Membrane integration of Na,K-ATPase alpha-subunits and beta-subunit assembly

The control of membrane insertion of polytopic proteins is still poorly understood. We carried out in vivo translation/insertion experiments in Xenopus oocytes with combined wild type or mutant membrane segments of the alpha-subunit of the heterodimeric Na, K-ATPase linked to a glycosylation reporter sequence. We confirm that the four N-terminal hydrophobic segments of the alpha-subunit behave as alternating signal anchor/stop transfer motifs necessary for two lipid-inserted membrane pairs. For the six C-terminal membrane segments, however, proper packing depends on specific sequence information and association with the beta-subunit. M5 is a very inefficient signal anchor sequence due to the presence of prolines and polar amino acids. Its correct membrane insertion is probably mediated by posttranslational hairpin formation with M6, which is favored by a proline pair in the connecting loop. M7 has partial signal anchor function, which may be mediated by the presence of glycine and glutamine residues. The formation of a transmembrane M7/M8 pair requires the association of the beta-subunit, which induces a conformational change in the connecting extracytoplasmic loop that favors M7/M8 packing. The formation of the M9/M10 pair appears to be predominantly mediated by the efficient stop transfer function of M10. Mutations that provide signal anchor function to M5, M7, and M9 abolish or impede the transport activity of the enzyme. These data illustrate the importance of specific amino acids near or within hydrophobic regions as well as of subunit oligomerization for correct topographical alignment that is necessary for proper folding and/or activity of oligomeric membrane proteins.     

Differential expression of Na,K-ATPase alpha-isoform mRNAs in aging rat cerebellum

Age-dependent changes in the expression of Na,K-ATPase alpha 1- and alpha 3-mRNAs were analyzed in the rat cerebellum by in situ hybridization. In young rats, alpha 1-mRNA showed prominent labeling in the granular layer (GL) with moderate fine distribution in the molecular layer (ML), Purkinje cell layer (PCL), and white matter (WM) but no clusters over Purkinje cells (PCs). In old rats, alpha 1-mRNA remained unchanged in ML and PCL, but declined by 43% (P < 0.0001) in GL and increased by 624% (P < 0.0001) in WM. alpha 3-mRNA in young rats showed large clusters of label on stellate, basket, Golgi, and PCs and fine grains diffusely in ML, GL, and WM. In old rats, alpha 3-mRNA declined by 87% in ML, 83% in PCL, 84% per PC, and 89% in GL and increased by 111% in WM (all values P < 0.0001) relative to young rats. PC numbers were reduced by 30%, but the average area of PC profiles did not change significantly. In old rats, the specific cluster-like label related to alpha 3-mRNA on PCs, stellate, basket, and Golgi cells was lost. Immunocytochemistry of cerebellum and hippocampus showed no age-related change in the distribution and density of total catalytic polypeptide. Thus, the discordance between changes in the levels of mRNAs in neuronal layers and WM in the face of constant polypeptide levels indicates age-related changes in polypeptide turnover. Cell- and isoform-specificity of alpha-isoform mRNAs in aging rat cerebellum may reflect differential regulation underlying age-related impairments in signal transduction and motor learning.     

Plasticity of Na,K-ATPase isoform expression in cultures of flat astrocytes: species differences in gene expression

The sella and parasellar region may be affected by a variety of disease states. Diseases of this region often result in visual disturbances because of the proximity of the sella to the optic pathways and cranial nerves. Knowledge of the pathological conditions affecting the sella and surrounding structures is important for the orbital imager.     

E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.1 s-1 at 20 degreesC. However, whereas dephosphorylation of E2P formed from ATP was strongly stimulated by K+, dephosphorylation of E'2P formed from Pi in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'2P could be demonstrated with RH421 fluorescence. It appears, therefore, that in pig kidney enzyme the rapid binding of K+ to E'2P was followed by a slow transition to a nonfluorescent form. For shark enzyme the K+-induced decrease of RH421 fluorescence of Pi phosphorylated enzyme was due to K+ binding to the dephosphoenzyme (E1), thus shifting the equilibrium away from E'2P. When Pi phosphorylation was performed with enzyme equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Na+ or Li+, both the phosphorylation and the dephosphorylation rates were considerably increased. This indicates that binding of cations modifies the substrate site in a cation-specific way, suggesting an allosteric interaction between the conformation of the cation-binding sites and the phosphorylation site of the enzyme.     

