Tetrodotoxin-resistant sodium channels

Summary 1. Tetrodotoxin (TTX) has been widely used as a chemical tool for blocking Na⁺ channels. However, reports are accumulating that some Na⁺ channels are resistant to TTX in various tissues and in different animal species. Studying the sensitivity of Na⁺ channels to TTX may provide us with an insight into the evolution of Na⁺ channels.2. Na⁺ channels present in TTX-carrying animals such as pufferfish and some types of shellfish, frogs, salamanders, octopuses, etc., are resistant to TTX.3. Denervation converts TTX-sensitive Na⁺ channels to TTX-resistant ones in skeletal muscle cells, i.e., reverting-back phenomenon. Also, undifferentiated skeletal muscle cells contain TTX-resistant Na⁺ channels. Cardiac muscle cells and some types of smooth muscle cells are considerably insensitive to TTX.4. TTX-resistant Na⁺ channels have been found in cell bodies of many peripheral nervous system (PNS) neurons in both immature and mature animals. However, TTX-resistant Na⁺ channels have been reported in only a few types of central nervous system (CNS). Axons of PNS and CNS neurons are sensitive to TTX. However, some glial cells have TTX-resistant Na⁺ channels.5. Properties of TTX-sensitive and TTX-resistant Na⁺ channels are different. Like Ca²⁺ channels, TTX-resistant Na⁺ channels can be blocked by inorganic (Co²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Zn²⁺, La³⁺) and organic (D-600) Ca²⁺ channel blockers. Usually, TTX-resistant Na⁺ channels show smaller single-channel conductance, slower kinetics, and a more positive current-voltage relation than TTX-sensitive ones.6. Molecular aspects of the TTX-resistant Na⁺ channel have been described. The structure of the channel has been revealed, and changing its amino acid(s) alters the sensitivity of the Na⁺ channel to TTX.7. TTX-sensitive Na⁺ channels seem to be used preferentially in differentiated cells and in higher animals instead of TTX-resistant Na⁺ channels for rapid and effective processing of information.8. Possible evolution courses for Na⁺ and Ca²⁺ channels are discussed with regard to ontogenesis and phylogenesis.     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

Effect of pyrithiamine treatment of potassium ion fluxes in rat cortical slices

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enchanced Na⁺/K⁺ pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K⁺ was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K⁺ was monitored directly with a K⁺-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K⁺ fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K⁺ upon tetrodotoxin addition was inversely related to the extracellular Ca²⁺ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.     

Biochemical evidence for pharmacological similarities between alpha-adrenoreceptors and voltage-dependent Na+ and Ca++ channels

The α-adrenergic antagonists yohimbine, prazozin and phentolamine, but not the α-adrenergic agonists, block voltage-dependent Na⁺ channels of rat brain synaptosomes. The lipid-soluble neurotoxins (veratridine, aconitine, grayanotoxins and ceveratrum alkaloids), which cause a permanent activation of the Na⁺ channels by acting at the same receptor site as yohimbine, compete with [³H]yohimbine for its binding to rat brain α₂-adrenoreceptors. The calcium channel inhibitors verapamil and D₆₀₀ also block the Na⁺ channel and recognize α₂-adrenoreceptors.     

Sodium channel activity in brain membrane fractions isolated from rats of different ages

The Na⁺ channel activity (tetrodotoxin sensitive ²²Na⁺ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79–87) from rats 5, 10, 30 and 60 days old. The ²²Na⁺ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 μM) increases the ²²Na⁺ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 μM) causes an increment of the ²²Na⁺ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 μM), the $\text{K}{}_{0.5}$ for veratridine is diminished and the maximum ²²Na⁺ flux is increased. The increments of ²²Na⁺ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin ($\text{K}{}_{0.5}$ approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na⁺ chennel activity. The other subfraction is enriched in myelin and shows no Na⁺ channel actiivty. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na⁺ channel activity.     

