Amyloid beta-peptide inhibits Na+-dependent glutamate uptake

The purpose of this review is to summarize much of the work on the inhibition of the astroglial glutamate transporter in relation to excitotoxic neurodegeneration, in particular, inhibition of uptake by the beta-amyloid peptide (A beta) found in the Alzheimer's disease (AD) brain. There is evidence for oxidative stress in the AD brain, and A beta has been found to generate reactive oxygen species (ROS), thus adding to the stress or possibly initiating it. The oxidative inhibition of the glutamate transporter protein by A beta increases the vulnerability of glutamatergic neurons, and by rendering them susceptible to the excitotoxic insult that results from impaired glutamate uptake, A beta can be directly connected to the neurodegeneration that follows.     

Mutational analysis of Glu-327 of Na+-K+-ATPase reveals stimulation of 86Rb+ uptake by external K+

A competition assay of⁸⁶Rb⁺uptake in HeLa cells transfected with ouabain-resistantNa⁺-K⁺-ATPasemutants revealed a stimulation of⁸⁶Rb⁺uptake at low external concentrations (1 mM) of competitor(K⁺). Of the models that weretested, those that require that two K⁺ be bound before transportoccurs gave the worst fits. Random and ordered binding schemesdescribed the data equally well. General models in which both bindingand transport were allowed to be cooperative yielded parameter errorslarger than the parameters themselves and could not be utilized. Modelsthat assumed noncooperative transport always showed positivecooperativity in binding. E327Q and E327L mutated forms of rat₂ had lower apparent affinities for the first K⁺ bound than didwild-type rat ₂ modified to beouabain resistant. The mutations did not affect the apparent affinityof the second K⁺ bound. Modelsthat assumed noncooperativity in binding always showed positivelycooperative transport, i.e., enzymes with two K⁺ bound had a higher flux thanthose with one K⁺ bound. Increasesin external Na⁺ decreased theapparent affinity for K⁺ for allmodels and decreased the ratio of the apparent influx rate constantsfor E327L.

     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

The dependence on energy and K+ ions of the accumulation of glutamate by rat liver mitochondria

The time courses of the changes of glutamate content in rat liver mitochondria have been followed after either removing the energy supply or applying an energy supply. When the energy supply is inhibited, or when energy is dissipated by uncoupling, the glutamate is lost in a few minutes. The poison avenaciolide also causes glutamate to be lost. Uptake of glutamate requires an energy supply and proceeds as a first-order process with rate constants 0.059 min^?1 at 10 °C, 0.16 min^?1 at 20 °C, and 0.30 min^?1 at 30 °C. The glutamate distribution between mitochondria and medium in presence of an energy supply approaches the distribution holding for pyruvate. The uptake of glutamate appears to have a requirement for K⁺ ions in the medium.     

The effects of external K+ and Na+ on the chemotaxis of rabbit peritoneal neutrophils.

Extracellular K+ enhances the chemotactic responsiveness and spontaneous movememt of rabbit peritoneal neutrophils but is not required for these functions. Other monovalent cations act the same; the rank order of their effectiveness is K+ = NH4 greater than Rb+ Cs+ greater than Li = Na. The K+ specific ionophore, valinomycin (10-7 M) inhances chemotaxis in the presence of K+ but not in its absence; another K+ specific ionophore, nigericin (10-7 M) inhibits chemotaxis in the absence of K+ but not in its presence. Ouabain (5 x 10-6 M) prevents the enhancing effects of K+ on chemotaxis. Removing the Na+ of the buffer and substituting it with K+, choline or glucose greatly enhances spontaneous motility but depresses chemotactic activity. One hypothesis suggested by the above results is that as a part of their action, chemotactic factors stimulate a net influx of K+ into the neutrophil; an alternative or additional hypothesis is that chemotactic factors stimulate a net efflux of Na+ from the neutrophil.     

Relief of Na+ block of Ca2+-activated K+ channels by external cations

The flickery block of single Ca2+-activated K+ channels that is produced by internally applied Na+ can be relieved by millimolar concentrations of external K+. This effect of K+ on the kinetics of Na+ block was studied by the method of amplitude distribution analysis described in the companion paper (Yellen, G., 1984b, J. Gen. Physiol., 84:157-186). It appears that K+ relieves block by increasing the exit rate of the blocking ion from the channel, not by competitively slowing its entrance rate. This suggests that a K ion that enters the channel from the outside can expel the blocking Na ion, which entered the channel from the inside. Cs+, which cannot carry current through the channel, and Rb+, which carries a reduced current through the channel, are just as effective as K+ in relieving the block by internal Na+. The kinetics of block by internal nonyltriethylammonium (C9) are unaffected by the presence of these ions in the external bathing solution.     

