Insect Na(+)/K(+)-ATPase

Na(+)/K(+)-ATPase (sodium/potassium pump) is a P-type ion-motive ATPase found in the plasma membranes of animal cels. In vertebrates, the functions of this enzyme in nerves, heart and kidney are well characterized and characteristics a defined by different isoforms. In contrast, despite different tissue distributions, insects possess a single isoform of the alpha-subunit. A comparison of insect and vertebrate Na(+)/K(+)-ATPases reveals that although the mode of action and structure are very highly conserved, the specific roles of the enzyme in most tissues varies. However, the enzyme is essential for the function of nerve cells, and in this respect Na(+)/K(+)-ATPase appears to be fundamental in metazoan evolution.     

Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P.     

Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.     

V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 immunoreactivity in the gill epithelium of the Pacific hagfish (Epatretus stoutii)

We report the presence of the ion transporting proteins V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in the gill epithelium of the Pacific hagfish Epatretus stoutii. Heterologous antibodies recognized single bands of the appropriate sizes for the three transporters in western blots. Immunohistochemical staining revealed that the distribution of labeled cells in the gill epithelium was identical for the three proteins. Immunopositive cells were most abundant in the primary filament from the afferent side of the gill pouch, and their number diminished towards the lamella. Na(+)/K(+)-ATPase-like immunoreactivity (L-IR) occurred throughout the cell cytoplasm, probably associated to the basolateral tubular system. V-H(+)-ATPase L-IR was similar to Na(+)/K(+)-ATPase, although some cells had slightly heavier staining in either the supra- or infra-nuclear region. NHE2 L-IR was also generally cytoplasmic, but a minority of the cells had stronger immunoreactivity in the apical region. In general, all three ion transporting proteins were localized in the same cells, as estimated from 4-microm immunostained consecutive sections. We hypothesize that these putative ion-transporting cells are involved in systemic acid/base regulation and discuss other possible roles. This is the first report of V-H(+)-ATPase in myxinoids, and the first NHE2 report in the Pacific hagfish.     

用荧光标记钠钾ATP酶显示气道感受器结构

由于普通光学和荧光显微镜功能上的限制以及缺乏良好的染色方法,我们对于呼吸道感受器形态方面的认识进展缓慢,缺乏对感受器结构的认识,阻碍了对其基本生理活动的探讨,随着共聚焦显微镜的产生与图像处理技术的进展,不但提高了对荧光结构的分辨率,还可通过三维重组而展现微小物体的整体结构。本文阐述了一种新颖方法,利用上述技术并结合免疫组织化学方法,采用Na^ /K^ -ATPase作为标记,对家兔呼吸道感受器进行了观察。本文中经过处理的气道组织结构,背景清晰,感受器部位着色强,观察到的整个感受器,结构复杂,呈树枝状,感受器末梢膨大,形成叶片状,本文首次以高清晰度展示了呼吸气道中单个感受器的整体结构,改变了需要以手绘来刻画感受器的现状,无疑,这种新方法能促进了解感受器的结构及其生理活动的机理。     

Structural basis of Na(+)/K(+)-ATPase adaptation to marine environments

Throughout evolution, enzymes have adapted to perform in different environments. The Na(+)/K(+) pump, an enzyme crucial for maintaining ionic gradients across cell membranes, is strongly influenced by the ionic environment. In vertebrates, the pump sees much less external Na(+) (100-160 mM) than it does in osmoconformers such as squid (450 mM), which live in seawater. If the extracellular architecture of the squid pump were identical to that of vertebrates, then at the resting potential, the pump's function would be severely compromised because the negative voltage would drive Na(+) ions back to their binding sites, practically abolishing forward transport. Here we show that four amino acids that ring the external mouth of the ion translocation pathway are more positive in squid, thereby reducing the pump's sensitivity to external Na(+) and explaining how it can perform optimally in the marine environment.     

Inhibitors of Na(+)/H(+) and Na(+)/Ca(2+) exchange potentiate methamphetamine-induced dopamine neurotoxicity: possible role of ionic dysregulation in methamphetamine neurotoxicity