Interaction of pyrethroids with the Na channel in mammalian neuronal cells in culture

The interaction of a series of pyrethroids with the Na⁺ channel of mouse neuroblastoma cells has been followed using both an electrophysiological and a ²²Na⁺ influx approach. By themselves, pyrethroids do not stimulate ²²Na⁺ entry through the Na⁺ channel (or the stimulation they give is too small to be analyzed). However, they stimulate ²²Na⁺ entry when used in conjuction with other toxins specific for the gating system of the channel. These include batrachotoxin, veratridine, dihydrograyanotoxin II or polypeptide toxins like sea anemone and scorpion toxins. This stimulatory effect is fully inhibited by tetrodotoxin with a dissociation constant of 1.6 nM for the tetrodotoxin-receptor complex. Half-maximum saturation of the pyrethroid receptor on the Na⁺ channel is observed in the micromolar range for the most active pyrethroids, Decis and RU 15525. The synergism observed between the effect of pyrethroids on ²²Na⁺ influx on the one hand, and the effects of sea anemone toxin II, Androctonus scorpion toxin II, batrachotoxin, veratridine and dihydrograyanotoxin II on the other, indicates that the binding component for pyrethroids on the Na⁺ channel is distinct from the other toxin receptors. It is also distinct from the tetrodotoxin receptor.Some of the pyrethroids used in this study bind to the Na⁺ channel but are unable to stimulate ²²Na⁺ entry. These inactive compounds behave as antagonists of the active pyrethroids.An electrophysiological approach has shown that pyrethroids by themselves are active on the Na⁺ channel of mammalian neurones, and essentially confirm the conclusions made from ²²Na⁺ flux measurements.Pyrethroids are also active on C9 cells in which Na⁺ channels are ‘silent’, that is, not activatable by electrical stimulation. Pyrethroids chemically activate the silent Na⁺ channel in a manner similar to that with veratridine, batrachotoxin, or polypeptide toxins, which are known to slow down the inactivation process of a functional Na⁺ channel.     

Vinpocetine Selectively Inhibits Neurotransmitter Release Triggered by Sodium Channel Activation

The effects of vinpocetine on internal Na⁺ (Na_i), cAMP accumulation, internal Ca²⁺ (Ca_i) and excitatory amino acid neurotransmitters release, under resting and under depolarized conditions, was investigated in rat striatum synaptosomes. Veratridine (20 M) or high K⁺ (30 mM) were used as depolarizing agents. Results show that vinpocetine in the low M range inhibits the elevation of Na_i, the elevation of Ca_i and the release of glutamate and aspartate induced by veratridine depolarization. In contrast, vinpocetine fails to inhibit the rise of Ca_i and the neurotransmitter release induced by high K⁺, which are both TTX insensitive responses. Results also show that the inhibition exerted by vinpocetine on all the above veratridine-induced responses is not reflected in PDE activity. Our interpretation of these results is that vinpocetine inhibits neurotransmitter release triggered by veratridine activation of voltage sensitive Na⁺ channels, but not that triggered by a direct activation of VSCC. Thus, the main mechanism involved in the neuroprotective action of vinpocetine in the CNS is unlikely to be due to a direct inhibition of Ca²⁺ channels or PDE enzymes, but rather the inhibition of presynaptic Na⁺ channel-activation unchained responses.     

The sodium channel in non-impulsive cells Interaction with specific neurotoxins

The cell line C₉ used in this paper has a resting potential of ?50 mV (±10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na⁺ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstrated by electrophysiological techniques and by ²²Na⁺ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. ²²Na⁺ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of ²²Na⁺ influx into the C₉ cell. The stimulation of ²²Na⁺ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of ²²Na⁺ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

Energetics of Functional Activation in Neural Tissues

Glucose utilization (lCMR_glc) increases linearly with spike frequency in neuropil but not perikarya of functionally activated neural tissues. Electrical stimulation, increased extracellular [K⁺] ([K⁺]₀), or opening of Na⁺ channels with veratridine stimulates 1CMR_glc in neural tissues; these increases are blocked by ouabain, an inhibitor of Na⁺,K⁺-ATPase. Stimulating Na⁺,K⁺-ATPase activity to restore ionic gradients degraded by enhanced spike activity appears to trigger these increases in lCMR_glc. Cultured neurons behave similarly. Astrocytic processes that envelop synapses in neuropil probably contribute to the increased lCMR_glc. lCMR_glc in cultured astroglia is unaffected by elevated [K⁺]₀ but is stimulated by increased intracellular [Na⁺] ([Na⁺]_i), and this stimulation is blocked by ouabain or tetrodotoxin. L-Glutamate also stimulates lCMR_glc in astroglia. This effect is unaffected by inhibitors of NMDA or non-NMDA receptors, blocked by ouabain, and absent in Na⁺-free medium; it appears to be mediated by increased [Na⁺]_i due to combined uptake of Na⁺ with glutamate via Na⁺/glutamate co-transporters.     