Interleukin-1 stimulates glutamate uptake in glial cells by accelerating membrane trafficking of Na+/K+-ATPase via actin depolymerization

Interleukin-1 (IL-1) is a mediator of brain injury induced by ischemia, trauma, and chronic neurodegenerative disease. IL-1 also has a protective role by preventing neuronal cell death from glutamate neurotoxicity. However, the cellular mechanisms of IL-1 action remain unresolved. In the mammalian retina, glutamate/aspartate transporter (GLAST) is a Na(+)-dependent, major glutamate transporter localized to Müller glial cells, and loss of GLAST leads to glaucomatous retinal degeneration (T. Harada, C. Harada, K. Nakamura, H. A. Quah, A. Okumura, K. Namekata, T. Saeki, M. Aihara, H. Yoshida, A. Mitani, and K. Tanaka, J. Clin. Investig. 117:1763-1770, 2007). We show here that IL-1 increases glutamate uptake in Müller cells by a mechanism that involves increased membrane Na(+)/K(+)-ATPase localization, required for counteracting the Na(+)-glutamate cotransport. IL-1 activated the p38 mitogen-activated protein kinase (MAPK)/capase 11 pathway, which destabilizes the actin cytoskeleton allowing Na(+)/K(+)-ATPase membrane redistribution. Furthermore, pretreatment with IL-1 protected retinal neurons from glutamate neurotoxicity through p38 MAPK signaling. Our observations suggested that IL-1 acts as a potential neuroprotective agent by modulating the functions of the glia-neuron network.     

Comparative temperature dependence of (Na+ + K+)-ATPase.

The temperature dependence of (Na+ + K+)-ATPase was measured, utilizing preparations of enzyme from heat and kidney of rats, hamsters, guinea pigs, ground squirrels, turtles, chickens, and ducks. The two hibernating species, hamsters and ground squirrels, were studied awake at normothermia and hibernating at 4 degrees C. The results for every species except the turtles showed the same temperature dependence established for (Na++K+)-ATPase from rabbit kidney with a quasi-linear dependence above 15 degrees C and little or no activity below 15 degrees C. Turtle enzymes showed a broad activity versus temperature curve with a fall-off at high and low temperatures. The data in all cases, including the turtle data, may be fitted by a previously described thermodynamic kinetic model. Further, the model will fith the turnover or decrease in enzyme activity at higher temperatures observed in a number of cases. These results do not support the widely imputed ion pumping role for (Na++K+)-ATPase.     

The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy.     

Na+ and K+ uptake and exchange by the amphibian oocyte during the first meiotic division

The uptake and efflux of ²²Na and ⁴²K were studied in denuded Rana pipiens oocytes following progesterone induction of the resumption of meiotic maturation. Coincident with the breakdown of the large nucleus, or germinal vesicle, there is a virtual disappearance of K⁺ permeability of the oocyte plasma membrane. Only about 1–2% of the total [K⁺]_i is exchanged by completion of nuclear breakdown (8–10 hr) and accounts for the finding that there is no detectable change in total [K⁺]_i during the first meiotic division (20–24 hr). In the case of Na⁺, influx, exchange, and efflux kinetics were unchanged during the first meiotic division, with 20 and 35% of the total oocyte Na⁺ exchanging by the completion of nuclear breakdown and first meiotic division, respectively. Removal of Na⁺ from the incubation medium produced and earlier nuclear breakdown, whereas a K-free medium delayed breakdown. There was no effect of 10 μm/ml tetrodotoxin or 10^?5M strophanthidin on the time course of nuclear breakdown. Thus one action of progesterone appears to be a selective turning off of “K channels” in the oocyte plasma membrane. The disappearance of K selectivity of the oocyte plasma membrane coincides with plasma membrane depolarization, as well as nuclear swelling and breakdown.     

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.     

