Inhibition of rat synaptic membrane Na⁺/K⁺-ATPase and ecto-nucleoside triphosphate diphosphohydrolases by 12-tungstosilicic and 12-tungstophosphoric acid

The in vitro influence of Keggin structure polyoxotungstates, 12-tungstosilicic acid, H(4)SiW(12)O(40) (WSiA) and 12-tungstophosphoric acid, H(3)PW(12)O(40) (WPA), and monomer Na(2)WO(4) × 2H(2)O on rat synaptic plasma membrane (SPM) Na(+)/K(+)-ATPase and E-NTPDase activity was studied, whereas the commercial porcine cerebral cortex Na(+)/K(+)-ATPase served as a reference. Dose-dependent Na(+)/K(+)-ATPase inhibition was obtained for all investigated compounds. Calculated IC(50) (10 min) values, in mol/l, for SPM/commercial Na(+)/K(+)-ATPase, were: 3.4 × 10(-6)/4.3 × 10(-6), 2.9 × 10(-6)/3.1 × 10(-6) and 1.3 × 10(-3)/1.5 × 10(-3) for WSiA, WPA and Na(2)WO(4) × 2H(2)O, respectively. In the case of E-NTPDase, increasing concentrations of WSiA and WPA induced its activity reduction, while Na(2)WO(4) × 2H(2)O did not noticeably affect the enzyme activity at all investigated concentrations (up to 1 × 10(-3)mol/l). IC(50) (10 min) values, obtained from the inhibition curves, were (in mol/l): 4.1 × 10(-6) for WSiA and 1.6 × 10(-6) for WPA. Monolacunary Keggin anion was found as the main active molecular species present under physiological conditions (in the enzyme assays, pH 7.4), for the both polyoxotungstates solutions (1 mmol/l), using Fourier transform infrared (FT-IR) and micro-Raman spectroscopy. Additionally, commercial porcine cerebral cortex Na(+)/K(+)-ATPase was exposed to the mixture of Na(2)WO(4) × 2H(2)O and WSiA at different concentrations. Additive inhibition effect was achieved for lower concentrations of Na(2)WO(4) × 2H(2)O/WSiA (≤ 1 × 10(-3)/4 × 10(-6) mol/l), while antagonistic effect was obtained for all higher concentrations of the inhibitors.     

Differential expression of sodium channel activities during the development of chick skeletal muscle cells in culture

The expression of Na+ channels during differentiation of cultured embryonic chick skeletal muscle cells was investigated using saxitoxin (STX) and batrachotoxin (BTX), which previously have been shown to interact with distinct, separate receptor sites of the voltage-sensitive Na+ channel of excitable cells. In the present study, parallel measurements of binding of [3H]-STX (STX) and of BTX-activated 22Na+ uptake (Na influx) were made in order to establish the temporal relationship of the appearance of these two Na+ channel activities during myogenesis. Na influx was clearly measurable in 2-d cells; from day 3 to day 7 the maximum Na influx approximately doubled when measured with saturating BTX concentrations potentiated by Leiurus scorpion toxin, while the apparent affinity of BTX, measured without scorpion toxin, also increased. Saturable STX binding did not appear consistently until day 3; from then until day 7 the STX binding capacity increased about threefold, whereas the equilibrium dissociation constant (KD) decreased about fourfold. Although Na influx in cells of all ages was totally inhibited by STX or tetrodotoxin (TTX) at 10 microM, lower concentrations (2-50 nM) blocked the influx in 7-d cells much more effectively than that in 3-d cells, where half the flux was resistant to STX at 20-50 nM. Similar but smaller differences characterized the block by TTX. In addition, when protein synthesis is inhibited by cycloheximide, both Na influx and STX binding activities disappear more rapidly in 3-d than in 7-d cells, which shows that these functions are less stable metabolically in the younger cells.     

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

The Na/K-ATPase regulation by neurotransmitters in ontogeny

Activity of the Na/K-ATPase from rat brain synaptic membranes is inhibited by NA (noradrenaline). However, during fractionation of cytozole from nerve endings, two non-homogeneous peaks are found (SF(a), 60-100 kD and SF( i ),;10 kD), which influence the Na/K-ATPase activity, both directly and SF(a) NA-dependently. Joint action of NA and synaptic factors (SF(a) and SF(i)) on the Na/K-ATPase, represents a sum of four different processes: 1) NA, without synaptic factors, inhibits the Na/K-ATPase; 2) At low SF(a) concentrations NA-dependent Na/K-ATPase activatory mechanism is evident; 3) At high SF(a) concentrations NA-independent Na/K-ATPase is activated; 4) The low-molecular SF(i) protein inhibits the Na/K-ATPase. Regulation of the Na/K-ATPase activity by NA, SF(a) and SF( i), obtained in similar conditions from two weeks old and one year old rats, is different. In older rats SF(i) is characterized with strong Na/K-ATPase inhibition; in younger rats SF(i) does not change the Na/K-ATPase activity. The NA- and SF(i) -dependent inhibition and activation ratio is different in young and elder rats. In two week olds NA/SF(i) activatory mechanism is stronger, while in one year olds NA-dependent inhibition of the Na/K-ATPase is prevailing. These experimental data indicate that regulation of the Na/K-ATPase activity has an important role in synaptic transmission and that this process has noteworthy, albeit presently unknown, functional importance in integrative activity of the brain.     