Influence of inhaled nitric oxide and hyperoxia on Na,K-ATPase expression and lung edema in newborn piglets

This study was undertaken to examine the combined effect of nitric oxide (NO) and hyperoxia on lung edema and Na,K-ATPase expression. Newborn piglets were exposed to room air (FiO2 = 0.21), room air plus 50 ppm NO, hyperoxia (FiO2 >/= 0.96) or to hyperoxia plus 50 ppm NO for 4-5 days. Animals exposed to NO in room air experienced only a slight decrease in Na,K-ATPase alpha subunit protein level. Hyperoxia, in the absence of NO, induced both the mRNA and the protein level of Na,K-ATP-ase alpha subunit and significantly increased wet lung weight, extravascular lung water, and alveolar permeability. NO in hyperoxia decreased the hyperoxic-mediated induction of Na,K-ATPase alpha subunit mRNA and protein while wet lung weight, extravascular lung water, and alveolar permeability remained elevated. These results suggest that 50 ppm of inhaled NO may not improve hyperoxic-induced lung injury and may interfere with the expression of Na,K-ATPase which constitutes a part of the cellular defense mechanism against oxygen toxicity.     

Tissue-specific distribution and modulatory role of the gamma subunit of the Na,K-ATPase

The Na,K-ATPase comprises a catalytic alpha subunit and a glycosylated beta subunit. Another membrane polypeptide, gamma, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with alpha and beta in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-gamma antiserum to define the tissue specificity and topology of gamma and to address the question of whether gamma has a functional role. The trypsin sensitivity of the amino terminus of the gamma subunit in intact right-side-out pig kidney microsomes has confirmed that it is a type I membrane protein with an extracellular amino terminus. Western blot analysis shows that gamma subunit protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells, alpha1-transfected HeLa cells, all derived from the same (rat) species, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain insight into gamma function, the effects of the anti-gamma serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that gamma stabilizes E1 conformation(s) of the enzyme and that anti-gamma counteracts this effect: (i) anti-gamma inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E2(K) --> E1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-gamma indicating that the antibody shifts the E1 left and right arrow left and right arrow E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+ concentration reduced to levels ( --> E2P transition, anti-gamma is stimulatory. These observations taken together with evidence that the pig gamma subunit, which migrates as a doublet on polyacrylamide gels, is sensitive to digestion by trypsin, and that Rb+ ions partially protect it against this effect, indicate that the gamma subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E1. Accordingly, binding of anti-gamma disrupts alphabeta-gamma interactions and counteracts these modulatory effects of the gamma subunit.     

The gamma subunit of the Na,K-ATPase induces cation channel activity

The gamma subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The gamma subunit was expressed in Sf-9 insect cells and Xenopus oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the gamma subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human gamma subunit. Confocal microscopy demonstrates that the gamma subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that gamma colocalizes with alpha1 when these proteins are coexpressed. When Sf-9 cells were coinfected with alpha1 and gamma, antibodies to the gamma subunit were able to coimmunoprecipitate the alpha1 subunit, suggesting that gamma is able to associate with alpha1. The gamma subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in Xenopus oocytes. Evidence that the gamma subunit is a functional component was supported by experiments showing gamma-induced cation channel activity when expressed in oocytes and increases in Na+ and K+ uptake when expressed in Sf-9 cells.     

The role of Na,K-ATPase inhibitors in hypertension and end-stage renal disease

In Part I of this two-part study, the authors investigated heat production during osteotomy drilling at three different speeds, and determined that high-speed drilling produced the least heat when using 700 XL carbide burs. Part II of the study histologically examines the rate and quality of healing after drilling osteotomies at the three speeds in the mandible. Osteotomies were histologically examined 2, 4, and 6 weeks postoperatively. Histologic findings suggested that in the initial 6 weeks, the rate of healing and quality of new bone formation were higher after high-speed drilling than after low- or intermediate-speed drilling. These results, when considered with the results reported in Part I in which a 4.3 degrees C difference in heat production was observed between the speeds, seem to imply a relationship between heat production and healing for osteotomy drilling.     

Dynamics of Na,K-ATPase sites in lateral cochlear wall tissues of the rat

In order to study vertebral fractures in various study populations, we earlier prepared a database of vertebral dimensions derived from spinal radiographs of 191 normal women seen regularly over 25 years. In this report we have expanded the range of measurements to include vertebral levels T3 to L5. We report means and standard deviations on anterior and posterior heights, on wedge shape and on heights relative to adjacent vertebrae. When one or both of the latter two quantities are 'far' below the mean, a vertebra is called deformed. We also describe a more flexible way of expressing damage using the number of deformed vertebrae, the degree of deformity of individual vertebrae, or the total damage to the entire spine. In assessing damage we use criteria for deformity adjusted to the limits detected by an experienced diagnostician, replacing an earlier approach based on 95% probability limits of normal variation. The normal women from whom these variations are ascertained are a low-prevalence group with respect to vertebral deformity, with prevalence of 2.8%. When the criteria developed from these women were applied to a moderate-prevalence group (37%) the model had a sensitivity of 97%, a specificity of 89% and an accuracy of 92% as regards the identification of subjects with damaged vertebrae. When used epidemiologically for a moderate-prevalence group the model has a known overestimation of 15%. the model is compared with other schemes for identifying vertebral deformities.     