Veratridine blocks voltage-gated potassium current in human T lymphocytes and in mouse neuroblastoma cells

(i) Effects of veratridine on ionic conductances of human peripheral blood T lymphocytes have been investigated using the whole-cell patch-clamp technique, (ii) Veratridine reduces the net outward current evoked by membrane depolarizations. The reduction originates from block of a 4-aminopyridine-sensitive, voltage-gated K⁺ current, (iii) Human T lymphocytes do not appear to express voltage-gated Na⁺ channels, since inward currents are observed neither in control nor in veratridine- and bretylium-exposed lymphocytes. (iv) The effect of veratridine consists of an increase in the rate of decay of the voltage-gated K⁺ current and a reduction of the peak current amplitude. Both effects depend on veratridine concentration. Halfmaximum block occurs at 97 m and the time constant of decay is reduced by 50% at 54 m of veratridine. (v) Possible mechanisms of veratridine action are discussed. The increased rate of K⁺ current decay is most likely due to open channel block. The decrease of current amplitude may involve an additional mechanism. (vi) In cultured mouse neuroblastoma N1E-115 cells, veratridine blocks a component of voltage-gated K⁺ current, in addition to its effect on voltage-gated Na⁺ current. This result shows that the novel effect of veratridine is not confined to lymphocytes.We thank Jacobien Künzel of the Wilhelmina Hospital for Children, Utrecht, for providing the blood samples and Aart de Groot for technical assistance. The research was supported by a fellowship of the Royal Netherlands Academy of Arts and Sciences to M. Oortgiesen.     

Veratridine-Induced Depolarization Reduces the Palmitoylation of Brain and Myelin Glycerolipids

Abstract: In this study, we have investigated the effect of neuronal depolarization on the palmitoylation of myelin lipids. For this purpose, brain slices from 60-day-old rats were incubated with [³H]palmitate for 1 h in the presence or absence of various drugs. Veratridine (100 µM) reduced the incorporation of [³H]palmitate into all brain glycerolipids by 40–50%, whereas the labeling of sphingolipids was unaffected. Similar results were obtained by using [³H]glycerol as a precursor, demonstrating that veratridine also causes a reduction in the de novo synthesis of glycerolipids. Both tetrodotoxin (1 µM) and ouabain (1 mM) prevented the effect of veratridine, indicating that it is mediated through the opening of voltage-gated sodium channels and involves the stimulation of the Na⁺/K⁺ pump. Decreased levels of both ATP, due to activation of the Na⁺,K⁺-ATPase, and the precursor palmitoyl-CoA were found in the veratridine-treated slices, thus explaining the reduction in lipid synthesis. Neuronal depolarization also decreased the synthesis of lipids present in the myelin fraction. The relatively high specific radioactivity of myelin lipids and the results from both repeated purification experiments and mixing experiments ruled out the possibility that the radioactive lipids present in myelin could derive from contamination with other subcellular fraction(s). Because neither mature oligodendrocytes nor myelin is known to express voltage-dependent Na⁺ channels, it is conceivable that the effect of veratridine on myelin glycerolipid metabolism occurs by an indirect mechanism such as an increase in the extracellular [K⁺]. However, the presence of 60 mM KCl in the medium did not affect the acylation of either brain or myelin lipids. These results raise questions as to the absence of sodium channels in myelinating oligodendrocytes and/or myelin.     

