Phosphorylated intermediate of the ouabain-insensitive,Na-stimulated ATPase in rat kidney cortex and rainbow trout gills

Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Na⁺-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg²⁺ dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg²⁺-dependent, Na⁺-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH₂OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Na⁺ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1–E2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na·E1∼P to Na·E2-P.     

Na,K-ATPase structure/function relationships probed by the denaturant urea

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37 °C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K⁺ as well as the K⁺-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na⁺. Half-maximal inhibition is seen with about 1 M urea for shark enzyme and about 2 M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation.     

Mechanism of the Na,K-ATPase Inhibition by MCS Derivatives

The previously reported class of potent inorganic inhibitors of Na,K-ATPase, named MCS factors, was shown to inhibit not only Na,K-ATPase but several P-type ATPases with high potency in the sub-micromolar range. These MCS factors were found to bind to the intracellular side of the Na, K-ATPase. The inhibition is not competitive with ouabain binding, thus excluding its role as cardiac-steroid-like inhibitor of the Na,K-ATPase. The mechanism of inhibition of Na,K-ATPase was investigated with the fluorescent styryl dye RH421, a dye known to report changes of local electric fields in the membrane dielectric. MCS factors interact with the Na,K-ATPase in the E₁ conformation of the ion pump and induce a conformational rearrangement that causes a change of the equilibrium dissociation constant for one of the first two intracellular cation binding sites. The MCS-inhibited state was found to have bound one cation (H⁺, Na⁺ or K⁺) in one of the two unspecific binding sites, and at high Na⁺ concentrations another Na⁺ ion was bound to the highly Na⁺-selective ion-binding site.     

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb+

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K₂)Mg–MgF₄, an E2∙Pi-like state capable to occlude K⁺ (or Rb⁺). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb⁺ with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1–E2 conformational transition and the kinetics of Rb⁺ exchange between the medium and the E2(Rb₂)Mg–MgF₄ state. Our results show that both in the absence and in the presence of Rb⁺, simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg–MgF₄ complex, that is unable to shift to E1 upon addition of Na⁺. The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg–MgF₄ formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb⁺ caused by Mg^2 +. Na⁺-induced release of Rb⁺ from E2(Rb₂)Mg–MgF₄ occurs at the same rate as from E2(Rb₂) but is insensitive to ADP. The rate of Rb⁺ occlusion into the E2Mg–MgF₄ state is 5–8 times lower than that described for the E2Mg–vanadate complex. Since the E2Mg–MgF₄ and E2Mg–vanadate complexes represent different intermediates in the E2-P → E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.     

Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion

We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hank’s solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb⁺ in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb⁺ in the absence and in the presence of ouabain. The optimal conditions for Rb⁺ incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 °C, 60 min; rinsing time with cold saline, 5-10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC₅₀ was 100 μM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the β₁ subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH₄⁺ as activating cation. The enzyme was also stimulated by Na⁺, but in contrast to the rat enzyme, hardly by K⁺. SCH 28080 inhibited the NH₄⁺-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K⁺], and [Na⁺] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E₁P to E₂P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H⁺/Na⁺ and/or H⁺/NH₄⁺ transport but not in Na⁺/K⁺ transport.     

Differences between CTP and ATP as substrates for the (Na + K)-ATPase

CTP was a poorer substrate than ATP when substituted in the (Na + K)-ATPase reaction assay, not only in terms of K_m but also of V. CDP was a poorer inhibitor than ADP, so product inhibition cannot account for CTP being a poorer substrate. In the Na-ATPase reaction, which the enzyme also catalyzes, substituting CTP for ATP resulted in greater activity, arguing against CTP being less effective than ATP in forming the enzyme-phosphate intermediate common to both reactions. Ligands that favor the E₂ conformational state of the enzyme, K⁺, Mg²⁺, and Mn₂⁺, inhibited the (Na + K)-CTPase reaction more than the (Na + K)-ATPase. Conversely, Triton X-100, which favors the E₁ conformational state of the enzyme, K⁺, Mg²⁺, and Mn²⁺, inhibited the (Na + K)-CTPase ATPase reaction but stimulated the (Na + K)-CTPase. Although the (Na + K)-ATPase reaction sequence probably involves cyclical interconversion between E₁ and E₂ conformational states (and is thus inhibitable by ligands favoring either state), the K-phosphatase reaction catalyzed by the enzyme apparently functions entirely in the E₂ state. This reaction is better stimulated by CTP plus Na⁺ than by ATP plus Na⁺; moreover, CTP lessens inhibition by Triton X-100, and ATP lessens inhibition by inorganic phosphate (which reacts with the E₂ state). These observations indicate that CTP is a poorer substrate than ATP because it is less effective in promoting conversion of E₂ to E₁, essential for the (Na + K)-dependent reaction mechanism. However, contrary to this rationale, dimethyl sulfoxide stimulated the (Na + K)-CTPase reaction although by other criteria, including inhibition of the (Na + K)-ATPase, the reagent appears to favor the E₂ over the E₁ conformational state.     

Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K_0.5 values for Na⁺ with minor alterations in K_0.5 values for K⁺ and NH₄⁺, causing a decrease in maximal velocities under saturating Na⁺, K⁺ and NH₄⁺ concentrations. Phosphorylation measurements in the presence of 20 µM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na⁺, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na⁺, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na⁺ at the Na⁺-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.     

Transport of monovalent thallium across the membrane of oocyte of the lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

Mechanisms of transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis were studied by using ²⁰⁴Tl. The Tl⁺ transport in lamprey oocytes has been shown to be realized by at least two pathways: through Na/K-pump and by mechanisms of Na,K,Cl-cotransport. In the standard Ringer solution (mM): 4 KCl, 140 NaCl, 0.5 CaCl₂, 5 glucose, 10 Tris-HCl-in the presence of ouabain, the coefficient of the ²⁰⁴Tl stationary distribution (cell/medium) was within the range of 2.3–2.5, while the time necessary to reach its 50% value amounted to 40–45 min at 20°C. In potassium-free media, transport of ²⁰⁴Tl via Na/K-pump was described by simple kinetics with saturation and was characterized by the value V _max = 520 pmol/(cell h) and K _M = 0.3 mM. In the presence of 4 mM K⁺ and 0.1 mM/1 Tl⁺, the ouabain-sensitive Tl⁺ flux decreased to 75 pmol/(cell h). At activation of the mechanism of Na,K,Cl-cotransport by the outer Na⁺ (in Na-NMDG media of different composition) the total influx of Tl⁺ reached 193 ± 20 pmol/(cell h), while the bumetanide-sensitive component—119 ± 12 pmol/(cell h) with K _M for Na⁺ about 20 mM. In the incubation media with variable concentration of chloride ions (replacement of Cl⁻ by NO₃) the total Tl⁺ flux reached 220 ± 21, while via the mechanisms of Na,K,Cl-cotransport—87 ± 8 pmol/(cell h). Under our experimental conditions, mechanisms of active transport and Na,K,Cl-cotransport accounted for 94% of the Tl⁺ influx. The potassium channels that usually are permeable also to monovalent thallium ions were not revealed.     

Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the <Emphasis Type="Italic">N</Emphasis>-glycans of their <Emphasis Type="Italic">β</Emphasis> subunits

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P₂-type pumps that catalyze active transport of Na⁺ in exchange for K⁺ (Na,K-ATPase) or H⁺ in exchange for K⁺ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β ₁-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β ₁-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β ₁-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β ₁–β ₁ interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β ₂-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β ₂-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit. Supported in part by NIH grants DK46917, DK58333, D53642, and USVA     

Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.     

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.     

Mechanism of high K+ and Tl+ uptake in cultured human glioma cells

Summary 1. The aim of this study was to elucidate if the K⁺ uptake was higher in cultured human glioma cells than in cells from other malignant tumors and to analyze the importance of membrane potential and K⁺ channels for the uptake.2. K⁺ transport properties were studied with the isotopes⁴²K and the K-analogue²⁰¹Tl.3. Comparison with cultured cells from other malignant tumors showed that the specific steady-state accumulation of Tl⁺ was significantly higher in glioma cells (U-251MG and Tp-378MG).4. In Ringer's solution at 37°C the rates of K⁺ and Tl⁺ uptake were both inhibited by about 55% in ouabain and 60% in furosemide, bumetanide, or Na⁺-or Cl^–-free medium. This indicated that the routes for K⁺ and Tl⁺ uptake were similar and due to Na,K-ATPase-dependent transport and to Na-K-Cl cotransport.5. About 10% of the uptake was neither ouabain nor bumetanide sensitive. Ba²⁺, which is known to block inward-rectifying K⁺ channels and to depolarize glial cells, and other K⁺ channel blockers (Cs⁺ and bupivacaine), had no effect on Tl⁺ uptake.6. Metabolic inhibition with dinitrophenol reduced the uptake rate to 17%.7. The washout of Tl⁺ was unaffected by bumetanide and K⁺ channel blockers, but dinitrophenol caused a transient increase of 75%, an effect which persisted in the presence of K⁺ channel blockers.8. It was concluded that the high specific K⁺ and Tl⁺ accumulation in cultured human glioma cells was due not to the presence of inwardly rectifying K⁺ channels or other identified K⁺ channels, but to Na,K-ATPase dependent transport and Na-K-Cl cotransport.     

Solubilization and further chromatographic purification of highly purified,membrane-bound Na,K-ATPase

Highly purified membrane-bound Na,K-ATPase from pig kidney outer medulla was dissolved in the non-ionic detergent C₁₂E₈. Chromatography of the dissolved material on a DEAE matrix yielded enzymatical material having a ouabain-binding capacity of 6.9 nmoles per mg protein (measured according to Lowry et al., with bovine serum albumin as standard). This material, which after addition of lipids had the same K⁺-phosphatase turnover as the membrane-bound enzyme, could consist entirely of live molecules with a molecular weight of 145 kDa, a value close to that expected for αβ-protomers of Na,K-ATPase.     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.     