Inhibition of epithelial Na+ currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator

Mucormycosis historically has caused substantial morbidity with high mortality in renal transplant patients with disseminated and/or rhinocerebral infection and in patients with gastrointestinal illness regardless of predisposing conditions. We report the first successful treatment of gastric mucormycosis in a renal transplant recipient and review presumed pathogenic mechanisms of mucormycosis in renal transplant recipients as well as historical data.     

Zebrafish vimentin: molecular characterization, assembly properties and developmental expression

In order to determine both clinical and spirometric changes due to high environmental concentrations of wheat dust at a wheat processing plant mill, 48 exposed men and 48 age and antroprometrically-matched, non-exposed apparently healthy men were studied. In both groups a medical and occupational history were taken, and spirometric measurements were carried out, that included Forced Vital Capacity (FVC), Forced Expiratory Volume at the first second (FEV1), Peak Flow Rate (PFR), Forced Percentual Expiratory Volume (FEV%), Forced Percentual Vital Capacity (FVC%), Forced Expiratory Flow at 25% (FEV25%), at 50% (FEV50%) and at 75% (FEV75%) of their Forced Vital Capacity, which were analyzed through Corzo's predictive equations and the lung deterioration's criteria by USA's Thoracic Association. The environmental wheat dust was determined by gravimetry and its concentration was higher than the legally admitted (3/5, 60%). There was a decrease in the PFR, FEV%, FEV25% and FEV75%. (p < 0.05). In addition, 4 restrictive and 1 obstructive syndrome were detected in the exposed workers and none in the control group. The spirometric values diminished in a positive correlation with the time of exposure and smoking habits. There was no correlation between the clinical findings and the dust concentration but it did exist with the spirometric values. It is concluded that in this plant, the wheat dust exposed workers have a diminished spirometric values.     

Changes in steady-state conformational equilibrium resulting from cytoplasmic mutations of the Na,K-ATPase alpha-subunit

Mutations comprising either deletion of 32 amino acids from the NH2 terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase result in a shift in the steady-state E1 left arrow over right arrow E2 conformational equilibrium toward E1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant alpha1M32E233K double mutant, growth was markedly reduced unless the K+ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (approximately 10-fold) K+ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in Vmax and a compensatory approximately 3-fold increase in expressed alpha1M32E233K protein. In contrast to the behavior of either alpha1M32 or E233K, alpha1M32E233K also showed alterations in apparent cation affinities. K'Na was decreased approximately 2-fold and K'K was increased approximately 2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233 substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.     

Synthesis, assembly, and intracellular transport of the platelet glycoprotein Ib-IX-V complex

The platelet glycoprotein Ib-IX-V complex plays critical roles in adhering platelets to sites of blood vessel injury and in platelet aggregation under high fluid shear stress. The complex is composed of four membrane-spanning polypeptides: glycoprotein (GP) Ibalpha, GP Ibbeta, GP IX, and GP V. Glycoprotein Ibalpha contains a binding site for von Willebrand factor through which it mediates platelet adhesion; GP V is required for the complex to bind thrombin with high affinity; and both GP Ibbeta and GP IX are necessary for efficient plasma membrane expression of the complex. To further define the roles of the individual polypeptide subunits in the biosynthesis and intracellular transport of the GP Ib-IX-V complex, we studied full and partial complexes expressed in heterologous mammalian cells. We found that the full complex was formed within minutes in the endoplasmic reticulum before being transported into the Golgi cisternae. Approximately 160 min were required for the complex to be fully processed and to appear on the plasma membrane. About 25% of GP Ibalpha expressed as part of either a GP Ib-IX complex or a GP Ib-IX-V complex was degraded through a nonlysosomal pathway. Over 60% of GP Ibalpha, however, was degraded when it was expressed in partial complexes with only GP Ibbeta or GP IX. The increased degradation was blocked by treating cells either with brefeldin A to prevent the transport of proteins from the endoplasmic reticulum to the Golgi or with lysosomal inhibitors, indicating that GP Ibalpha expressed in partial complexes was targeted to the lysosomes for degradation. These results indicate that the presence of both GP Ibbeta and GP IX, but not the presence of GP V, is required for efficient processing and targeting of GP Ibalpha to the plasma membrane. Absence of either GP Ibbeta or GP IX increased the rate of GP Ibalpha degradation, providing an explanation for why mutation of their genes leads to deficient GP Ibalpha expression and platelet adhesion in Bernard-Soulier syndrome, the deficiency disorder of the complex.     