Ionic channels through the axon membrane (a review)

Ionic channels are discrete sites at which the passive movement of ions takes place during nervous excitation. Three types of channels are distinguished. 1. Leakage channels that are permanently open to various cations. 2. Na channels that open promptly on depolarization but slowly close again (inactivate) on sustained depolarization and that are predominantly permeable to Na⁺ ions. 3. K channels that on depolarization open after some delay but stay open and that are mainly passed by K⁺ ions. The selectivity sequence of the Na channels of the squid axon (or frog nerve) is as follows: Na⁺ ≈ Li⁺>(T1+)>NH⁺ ₄?K⁺> Rb⁺, Cs⁺; that of K channels is: (T1+)>K⁺>Rb⁺>NH⁺ ₄?Na⁺, Cs⁺, Na channels are selectively blocked by tetrodotoxin (TTX) or saxitoxin (STX), K channels by tetraethylammonium ions (TEA). Either channel type is reversibly blocked when one drug molecule binds to one site per channel, the equilibrium dissociation constant of these reactions being about 3×10^?9 MTTX (or STX) and 4×10^?4 M TEA, respectively. Because of their specificity and high affinity, TTX and STX are used to “titrate” the Na channels whose density appears to be of the order of 100/Μm². The “gates” of the channels operate as a function of potential and time but independent of the permeating ion species. Drugs (e.g. veratridine) and enzymes (e.g. pronase, applied intraaxonally) cause profound changes in the gating function of the Na channels without influencing their selectivity. This points to separate structures for gating and ion discrimination. The latter is thought to be, in part, brought about by a “selectivity filter” of which detailed structural ideas exist. Recent experiments suggest that the gates of the Na channels are controlled by charged particles moving within the membrane under the influence of the electrical field.     

RAPID EFFECTS OF VERATRIDINE, TETRODOTOXIN, GRAMICIDIN D, VALINOMYCIN AND NaCN ON THE NA+, K+ AND ATP CONTENTS OF SYNAPTOSOMES

Abstract— The effects of brief exposures of a number of depolarizing agents on ²⁴Na⁺ influx and on the Na⁺, K⁺ and ATP contents of synaptosomes were studied using a Millipore filtration technique to terminate the reaction. When synaptosomes were incubated in normal medium, there was a rapid influx of ²⁴Na⁺ and a gain in Na’contents; neither the ²⁴Na⁺ influx nor the Na⁺ gain were blocked by tetrodotoxin suggesting that this Na⁺ entry did not involve Na⁺-channels. Veratridine markedly increased the rate of ²⁴Na⁺ influx into synaptosomes and also increased the Na⁺ content and decreased the K⁺ content of synaptosomes within the first 10s of exposure. The normal ion contents were reversed by 1 min. The effects of veratridine on Na⁺ influx and on synaptosomal ion contents were prevented by tetrodotoxin and required Na⁺ in the medium. The ionophores gramicidin D and valinomycin also rapidly reversed the Na⁺ and K⁺ contents of synaptosomes, but these effects could not be blocked by tetrodotoxin. The reducing effect of gramicidin D on synaptosomal K⁺ content required Na’in the medium, whereas valinomycin caused a fall in the K⁺ content of synaptosomes in a Na⁺-free medium. Veratridine and gramicidin D, at concentrations known to reverse the synaptosomal ion contents, did not affect synaptosomal ATP levels. In contrast, valinomycin and NaCN caused an abrupt fall in synaptosomal ATP levels. The above findings suggest that veratridine quickly alters synaptosomal Na⁺ and K⁺ contents by opening Na ⁺-channels in the presynaptic membrane, and provide direct evidence for the existence of Na⁺-channels in synaptosomes. In contrast, gramicidin D and valinomycin appear to act independently of Na ⁺-channels, possibly by their ionophoric effects and, in the case of valinomycin, by diminishing synaptosomal ATP contents and hence diminishing Na⁺-pump activity. The rapid reversals of Na⁺ and K⁺ contents by these drugs could affect the resting membrane potentials, Na⁺-Ca²⁺ exchange across the synaptosomal membrane, and the release, synthesis and uptake of neurotransmitters by synaptosomes.     

Sodium-dependent efflux of K+ and Rb+ through the activated sodium channel of neuroblastoma cells

Passive efflux of⁴²K or⁸⁶Rb from differentiated mouse neuroblastoma cells in culture was stimulated up to 8-fold by 10^?4 M veratridine. The increased efflux could be blockedby low concentrations of tetrodotoxin (K_i = 4×10^?9 g/ml), and did not occur with other cell types lacking an excitable membrane. The temperature sensitivity of the activated component was much higher than that of the normal passive outflow. It is suggested that the veratridine-dependent, tetrodotoxin-sensitive efflux represents passage of ions through the excitable Na⁺ channel. Replacement of extracellular Na⁺ by Tris⁺ abolished the activation by veratridine. Titration of the Na⁺ requirement resulted in a hyperbolic relationship between external Na⁺ concentration and efflux rate, with an apparent K_m of 66.7 mM for Na⁺. This phenomenon may reflect an interaction between extracellular ions and a regulatory site on the Na⁺ channel.     

