase, which involve changes in the occupancy of the two principle conformational states, E1P and E2P. From these experiments, evidence is provided that the beta1-S62C mutant can be directly used to monitor the conformational state of the enzyme, while the F64C mutant reveals a relaxation process that is triggered by sodium transport but evolves on a much slower time scale. Finally, shifts in voltage dependence and kinetics observed for mutant K65C show that this charged lysine residue, which is conserved in beta1 isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release.

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Species referenced: Xenopus laevis
Genes referenced: tbx2 tff3.7

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	Figure 1. . (A) Albers-Post scheme for the Na+/K+ ATPase reaction cycle. (B) Scheme of the experimental setup used for voltage-clamp fluorometry. (C) Schematic diagram of the transmembrane domain and adjacent residues of the sheep Î²1 subunit of the Na+/K+-ATPase. The diagram depicts residues Arg-27 to Ala-73; in bold are shown the 11 residues Met-57 to Tyr-67, which were individually replaced by cysteines for the purpose of site-directed fluorescence labeling. Three residues that demonstrate significant fluorescence changes in response to voltage pulses are colored red.
	Figure 2. . Stationary current and fluorescence measurements of Na+/K+-ATPase Î±/Î² complexes containing different Î² subunit constructs upon expression in Xenopus oocytes. (A) Stationary pump currents of the Na+/K+-ATPase expressed with wild-type and single cysteine mutants of the Î² subunit at 0 mV holding potential in response to 10 mM K+. Data originated from 5â11 oocytes; values are means Â± SEM. (B) Parallel recording of pump current (top) and fluorescence change (bottom) from an oocyte coinjected with Na+/K+-ATPase sNaKÎ±1, ÃCys and sÎ²1 -F64C cRNA in response to 10 mM K+ and 10 mM ouabain at 0 mV holding potential.
	Figure 3. . Voltage pulseâinduced fluorescence responses of the Na+/K+-ATPase with different single cysteine mutants of the Î² subunit under K+-free (Na+/Na+ exchange) conditions. Recordings originated from oocytes coexpressing the Î± subunit construct sNaKÎ±1, ÃCys together with sÎ²1-S62C (A), sÎ²1-F64C (B), sÎ²1-K65C (C). D shows the applied voltage protocol. Voltage jumps were performed from a holding potential of â80 mV to values between +60 mV and â200 mV in 20-mV steps. Since hyperpolarizing potentials result in an increase in fluorescence, negative potentials are shown at the top and positive potentials at the bottom of the voltage protocol. Note the expanded time scale for the recording in B. Voltage jumpâinduced transient currents, obtained as ouabain-sensitive difference currents (see materials and methods) recorded in parallel to fluorescence change signals for S62C (E), F64C (F), and K65C (G).
	Figure 4. . Voltage dependence and kinetics of voltage jumpâinduced fluorescence changes and comparison with properties of the corresponding transient charge movements under K+-free (Na+/Na+ exchange) conditions. Data originated from oocytes coexpressing the Î± subunit construct sNaKÎ±1, ÃCys together with sÎ²1-S62C (A and D), sÎ²1-F64C (B and E), sÎ²1-K65C (C and F). Left panels show the voltage dependences of normalized fluorescence saturation values (âª) obtained from mono- (A and C) and biexponential fits (B) to the data, and the corresponding normalized values for the translocated charge (â¡) obtained from integration of the transient currents recorded in parallel. Curve parameters are summarized in Table I. Right panels show the voltage dependence of reciprocal time constants obtained from monoexponential fits of the transient current traces before (âª) and after (â¡) labeling of oocytes with TMRM, together with reciprocal time constants from fits of fluorescence signals under K+-free conditions (â´) or in presence of K+ (99.9 mM Na+/0.1 mM K+, â; 99.5 mM Na+/0.5 mM K+, â¢). Data are means Â± SEM from six oocytes.