Chronic K-supplementation decreases myocardial [Na,K-ATPase] and net K-uptake capacity in rodents

The effects of high K intake on plasma K, myocardial K content and Na,K-ATPase concentration and on myocardial K uptake during KCl infusion were evaluated in rodents. Myocardial Na,K-ATPase was quantified in crude homogenates by K-dependent pNPPase activity in rats, and in intact samples by3H-ouabain binding in guinea pigs. Na, K-ATPase alpha isoform distribution was assessed by immunoblotting. Plasma K was monitored in anesthetized rats during intravenous infusion of 0.75 mmol KCl/100 g body weight/h. A significant increase in plasma K was observed after 2 days of K supplementation, 4.9+/-0.2 (mean+/-s.e.m.)v 3.0+/-0.2 mmol/l in weight matched controls ( P<0.01,n=5) and this difference remained stable. After 1 day, a significant myocardial K content increase was obtained, 86. 2+/-3.0v 76.7+/-1.9 micromol/g wet weight (P<0.05, n=5); after 4 days myocardial K stabilized 4.9+/-1.2 micromol/g wet weight above control level (P<0.05,n=5). From the 4th day, a significant decrease in myocardial K-dependent pNPPase activity was observed, 1.18+/-0.04v 1. 31+/-0.01 micromol/min/g wet weight in weight matched controls (P<0. 05,n=5); after 2 weeks the decrease was 29% (P<0.05,n=5), with a reduction in alpha1-isoform abundance by 24% (P<0.05,n=5), and a tendency to a decrease in alpha2 of 10% (n.s.,n=5). The measurements were validated by 3H-ouabain binding to myocardial samples from guinea pigs K-supplemented for 2 weeks, showing a decrease of 21% (P<0.05,n=5). During KCl infusion, the myocardial K content increase rate was reduced by 52% (P<0.05) in the K-supplemented rats. The observed effects of K-supplementation on plasma K, myocardial K content and myocardial K-dependent pNPPase activity were abolished within 2 days after reallocation to chow with normal K content. In conclusion, high K-intake is associated with significantly and reversible increased plasma and myocardial K content, and decreased myocardial Na,K-ATPase concentration and net myocardial K uptake capacity. Thus, the heart is protected from major increases in intracellular K concentrations during chronically-high K-intake.     

Cell cycle-dependent and kinase-specific regulation of the apical Na/H exchanger and the Na,K-ATPase in the kidney cell line LLC-PK1 by calcitonin

Calcitonin (CT), which regulates serum calcium through its actions in bone and the kidney tubule, also has a potent natriuretic effect in vivo. Na reabsorption in the proximal kidney tubule is mostly dependent on the activity of the Na,K-ATPase and the apical Na/H exchanger. We have previously shown that CT regulates the activity of the Na,K-ATPase in the proximal kidney tubule cell line LLC-PK1 in a cell cycle-dependent manner. We report here that, in the same cells, CT also regulates the Na/H exchanger through a cell cycle-specific activation of the Ca/calmodulin-dependent protein kinase II. In G2 phase, no changes in ethylisopropyl amiloride-sensitive 22Na uptake is observed, despite an increase in cAMP. In contrast, the hormone inhibits the apical exchanger when the cells are in S phase, resulting in an 80% inhibition of 22Na uptake. These results demonstrate that CT affects the activity of the two major proximal tubule Na transport systems and may help clarify the mechanisms by which CT regulates Na+ reabsorption.     

Rat and human glutamate transporter GLAST1 stable heterologous expression, biochemical and functional characterization

Human embryonic kidney cell lines (HEK293) which express heterologously and permanently the human and rat GLAST1 (high affinity, Na(+)-dependent, CNS-specific L-glutamate transporter) have been established by the transfer of the two minigenes under the control of the cytomegalovirus promoter by electroporation. The transfected HEKh GLAST1 (human) and HEKrGLAST1 (rat) cell lines strongly express the glutamate uptake system which exhibits all biochemical and electrophysiological properties determined so far in the transiently expressing Xenopus oocyte system except the K+ dependence. These cell lines are a valuable tool for further biochemical, physiological, and pharmacological studies on this uptake system of the most important excitatory neurotransmitter.     

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.