Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents

Summary Nonstationary pump currents which have been observed in K⁺-free Na⁺ media after activation of the Na,K-ATPase by an ATP-concentration jump (see the preceding paper) are analyzed on the basis of microscopic reaction models. It is shown that the behavior of the current signal at short times is governed by electrically silent reactions preceding phosphorylation of the protein; accordingly, the main information on charge-translocating processes is contained in the declining phase of the pump current. The experimental results support the Albers-Post reaction scheme of the Na,K-pump, in which the translocation of Na⁺ precedes translocation of K⁺. The transient pump current is represented as the sum of contributions of the individual transitions in the reaction cycle. Each term in the sum is the product of a net transition rate times a dielectric coefficient describing the amount of charge translocated in a given reaction step. Charge translocation may result from the motion of ion-binding sites in the course of conformational changes, as well as from movement of ions in access channels connecting the binding sites to the aqueous media. A likely interpretation of the observed nonstationary currents consists in the assumption that the principal electrogenic step is the E₁-P/P-E₂ conformational transition of the protein, followed by a release of Na⁺ to the extracellular side. This conclusion is supported by kinetic data from the literature, as well as on the finding that chymotrypsin treatment which is known to block the E₁-P/P-E₂ transition abolishes the current transient. By numerical simulation of the Albers-Post reaction cycle, the proposed mechanism of charge translocation has been shown to reproduce the experimentally observed time behavior of pump currents.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Conformational transitions and charge translocation by the Na,K pump: Comparison of optical and electrical transients elicited by ATP-concentration jumps

Summary The electrogenic properties of the Na,K-ATPase were studied by correlating transient electrical events in the pump molecule with conformational transitions elicited by an ATP-concentration jump. Flat membrane fragments containing a high density (8000 m^–2) of oriented Na,K-ATPase molecules were bound to a planar lipid bilayer acting as a capacitive electrode. ATP was released in the medium from a photolabile inactive ATP derivative (caged ATP) by a 40-sec light flash. Electrical signals resulting from transient charge movements in the protein under single-turnover conditions were recorded in the external measuring circuit. In parallel experiments carried out under virtually identical conditions, the fluorescence of membrane fragments containing Na,K-ATPase with covalently-bound 5-iodoacetamido-fluorescein (5-IAF) was monitored after the ATP-concentration jump. When the medium contained Na⁺, but no K⁺, the fluorescence of the 5-IAF-labeled protein decreases monotonously after release of ATP. In the experiments with membrane fragments bound to a planar bilayer, a transient pump current was observed which exhibited virtually the same time behavior as the fluorescence decay. This indicates that optical and electrical transients are governed by the same rate-limiting reaction step. Experiments with chymotrypsin-modified Na,K-ATPase suggest that both the fluorescence change as well as the charge movement are associated with the deocclusion of Na⁺ and release to the extracellular side. In experiments with Na⁺-free K⁺ media, a large inverse fluorescence change is observed after the ATP-concentration jump, but no charge translocation can be detected. This indicates that deocclusion of K⁺ is an electrically silent process.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Neurochemical Studies on the Cerebellar Hypoplasia of Gunn Rat (Hereditary Hyperbilirubinemic Rat)

The cerebellar hypoplasia induced by hereditary hyperbilirubinemia in the Gunn rat was analyzed neurochemically and immunohistochemically. The antiserum against myelin basic protein was used to visualize the arborization of the fibers in the cerebellum. Arborization was very scarce in the affected lobes of the homozygous (jj) cerebellum. Na,K-ATPase activity did not show significant differences between the jj and the control (Jj) cerebellum. The concentration of norepinephrine in the jj cerebellum was about 1.5 times that of the control. However, the activation ratio of the Na,K-ATPase by norepinephrine and other catecholamines such as dopamine and isoproterenol was about twice as high as the basal activity, and no significant difference was observed between the jj and the Jj cerebella. The glutamic acid decarboxylase activity of the jj cerebellum did not differ significantly from that of the control.     

Partial Inactivation of Na,K-ATPase in Cortical Brain Slices Incubated in Normal Krebs-Ringer Phosphate Medium at 1 and at 10 Atm Oxygen Pressures

Na,K-ATPase (ATP phosphohydrolase EC 3.6.1.3) activity was determined in homogenates of cortical brain slices after incubation in normal Krebs-Ringer phosphate medium at 1 atm oxygen pressure. After 10 min of incubation Na,K-ATPase activity was reduced by approximately 50%. Longer incubation did not cause further change in activity. The presence of 0.1 mM-MnCl2 in the medium offered significant protection, while an excursion to 10 atm oxygen pressure caused further inactivation. Measurements of malonaldehyde levels suggest that the inhibition of Na,K-ATPase is a result of lipid peroxidation. The evidence indicates that brain slices incubated under standard conditions suffer considerable oxidative damage.     

A Structural View on the Functional Importance of the Sugar Moiety and Steroid Hydroxyls of Cardiotonic Steroids in Binding to Na,K-ATPase

The Na,K-ATPase is specifically inhibited by cardiotonic steroids (CTSs) like digoxin and is of significant therapeutic value in the treatment of congestive heart failure and arrhythmia. Recently, new interest has arisen in developing Na,K-ATPase inhibitors as anticancer agents. In the present study, we compare the potency and rate of inhibition as well as the reactivation of enzyme activity following inhibition by various cardiac glycosides and their aglycones at different pH values using shark Na,K-ATPase stabilized in the E2MgP_i or in the E2BeF_x conformations. The effects of the number and nature of various sugar residues as well as changes in the positions of hydroxyl groups on the β-side of the steroid core of cardiotonic steroids were investigated by comparing various cardiac glycoside compounds like ouabain, digoxin, digitoxin, and gitoxin with their aglycones. The results confirm our previous hypothesis that CTS binds primarily to the E2-P ground state through an extracellular access channel and that binding of extracellular Na⁺ ions to K⁺ binding sites relieved the CTS inhibition. This reactivation depended on the presence or absence of the sugar moiety on the CTS, and a single sugar is enough to impede reactivation. Finally, increasing the number of hydroxyl groups of the steroid was sterically unfavorable and was found to decrease the inhibitory potency and to confer high pH sensitivity, depending on their position on the steroid β-face. The results are discussed with reference to the recent crystal structures of Na,K-ATPase in the unbound and ouabain-bound states.     

Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.