Membrane regulation of brain Na,K-ATPase inhibition by calcium ions

In contrast to the purified enzyme. Na, K-ATPase from intact synaptic membranes is inhibited by Ca2+ according to a biphase pattern at Ca2+ concentrations of 10(-6) to 10(-3) M. The membrane damage after three washings with bidistilled water results in elimination of low cocentration phase. Recombination of the sediment and the supernatant restores the initial shape of the inhibition curve. Dithiothreitol greatly increases the inhibition by low Ca concentrations. This effect is absent in the purified enzyme preparation and is considerably reduced after the membrane damage. Recombination restores the dithiothreitol effect. It is suggested that the sensitivity of membrane Na,K-ATPase to low concentrations of Ca2+ is controlled by the components (most likely, peripheral proteins), which are loosely bound to the membrane, this process being dependent on the degree of the SH-group reduction.     

Poneratoxin, a novel peptide neurotoxin from the venom of the ant, Paraponera clavata.

1. At concentrations varying from 10(-8) to 10(-6) M synthetic poneratoxin (PoTX) is a strong, but very slowly acting agonist for smooth muscles and its blocks synaptic transmission in the insect CNS in a concentration-dependent manner and depolarizes giant interneurons. 2. However, in isolated dorsal unpaired median cells 10(-6) M PoTX causes only a reversible hyperpolarization of about 5 mV. 3. At concentrations from 10(-8) to 10(-6) M PoTX affects the electrical activity of isolated cockroach axons, as well as isolated frog and rat skeletal muscle fibres. 4. PoTX prolongs action potentials and induces slow automatic activity, due to a slow Na(+)-current activation at very negative values of potential and due to slow deactivation.     

Sodium ions regulate a specific population of acidic amino acid receptors in synaptic membranes

The regulatory effects of Na+ on C1-/Ca2+-dependent and C1-/Ca2+-independent L-glutamate binding sites were examined. In Tris-C1-/Ca2+ buffer, the binding of L-[3H]-glutamate to rat brain synaptic membranes was 5-fold higher than in Tris-acetate buffer. Low concentrations of Na+ (less than 5 mM) markedly depressed L-glutamate binding when assayed in Tris-C1/Ca2+ buffer, and this effect was attenuated by the selective blocker of C1-/Ca2+-dependent binding sites, DL-2-amino-4-phosphonobutyrate (APB). Scatchard analyses indicated that the effect of Na+ was due to a decrease in the number of C1-/Ca2+-dependent binding sites with no change in affinity. In Tris-acetate buffer, low concentrations of Na+ had little effect on L-glutamate binding. Dose-response curves for the inhibition of L-glutamate binding by DL-APB indicated a predominant high-affinity (Ki 5-10 microM) inhibitory component in Tris-C1-/Ca2+ buffer, but mainly a low-affinity component (Ki 1-2 mM) in Tris-acetate buffer and in Tris-C1-/Ca2+ buffer containing Na+. These data indicate that low concentrations of Na+ regulate specifically the C1-/Ca2+-dependent, APB-sensitive class of L-glutamate binding sites.     

Anemone toxin II receptor site of the lobster nerve sodium channel. Studies in membrane vesicles and in proteoliposomes

The receptor-site for the sea anemone toxin II from Anemonia sulcata (ATX) and its functional relationship with the Na+ channel were studied in plasma membrane preparations from lobster walking leg nerves. The modification of the 22Na influx by ATX was determined in membrane vesicles and in proteoliposomes prepared by reconstitution of detergent-extracted, unfractionated membrane particles into soybean liposomes. The effects of two other toxins, veratridine (VER) and tetrodotoxin (TTX), which bind to Na+ channel receptor-sites other than that for polypeptide toxins, were also studied, ATX and VER stimulated 22Na flux into membrane vesicles with K0.5 values in the order of 10(-7) and 10(-5) M, respectively. Positive cooperativity among these toxins was also seen; ATX displaces the K0.5 for VER towards lower VER concentrations. TTX abolishes the 22Na influx increment caused by ATX and/or VER with a K0.5 in the order of 10(-8) M. In proteoliposomes, in contrast, ATX modified the 22Na influx only at high concentrations (greater than 1 microM) and in the presence of VER. VER stimulation and TTX inhibition of the VER and the VER plus ATX modified fluxes, had the same characteristics as in the vesicle preparations. Measurable ATX and VER toxin effects were only seen in the presence of an outwardly directed K+ gradient for both vesicles and proteoliposomes. Detergent treatment and the reconstitution procedure seem to affect the functional properties of the ATX receptor site whereas the VER and the TTX sites remain unaltered.     