K+Nutrition and Na+Toxicity: The Basis of Cellular K+/Na+Ratios

The capacity of plants to maintain a high cytosolic K⁺/Na⁺ratiois likely to be one of the key determinants of plant salt tolerance.Important progress has been made in recent years regarding theidentification and characterization of genes and transportersthat contribute to the cytosolic K⁺/Na⁺ratio. For K⁺uptake,K⁺efflux and K⁺translocation to the shoot, genes have been isolatedthat encode K⁺uptake and K⁺release ion channels and K⁺carriersthat are coupled to either a H⁺or Na⁺gradient. Although thepicture is less clear for the movement of Na⁺, one pathway,in the form of non-selective ion channels, is likely to playa role in Na⁺uptake, whereas Na⁺efflux and compartmentationare likely to be mediated by H⁺-coupled antiport. In addition,several proteins have been characterized that play prominentroles in the regulation of K⁺and/or Na⁺fluxes. In this BotanicalBriefing we will discuss the functions and interactions of thesegenes and transporters in the broader context of K⁺nutritionand Na⁺toxicity. Copyright 1999 Annals of Botany Company Salinty, K⁺/N⁺ratio, transporter, membrane.     

ATP-dependent proton uptake by proteoliposomes reconstituted with purified Na+,K+-ATPase

Proton transport catalyzed by the sodium pump was demonstrated using proteoliposomes reconstituted with purified pig kidney Na+,K+-ATPase. Intravesicular pH was monitored with fluorescence from fluorescein isothiocyanate dextran introduced into the vesicles. An ATP-induced ouabain-sensitive acidification of the intravesicular medium was observed, when the vesicles were incubated with ATP and without Na+. The ATP-induced acidification was blocked by either extravesicular Na+ or pretreatment of the enzyme with ouabain before reconstitution. Protonophores, X-537A or carbonyl cyanide m-chlorophenylhydrazone, abolished the intravesicular acidification. The acidification was not inhibited by 3 mM tetra-n-butylammonium. The initial rate of the H+ uptake was increased with a decrease in pH of the extravesicular medium, and the maximum rate was obtained at pH 5.5-5.6. It is concluded that H+ can be transported in place of Na+ by the sodium pump.     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Stimulation of the Na+/K+ pump by external [K+] is regulated by voltage-dependent gating.

Wild-type and mutants with alpha-subunits truncated at the N terminus of Na+/K+ pumps of Torpedo electroplax were expressed in Xenopus oocytes by injection of cRNAs encoding for one of the alpha-subunits and for the beta-subunit. Currents generated by the pump were investigated under voltage clamp in Na(+)-free solution, a condition where stimulation by external [K+] is the only voltage-dependent and rate-determining step in the pump cycle (Rakowski, R. F., Vasilets, L. A., LaTona, J., and Schwarz, W. (1991) J. Membr. Biol. 121, 177-187). Voltage dependence of the apparent Km value for pump stimulation and of maximum transport activity was investigated. Truncation of the intracellular N-terminal end of the alpha-subunit at the trypsin-accessible site (alpha delta K37, leaving Lys37) leads to nearly complete inhibition of pump current at physiological potentials, whereas ouabain binding capacity is retained indicating an essential involvement of the N-terminal end in the process of ion translocation. Truncation at the N-terminal end leaving Lys28 (alpha delta K28) or Thr29 (alpha delta T29) leads to removal of 6 or 7 lysine residues, respectively, and has no effect on maximum transport activity. On the other hand, the mutated pumps with alpha delta K28 or alpha delta T29 exhibit more pronounced voltage dependences for stimulation of pump current by external [K+] compared with the wild-type Torpedo pump. In particular, a pronounced increase in voltage dependence of the apparent affinity of pump stimulation is obtained by the removal of the Lys28. The results support the view that the lysine-rich region in the N-terminal end affects the cation binding to the pump molecule and that Lys28 is important.     

K+-independent active transport of Na+ by the (Na+ and K+)-stimulated adenosine triphosphatase

The (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from canine kidney reconstituted into phospholipid vesicles showed an ATP-dependent, ouabain-inhibited uptake of 22Na+ in the absence of added K+. This transport occurred against a Na+ concentration gradient, was not affected by increasing the K+ concentration to 10 microM (four times the endogenous level), and could not be explained in terms of Na+in in equilibrium Na+out exchange. K+-independent transport occurred with a stoichiometry of 0.5 mol of Na+ per mol of ATP hydrolyzed as compared with 2.9 mol of Na+ per mol of ATP for K+-dependent transport.