The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease.

The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

Tuning of the Na,K-ATPase by the beta subunit.

The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αß enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the ß subunit isoforms are less clear, though ß2 is essential for motor physiology in mammals. Here, we show that compared to ß1 and ß3, ß2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the ß transmembrane helix correlates with its functional effect, suggesting that the relative orientation of ß modulates ion binding at the α subunit. ß2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na(+) and K(+) gradients.

Electrophysiological Characterization of Na,K-ATPases Expressed in Xenopus laevis Oocytes Using Two-Electrode Voltage Clamping.

The transport of three Na(+) per two K(+) means that the Na,K-ATPase is electrogenic, and though the currents generated by the ion pump are small compared to ion channel currents, they can be measured using electrophysiology, both steady-state pumping and individual steps in the transport cycle. Various electrophysiological techniques have been used to study the endogenous pumps of the squid giant axon and of cardiac myocytes from for example rabbits. Here, we describe the characterization of heterologously expressed Na,K-ATPases using two-electrode voltage clamping (TEVC) and oocytes from the Xenopus laevis frog as the model cell. With this system, the effects of particular mutations can be studied, including the numerous mutations that in later years have been found to cause human diseases.

Development of a Thermofluor assay for stability determination of membrane proteins using the Na(+)/H(+) antiporter NhaA and cytochrome c oxidase.

Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.

Resonance Raman characterization of the ammonia-generated oxo intermediate of cytochrome c oxidase from Paracoccus denitrificans.

A novel oxo state of cytochrome c oxidase from Paracoccus denitrificans generated by successive addition of excess H2O2 and ammonia was investigated using resonance Raman (RR) spectroscopy. Addition of ammonia to the H2O2-generated artificial F state resulted in an upshift of the oxoferryl stretching vibration from 790 to 796 cm(-1), indicating that ammonia influences ligation of the heme-bound oxygen in the binuclear center. Concomitantly performed RR measurements in the high-frequency region between 1300 and 1700 cm(-1) showed a high-spin to low-spin transition of heme a3 upon generation of the F state that was not altered by addition of ammonia. Removal of H2O2 by addition of catalase resulted in the disappearance of the oxoferryl stretching vibration and major back transformation of heme a3 into the high-spin state. The ratio of high-spin to low-spin states was identical for intermediates created with and without ammonia, leading to the conclusion that ammonia does not interact directly with heme a3. Only for the ammonia-created state was a band at 612 nm observed in the UV-visible difference spectrum that was shifted to 608 nm after addition of catalase. Our results support the hypothesis by von der Hocht et al. [von der Hocht, I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 3964-3969] that addition of ammonia creates a novel oxo intermediate state called PN where ammonia binds to CuB once the oxo intermediate F state has been formed.

Subunit δ is the key player for assembly of the H(+)-translocating unit of Escherichia coli F(O)F1 ATP synthase.

The ATP synthase (F(O)F1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γε and the stator ab2α3ß3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F(O)F1 complexes by affinity chromatography with the use of mutants synthesizing different sets of F(O)F1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F(O)F1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H(+)-translocating unit as intermediate product. Surprisingly, during the biogenesis of F(O)F1, F1 subunit δ is the key player in generating stable F(O). Subunit δ serves as clamp between ab2 and c10α3ß3γε and guarantees that the open H(+) channel is concomitantly assembled within coupled F(O)F1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H(+)-translocating enzymes.

The torque of rotary F-ATPase can unfold subunit gamma if rotor and stator are cross-linked.

During ATP hydrolysis by F(1)-ATPase subunit γ rotates in a hydrophobic bearing, formed by the N-terminal ends of the stator subunits (αß)(3). If the penultimate residue at the α-helical C-terminal end of subunit γ is artificially cross-linked (via an engineered disulfide bridge) with the bearing, the rotary function of F(1) persists. This observation has been tentatively interpreted by the unfolding of the α-helix and swiveling rotation in some dihedral angles between lower residues. Here, we screened the domain between rotor and bearing where an artificial disulfide bridge did not impair the rotary ATPase activity. We newly engineered three mutants with double cysteines farther away from the C-terminus of subunit γ, while the results of three further mutants were published before. We found ATPase and rotary activity for mutants with cross-links in the single α-helical, C-terminal portion of subunit γ (from γ285 to γ276 in E. coli), and virtually no activity when the cross-link was placed farther down, where the C-terminal α-helix meets its N-terminal counterpart to form a supposedly stable coiled coil. In conclusion, only the C-terminal singular α-helix is prone to unwinding and can form a swivel joint, whereas the coiled coil portion seems to resist the enzyme's torque.

True wild type and recombinant wild type cytochrome c oxidase from Paracoccus denitrificans show a 20-fold difference in their catalase activity.

The four subunit (SU) aa(3) cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. Its binuclear active center, residing in SU I, contains heme a(3) and Cu(B). Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity. To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT strain was constructed, carrying the gene for SU I on a low copy number plasmid. This rec. WT showed no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential over-production of SU I might impair the correct insertion of heme a(3) and Cu(B) because of a deficiency in metal inserting chaperones. An altered distance between heme a(3) and Cu(B) and variations in protein structure are possible reasons for the observed increased catalase activity. The availability of chaperones was improved by cloning the genes ctaG and surf1c on the same plasmid as the SU I gene. The new rec. WT CcO showed in fact a reduced catalase activity. Using differential scanning calorimetry no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging the thermal stability of the rec. WT CcO was reduced compared to that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.

Interconversions of P and F intermediates of cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. This redox-driven proton pump catalyzes the four-electron reduction of molecular oxygen to water, one of the most fundamental processes in biology. Elucidation of the intermediate structures in the catalytic cycle is crucial for understanding both the mechanism of oxygen reduction and its coupling to proton pumping. Using CcO from Paracoccus denitrificans, we demonstrate that the artificial F state, classically generated by reaction with an excess of hydrogen peroxide, can be converted into a new P state (in contradiction to the conventional direction of the catalytic cycle) by addition of ammonia at pH 9. We suggest that ammonia coordinates directly to Cu(B) in the binuclear active center in this P state and discuss the chemical structures of both oxoferryl intermediates F and P. Our results are compatible with a superoxide bound to Cu(B) in the F state.

Domain compliance and elastic power transmission in rotary F(O)F(1)-ATPase.

The 2 nanomotors of rotary ATP synthase, ionmotive F(O) and chemically active F(1), are mechanically coupled by a central rotor and an eccentric bearing. Both motors rotate, with 3 steps in F(1) and 10-15 in F(O). Simulation by statistical mechanics has revealed that an elastic power transmission is required for a high rate of coupled turnover. Here, we investigate the distribution in the F(O)F(1) structure of compliant and stiff domains. The compliance of certain domains was restricted by engineered disulfide bridges between rotor and stator, and the torsional stiffness (kappa) of unrestricted domains was determined by analyzing their thermal rotary fluctuations. A fluorescent magnetic bead was attached to single molecules of F(1) and a fluorescent actin filament to F(O)F(1), respectively. They served to probe first the functional rotation and, after formation of the given disulfide bridge, the stochastic rotational motion. Most parts of the enzyme, in particular the central shaft in F(1), and the long eccentric bearing were rather stiff (torsional stiffness kappa > 750 pNnm). One domain of the rotor, namely where the globular portions of subunits gamma and epsilon of F(1) contact the c-ring of F(O), was more compliant (kappa congruent with 68 pNnm). This elastic buffer smoothes the cooperation of the 2 stepping motors. It is located were needed, between the 2 sites where the power strokes in F(O) and F(1) are generated and consumed.