Omega Conus geographus toxin: a peptide that blocks calcium channels

We previously reported that omega Conus geographus toxin (omega CgTX), blocks evoked-release of transmitter at synapses in frog and attenuates the Ca2+ component of the action potential of chick dorsal root ganglion neurons. We report here voltage-clamp experiments on cultured chick dorsal root ganglion neurons which demonstrate that omega CgTX produces a persistent block of voltage-gated Ca2+ currents. Thus, we conclude that omega CgTX inhibits synaptic transmission by blocking Ca2+ channels in the presynaptic nerve terminal. The toxin had no effect on K+ currents; however, in some but not all neurons, omega CgTX reduced Na+ currents by 10-25%. These findings suggest that omega CgTX should be useful as a probe to examine synaptic Ca2+ channels.     

Differential Effects of Tetanus Toxin on Inhibitory and Excitatory Neurotransmitter Release from Mammalian Spinal Cord Cells in Culture

The effect of tetanus toxin on depolarization-evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium-evoked release of [3H]glycine and [3H]glutamate in a time- and dose-dependent manner. Ninety minutes after the application of toxin (6 x 10(-10) M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150-210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin-exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin-induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization-evoked release of inhibitory versus excitatory neurotransmitter.     

Delta endotoxin is a potent inhibitor of the (Na,K)-ATPase

A 68-kDa protein, delta endotoxin, produced by Bacillus thuringiensis ssp. Kurstaki inhibits ion transport, (Na,K)-ATPase, and K+-p-nitrophenylphosphatase activity catalyzed by the Na+ pump. The Ki for inhibition of the K+-p-nitrophenylphosphatase activity of purified dog kidney (Na,K)-ATPase was approximately 0.37 microM. Delta endotoxin had a similar Ki for inhibition of (Na,K)-ATPase activity when assayed at low Na+ concentration (10 mM) but the inhibition was reversed when high concentrations of Na+ (100 mM NaCl) were added to the assay. Phosphorylation of the active site aspartyl residue with 32PO3-4 was also blocked by delta endotoxin. Ouabain-sensitive 86Rb+ uptake into intact human red blood cells was not inhibited by externally added toxin; however, strophanthidin-inhibitable 22Na+ uptake into inside-out vesicles from red blood cells was completely blocked by delta endotoxin (Ki = 0.73 microM). These data suggest that delta endotoxin must enter the cell before it can inhibit the Na+ pump.     

Leptinotoxin-h Action in Synaptosomes and Neurosecretory Cells: Stimulation of Neurotransmitter Release

Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+-containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 X 10(-11) M for synaptosomes, and 1.5 X 10(-10) M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+-free, EGTA-containing medium, provided that high (in the 10(-10) M range) concentrations were used; near-maximal responses were observed at 10(-5)M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 X 10(-5) M Ca2+, and was maximal at 2 X 10(-4) M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+-containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+-containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+-free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+-containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with alpha-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in Cationic Selectivity of the Nicotinic Channel at the Rat Ganglionic Synapse: A Role for Chloride Ions?

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.     

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.     

Properties of the interaction of the sodium channel with permeant monovalent cations

The use of sea anemone toxin, veratridine and scorpion toxin which specifically interact with the gating system of the sodium channel and maintain the channel in an open conformation has permitted a study of the mechanism of transport of monovalent cations through the selectivity filter of this channel. The initial rate of 22Na+ influx through the tetrodotoxin-sensitive Na+ channels of excitable cells is dependent upon the external concentrations of Na+ and Na+-substitutes with the following properties. (a) It is saturable at high Na+ concentrations and increases with the external Na+ concentration in a cooperative manner (nH = 1.6). (b) At low external Na+ concentrations (1 mM), it is activated and then inhibited by increasing external concentrations of monovalent cations such as Li+, guanidinium, hydrazinium, hydroxylamine and K+. The activating effect of these cations disappears at higher external Na+ concentrations (10 mM). The experimental data are consistent with a model involving at least two allosteric cation-binding sites per Na+ channel. The binding of monovalent cations to Na+ sites is characterized by a high positive homotropic cooperativity. Most of the work describes the properties of the Na+ channel in neuroblastoma cells. The mechanism has also been shown to be valid for excitable cells of other types and origins.     