Although the neurotoxic potential of methamphetamine (METH) is well established, underlying mechanisms have yet to be identified. In the present study, we sought to determine whether ionic dysregulation was a feature of METH neurotoxicity. In particular, we reasoned that if METH impairs the function of Na(+)/H(+) and/or Na(+)/Ca(2+) antiporters by compromising the inward Na(+) gradient [via prolonged DA transporter (DAT) activation and Na(+)/K(+) ATPase inhibition], then amiloride (AMIL) and other inhibitors of Na(+)/H(+) and/or Na(+)/Ca(2+) exchange would potentiate METH neurotoxicity. To test this hypothesis, mice were treated with METH alone or in combination with AMIL or one of its analogs; 1 week later, the animals were killed for studies of dopamine (DA) neuronal integrity. AMIL markedly potentiated the toxic effect of METH on DA neurons. Potentiation was not caused by increased core temperature, enhanced DAT activity or higher METH brain levels. The DAT inhibitor, WIN-35,428, protected completely against METH-induced DA neurotoxicity in AMIL pretreated animals, suggesting that the potentiating effects of AMIL require a METH/DAT interaction. Findings with METH and AMIL were extended to six other AMIL analogs (MIA, EIPA, DIMA, BENZ, BEP, DiCBNZ), another species (rats), and neuronal type (5-HT neurons). These results support the notion that ionic dysregulation may play a role in METH neurotoxicity.     

Eight amino acids form the ATP recognition site of Na(+)/K(+)-ATPase

Point mutations of a part of the H(4)-H(5) loop (Leu(354)-Ile(604)) of Na(+)/K(+)-ATPase have been used to study the ATP and TNP-ATP binding affinities. Besides the previously reported amino acid residues Lys(480), Lys(501), Gly(502), and Cys(549), we have found four more amino acid residues, viz., Glu(446), Phe(475), Gln(482), and Phe(548), completing the ATP-binding pocket of Na(+)/K(+)-ATPase. Moreover, mutation of Arg(423) has also resulted in a large decrease in the extent of ATP binding. This residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site, probably due to formation of a hydrogen bond with Glu(472). On the other hand, only some minor effects were caused by mutations of Ile(417), Asn(422), Ser(445), and Glu(505).     

Ca(2+)-dependent K(+) channels and Na(+)-K(+)-ATPase mediate H(2)O(2)- and superoxide-induced relaxations in canine trachealis.

We examined the ionic mechanisms underlying the responses of canine trachealis to superoxide (generated in vitro by using xanthine oxidase or added exogenously) and peroxide (generated spontaneously in vitro by the dismutation of superoxide or added exogenously). Although neither had any effect on resting tone, both triggered relaxations in carbachol-precontracted tissues. These relaxations were eliminated by catalase but were much less sensitive to the hydroxyl radical scavenger dimethylthiourea, indicating they were mediated primarily by peroxide. These relaxations were decreased in magnitude and/or slowed by nifedipine (10(-6) M), ouabain (10(-6) M), or tetraethylammonium (25 mM), but not by 4-aminopyridine (5 mM), and were small or absent in tissues precontracted with 30 mM KCl. Finally, peroxide triggered membrane hyperpolarization and elevated cytosolic concentration of Ca(2+) (primarily via release from the internal store). Thus peroxide-mediated relaxations seem to involve Ca(2+) release, opening of Ca(2+)-dependent K(+) channels, hyperpolarization, closure of Ca(2+) channels, and relaxation. In addition, some other free radical (hydroxyl radical?) may activate the Na(+)-K(+) pump, also hyperpolarizing the membrane and causing relaxation.     

Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons

To determine the role ofmembrane transporters in intracellular pH (pH_i) regulationunder conditions of low microenvironmental O₂, we monitoredpH_i in isolated single CA1 neurons using the fluorescentindicator carboxyseminaphthorhodafluor-1 and confocal microscopy. Aftertotal O₂ deprivation or anoxia (PO₂ 0 Torr), a large increase in pH_i was seen in CA1neurons in HEPES buffer, but a drop in pH_i, albeit small,was observed in the presence of HCO. Ionicsubstitution and pharmacological experiments showed that the largeanoxia-induced pH_i increase in HEPES buffer was totallyNa⁺ dependent and was blocked by HOE-694, stronglysuggesting the activation of the Na⁺/H⁺exchanger (NHE). Also, this pH_i increase in HEPES bufferwas significantly smaller in Na⁺/H⁺ exchangerisoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons,demonstrating that NHE1 is responsible for part of the pH_iincrease following anoxia. Both chelerythrine and H-89 partly blocked,and H-7 totally eliminated, this anoxia-induced pH_iincrease in the absence of HCO. We conclude that1) O₂ deprivation activatesNa⁺/H⁺ exchange by enhancing protein kinaseactivity and 2) membrane proteins, such as NHE, activelyparticipate in regulating pH_i during low-O₂states in neurons.