Cordil Reversibly Inhibits the Na,K-ATPase from Outside of the Cell Membrane. Role of K-dependent Dephosphorylation

Abstract

Cordil-LND796 is a new cardiotonic glycoside under development. In rat brain microsomes where three isoforms of the Na, K-ATPase with differential affinities for cardiac glycosides have been identified, Cordil had higher affinity for the α₃ (IC₅₀ = 0.02 μM) than for the α₂ (IC₅₀ = 0.6 μM) and the α₁ (IC₅₀ = 30 μM) isozymes. Cordil is potentially a selective inhibitor for both α₂ and α₃ Na, K-ATPase isoforms. Using inside out vesicles we have shown that Cordil binds to and inhibits Na, K-ATPase at an extracellular site. The dissociation kinetic rates (k_?1) from the ATPase and the phosphatase activity (K-dependent dephosphorylation) of the Na, K-ATPase were similar for Cordil. Despite these similarities to ouabain comparison of the kinetics of the Na, K-ATPase inhibition by ouabain and Cordil revealed marked differences in their association rates (k₊₁ = 0.7 1 mol¹ min^?1 and k₊₁ = 6 × 10_?3 1 mol_?1 min_?1 respectively) and their dissociation rates (k_?1 = 1.3 ± 0.2 × 10^?4 S^?1 and k_?1 = 69 ± 7 × 10^?4 s^?1 respectively). Both binding association and dissociation rates were enhanced for Cordil. These data are compatible with a stabilizing effect of Cordil on the E₂P conformational state of Na, K-ATPase.     

Water transport in epididymal and ejaculated rhesus monkey (Macaca mulatta) sperm during freezing

In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macacamulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 °C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 μm with a radius of 0.40 μm and an osmotically inactive cell volume, V_b, of 0.772V_o, where V_o is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of ∼22 μm⁻¹. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L_pg or L_pg[cpa] and the activation energy, E_Lp or E_Lp[cpa]) were found to range from: L_pg or L_pg[cpa] = 0.0020-0.0029 μm/min-atm; E_Lp or E_Lp[cpa]) = 10.6-18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, ; where the units of B_opt are °C/min, E_Lp or E_Lp[cpa] are kcal/mole, L_pg or L_pg[cpa] are μm/min-atm and SA/WV are μm⁻¹), we determined the optimal rates of freezing macaque sperm to be ∼23 °C/min (ejaculated sperm in the absence of CPAs), ∼29 °C/min (ejaculated sperm in the presence of glycerol), ∼24 °C/min (epididymal sperm in the absence of CPAs) and ∼24 °C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal macaque spermatozoa under corresponding cooling conditions.     

Na,K-ATPase expression in C2C12 cells during myogenesis: Minimal contribution of α2 Isoform to Na,K transport

Summary Cells of the murine skeletal muscle line, C₂C₁₂, undergo differentiation from mononuclear myoblasts to multinuclear myotubes that express a number of proteins associated with striated muscle. We examined the relationship between the abundance of the mRNAs encoding the fast-twitch Ca-ATPase and the α isoforms of Na,K-ATPase and the subsequent expression of their respective polypeptides. Both the mRNA and protein levels of the αl isoform remained constant throughout differentiation. In contrast, the content of mRNAs encoding the α2 isoform and fasttwitch Ca-ATPase increased coordinately with the abundance of their corresponding polypeptides during myotube development. Despite the dramatic increase in α2 expression, estimates of in vitro Na,K-ATPase activity and assessments of in vivo transport activity suggest that α2 contributes little to ionic homeostasis in C₂C₁₂ myotubes.     

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces V_max and turnover rate and raises K_0.5Na. The phosphomimetic mutants reverse these effects and reduce K_0.5Na below control K_0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K_0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E₂(2K)ATP → E₁(3Na)ATP but does not affect 3NaE₁P → E₂P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60–70) docks between the αN and αP domains in the E₂ conformation, but docking is weaker in E₁ (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na⁺-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.     

Na,K-ATPase as A Brownian Motor: Electric Field-InducedConformational Fluctuation Leads to Uphill Pumping of Cation inthe Absence of ATP

Na,K-ATPase uses chemical bond energy of ATP to pump K⁺ into, andNa⁺ out of a cell. Both are uphill transports. During the catalyticcycle the enzyme alternates between two conformational states, E₁ andE₂. This communication describes an experiment, which employs electricfield to drive oscillation or fluctuation of enzyme conformation betweenthe E₁ and the E₂ states. It is shown that the field-inducedconformational oscillation or fluctuation leads to uphill pumping of thecation by the enzyme without consumption of ATP. Biochemical specificityof the catalysis is preserved. Data indicate that Na,K-ATPase can harvestenergy from the applied electric field to perform chemical work, and aratchet mechanism is inherent in this energy transduction process. ATheory of Electroconformational Coupling (TEC) that embodies essentialfeatures of the Brownian Ratchet successfully simulates the field-frequencyand field-amplitude optima and other features of the ion pumping activity.A four-state TEC motor can achieve high efficiency of the energytransduction, asymptotically reaching 100% under the optimal condition.Pumping by ion rectification fails to reach high efficiency. The TECconcept is also mused to understand other biological motors and engines.