3H-5-hydroxytryptamine accumulation by rat brain synaptic vesicles in a membrane-impermeant medium, and selective reduction by 5,7-dihydroxytryptamine

In order to examine possible selectivity of amine uptake by synaptic vesicles, the ATP-stimulated accumulation of 3H-5-hydroxytryptamine (5HT) by synaptic vesicles from rat whole brain was examined in a medium comprised largely of membrane-impermeant anions (d-tartrate). Such media have previously been shown to stabilize vesicular accumulation of several neurotransmitters. Accumulation of 3H-5HT did not occur in tartrate medium alone, but was increased biphasically with increasing concentrations of both potassium phosphate and potassium bicarbonate. At optimal concentrations of each anion (10 mM), stable accumulation of 3H-5HT was observed at 37 degrees (26.1 +/- 1.2 pmol/mg protein; Km 6 X 10(-7) M), which was reduced by greater than 95% in the absence of K2ATP, at 4 degrees C, in the presence of 10(-6) M reserpine, or in the presence of the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Uptake was significantly antagonized by millimolar concentrations of Na+, Mg++ or Cl-, but was unaffected by ouabain (10(-5) M). Pretreatment of animals with 5,7-dihydroxytryptamine (5,7-DHT) (200 micrograms, intraventricular) 10 days prior to sacrifice reduced endogenous 5HT levels by 70%, while levels of endogenous norepinephrine (NE) and dopamine (DA) were unaffected. Accumulation of 3H-5HT, examined in the presence of 10(-6) M NE to block 3H-5HT accumulation by vesicles from noradrenergic nerve endings, was reduced by 40% in vesicles from treated animals. Vesicular accumulation of 3H-(-)-NE and 3H-DA was unaffected by 5,7-DHT treatment. The data suggest the possibility of preferential accumulation of 3H-5HT by vesicles arising from serotonergic nerve endings.     

Study of the endogenous cyclic nucleotide-dependent phosphorylation of synaptic membrane proteins

The highest activity of cyclic nucleotide-dependent (cAMP--2 X 10(-5) M, GMP--2 X 10(-4) M) phosphorylation of synaptic membrane proteins in vitro is revealed at equimolar concentrations of ATP and Mg2+ (10(-3)M) and depends on the ratio of the ATP concentration, protein amount in the assay and the period of exposure. At concentrations exceeding 10(-3) M ATP inhibits cyclic nucleotide-dependent phosphorylation. Optimal concentrations of ATR and Mg2+ to provide basal phosphorylation are found to be equal to 10(-2) M. Possible role of cyclic nucleotide-dependent phosphorylation in synaptic transmission is discussed.     

Na(+)-dependent high-affinity uptake of L-glutamate in cultured fibroblasts.

Uptake of 1 microM [3H]L-glutamate by cultured 3T3 fibroblasts was strongly dependent on extracellular Na+; it was reduced by elevated concentrations of K+ (60 mM) but it was not influenced by variations in the concentration of Ca2+ (0-9.6 mM). D- and L-Asparate, D- and L-threo-3-hydroxyaspartate DL-threo-3-methylaspartate and a few other glutamate derivatives and analogues inhibited the uptake but several close analogues of L-glutamate (including D-glutamate) had no effect, implying that the uptake system is highly structurally selective. The recently identified inhibitor of glutamate uptake in synaptosomal preparations, L-trans-pyrrolidine-2,4-dicarboxylate, was also among the inhibitors. Apparent Km of the uptake was found to be less than 10 microM. The present observations indicate that Na(+)-dependent 'high-affinity' uptake of L-glutamate may appear in structures which are apparently unrelated to glutamatergic synaptic transmission in the CNS.     

OD1, the first toxin isolated from the venom of the scorpion Odonthobuthus doriae active on voltage-gated Na+ channels

In this study, we isolated and pharmacologically characterized the first alpha-like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR. Using the two-electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage-gated Na+ channels expressed in Xenopus laevis oocytes (Na(v)1.2/beta1, Na(v)1.5/beta1, para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC50 = 80+/-14 nM) while Na(v)1.2/beta1 still was not affected at concentrations up to 5 microM. Na(v)1.5/beta1 was influenced at micromolar concentrations.     

The unique pharmacology of the scorpion alpha-like toxin Lqh3 is associated with its flexible C-tail

The affinity of scorpion alpha-toxins for various voltage-gated sodium channels (Na(v)s) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8-12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Na(v)s. We examined this hypothesis using Lqh3, an alpha-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Na(v)s compared with other alpha-toxins and its binding to insect Na(v)s is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other alpha-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9-10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Na(v)s expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling alpha-like toxins to affect a broad range of Na(v)s.