     

Ifenprodil restores GDNF-evoked Ca(2+) signalling and Na(+)/K(+) -ATPase expression in inflammation-pretreated astrocytes

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in neuroinflammatory and neuropathic pain conditions. Astrocytes produce and secrete GDNF, which interacts with its receptors to induce Ca(2+) transients. This study aimed first to assess intracellular Ca(2+) responses of astrocytes in primary culture when exposed to the neuroprotective and anti-inflammatory peptide GDNF. Furthermore, incubation with the inflammatory inducers lipopolysaccharide (LPS), NMDA, or interleukin 1-β (IL-1β) attenuated the GDNF-induced Ca(2+) transients. The next aim was to try to restore the suppressed GDNF responses induced by inflammatory changes in the astrocytes with an anti-inflammatory substance. Ifenprodil, an NMDA receptor antagonist at the NR2B subunit, was tested. It was shown to restore the GDNF-evoked Ca(2+) transients and increased the Na(+)/K(+) -ATPase expression. Ifenprodil seems to be a potent anti-inflammatory substance for astrocytes which have been pre-activated by inflammatory stimuli.     

Differential MAP kinase activation and Na(+)/H(+) exchanger phosphorylation by H(2)O(2) in rat cardiac myocytes

Bursts in reactive oxygen species productionare important mediators of contractile dysfunction duringischemia-reperfusion injury. Cellular mechanisms that mediatereactive oxygen species-induced changes in cardiac myocyte functionhave not been fully characterized. In the present study,H₂O₂ (50 µM) decreased contractility of adultrat ventricular myocytes. H₂O₂ caused aconcentration- and time-dependent activation of extracellularsignal-regulated kinases 1 and 2 (ERK1/2), p38, and c-JunNH₂-terminal kinase (JNK) mitogen-activated protein (MAP)kinases in adult rat ventricular myocytes. H₂O₂ (50 µM) caused transient activation of ERK1/2 and p38 MAP kinase thatwas detected as early as 5 min, was maximal at 20 min (9.6 ± 1.2- and 9.0 ± 1.6-fold, respectively, vs. control), and returned tobaseline at 60 min. JNK activation occurred more slowly (1.6 ± 0.2-fold vs. control at 60 min) but was sustained at 3.5 h. Theprotein kinase C inhibitor chelerythrine completely blocked JNKactivation and reduced ERK1/2 and p38 activation. The tyrosine kinaseinhibitors genistein and PP-2 blocked JNK, but not ERK1/2 and p38,activation. H₂O₂-inducedNa⁺/H⁺ exchanger phosphorylation was blocked bythe MAP kinase kinase inhibitor U-0126 (5 µM). These resultsdemonstrate that H₂O₂-induced activation of MAPkinases may contribute to cardiac myocyte dysfunction duringischemia-reperfusion.

     

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Physiological screening for target site insensitivity and localization of Na(+)/K(+)-ATPase in cardenolide-adapted Lepidoptera

Cardenolides are toxic plant compounds which specifically inhibit Na(+)/K(+)-ATPase, an animal enzyme which is essential for many physiological processes, such as the generation of action potentials. Several adapted insects feeding on cardenolide-containing plants sequester these toxins for their own defence. Some of these insects were shown to possess Na(+)/K(+)-ATPases with a reduced sensitivity towards cardenolides (target site insensitivity). In the present study we screened five species of arctiid moths feeding on cardenolide-containing plants for target site insensitivity towards cardenolides using an in vitro enzyme assay. The derived dose response curves of the respective Na(+)/K(+)-ATPases were compared to the insensitive Na(+)/K(+)-ATPase of the monarch butterfly (Danaus plexippus). Na(+)/K(+)-ATPases of all arctiid species tested were highly sensitive to ouabain, a water-soluble cardenolide which is most widely used in laboratory studies. Nevertheless, we detected substantial amounts of cardenolides in the haemolymph of two of the arctiid species. In caterpillars of the sequestering arctiid Empyreuma pugione and of D. plexippus we localized Na(+)/K(+)-ATPase by immunohistochemistry and western blot (in D. plexippus). Both techniques revealed strong expression of the enzyme in the nervous tissue and indicated weak expression or even absence in other tissues tested. We conclude that instead of target site insensitivity the investigated arctiid species use a different strategy to tolerate cardenolides. Most plausibly, the perineurium surrounding the nervous tissue functions as a barrier which prevents cardenolides from reaching Na(+)/K(+)-ATPase in the ventral nerve cord.     

Are the states that occlude rubidium obligatory intermediates of the Na(+)/K(+)-ATPase reaction?

In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.     

Assembly of a hybrid from the alpha subunit of Na+/K(+)-ATPase and the beta subunit of H+/K(+)-ATPase.

Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.