	Figure 5. . Voltage pulseâinduced fluorescence responses at different extracellular Na+ and K+ concentrations from oocytes coexpressing the Î± subunit construct sNaKÎ±1, ÃCys together with sÎ²1-S62C. (AâE) K+ titration in presence of Na+. Data were consecutively recorded from a single oocyte after changes to perfusion buffers with the following Na+/K+ contents: (A) 100 mM Na+ (no K+), (B) 99.9 mM Na+ and 0.1 mM K+, (C) 99.5 mM Na+ and 0.5 mM K+, (D) 99 mM Na+ and 1 mM K+, and (E) 95 mM Na+ and 5 mM K+. F shows the applied voltage protocol for all data traces shown in this figure. (GâJ) K+ titration in presence of NMDG. Voltage pulseâinduced fluorescence responses from a single oocyte experiment at different Na+/NMDG/K+ concentrations: (G) 100 mM Na+ (no K+, for control), (H) 99.9 mM NMDG and 0.1 mM K+, (I) 99.5 mM NMDG and 0.5 mM K+, and (J) 100 mM NMDG.
	Figure 6. . Voltage dependence values for fluorescence saturation amplitudes during K+ titrations, obtained from monoexponential fits to data traces from experiments as exemplified in Fig. 5. Top panels correspond to Î² subunit construct sÎ²1-S62C in (A) Na+-based and (B) NMDG-based solutions, bottom panels to Î² subunit construct sÎ²1-F64C, in (C) Na+-based and (D) NMDG-based solutions. Buffer compositions were as follows. (A and C) âª, 100 mM Na+; â¡, 99.9 mM Na+ and 0.1 mM K+; â´, 99.5 mM Na+ and 0.5 mM K+; â, 99 mM Na+ and 1 mM K+; â¢, 95 mM Na+ and 5 mM K+; and â, 100 mM Na+ and 10 mM ouabain. (B and D) âª, 100 mM Na+; â¡, 100 mM NMDG; â´, 99.9 mM NMDG and 0.1 mM K+; â, 99.5 mM NMDG and 0.5 mM K+; and â¢, 99 mM NMDG and 1 mM K+.
	Figure 7. . Determination of the apparent K0.5 for the shift of the steady-state distribution between E1 and E2 states by external K+ at +60 mV. Data were derived from fluorescence changes for the Na+/K+-ATPase containing the Î² subunit construct sÎ²1-S62C in the presence of NMDG-based (A) or Na+-based (B) buffers. Solid lines represent fits of a Hill equation to the data (where nH = 1), with K0.5 values as stated. Each dataset was obtained from three oocytes (means Â± SEM).
	Figure 8. . Model of the analyzed region of the Na+/K+-ATPase Î²1 subunit and possible orientation of residues with respect to the Î± subunit. (A) The analyzed region of the sheep Na+/K+-ATPase Î²1 subunit adjacent to the extracellular plasma membrane interface is represented as a helical wheel assuming an Î±-helical structure. The last residue of the transmembrane domain, M57, aligns with a putative tri-glycine helixâhelix interaction motif (see text), which points in the direction of the yellow arrow. The red arrow aligns with a cysteine residue (C45, not shown) within the transmembrane region of the Î²1 subunit that was shown to cross-link with helix M8 of the Î± subunit (Or et al., 1999; Ivanov et al., 2000). The blue arrow indicates the orientation of the F64 sidechain. (B) Putative orientation of the Na+/K+-ATPase Î²1 subunit's transmembrane helix with respect to the transmembrane domain of P2-ATPases. Views of the 10 transmembrane helices (M1 to M10) of the Na+/K+-ATPase Î± subunit perpendicular to the membrane plane from the cytoplasmic side are depicted as deduced from the SERCA crystal structure (Toyoshima et al., 2000). The Na+/K+-ATPase Î²1 subunit's transmembrane helix was oriented with C45 pointing towards M8 (in red) of the Î± subunit as suggested by cross-linking studies (Or et al., 1999; Ivanov et al., 2000), which allows positioning in two possible orientations according to Hasler et al. (2001). Arrows indicate the same directions as in A.

References [+] :

Albers, Biochemical aspects of active transport. 1967, Pubmed