Up-Regulation of Functional Voltage-Dependent Sodium Channels by Insulin in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [³H]saxitoxin ([³H]STX) binding in a time-dependent manner (t_1/2 = 26 h). Insulin (100 nM for 4 days) increased the B_max of [³H]STX binding by 49% without changing the K_D value and also augmented the maximal influx of ²²Na⁺ due to 560 µM veratridine by 39% without altering the EC₅₀ value of veratridine. The stimulatory effect of insulin on ²²Na⁺ influx was concentration-dependent with an EC₅₀ of 3 nM, whereas insulin-like growth factor (IGF)-I had little effect at 1 nM. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine (100 µM)-induced ²²Na⁺ influx by approximately twofold in both insulin-treated cells and untreated cells. Veratridine-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine-induced ²²Na⁺ influx via the nicotinic receptor-ion channel complex and high-K⁺ (direct activation of voltage-dependent Ca²⁺ channels)-induced ⁴⁵Ca²⁺ influx. Stimulatory effects of insulin on [³H]STX binding and veratridine-induced ²²Na⁺ influx were nullified by simultaneous treatment with either 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na⁺ channel α-subunit. These results suggest that the binding of insulin to insulin (but not IGF-I) receptors mediates the up-regulation of functional Na⁺ channel expression at plasma membranes; this up-regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).     

Effects of Levetiracetam,Carbamazepine, Phenytoin,Valproate, Lamotrigine,Oxcarbazepine, Topiramate,Vinpocetine and Sertraline on Presynaptic Hippocampal Na<Superscript>+</Superscript> and Ca<Superscript>2+</Superscript> Channels Permeability

Ion channels are targets of various antiepileptic drugs. In cerebral presynaptic nerve endings Na⁺ and Ca²⁺ channels are particularly abundant, as they control neurotransmitter release, including the release of glutamate (Glu), the most concentrated excitatory amino acid neurotransmitter in the brain. Several pre-synaptic channels are implicated in the mechanism of action of the pro-convulsive agent, 4-aminopyridine (4-AP). In the present study the effects of levetiracetam and other established and newer (vinpocetine) anti-epileptic drugs, as well as of the anti-depressant, sertraline on the increase in Ca²⁺ induced by 4-AP in hippocampal isolated nerve endings were investigated. Also the effects of some of the anti-seizure drugs on the selective increase in Ca²⁺ induced by high K⁺, or on the selective increase in Na⁺ induced by veratridine were tested. Sertraline and vinpocetine effectively inhibited the rise in Ca²⁺ induced by 4-AP, which was dependent on the out-in Na⁺ gradient and tetrodotoxin sensitive. Carbamazepine, phenytoin, lamotrigine and oxcarbazepine inhibited the rise in Ca²⁺ induced by 4-AP too, but at higher concentrations than sertraline and vinpocetine, whereas levetiracetam, valproic acid and topiramate did not. The three latter antiepileptic drugs also failed in modifying other responses mediated by the activation of brain presynaptic Na⁺ or Ca²⁺ channels, including Glu release. This indicates that levetiracetam, valproic acid and topiramate mechanisms of action are unrelated with a decrease in presynaptic Na⁺ or Ca²⁺ channels permeability. It is concluded that depolarized cerebral isolated nerve endings represent a useful tool to unmask potential antiepileptic drugs targeting presynaptic Na⁺ and/or Ca²⁺ channels in the brain; such as vinpocetine or the anti-depressant sertraline, which high effectiveness to control seizures in the animal in vivo has been demonstrated.     

RELEASE OF ATP FROM A SYNAPTOSOMAL PREPARATION BY ELEVATED EXTRACELLULAR K+ AND BY VERATRIDINE

A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K⁺ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made. Elevating the extracellular K⁺ concentration produced a graded release of ATP from synaptosomes. Rb⁺ also released ATP but Na⁺, Li⁺ and choline did not. The response to elevated K⁺ was not blocked by tetrodotoxin (TTX), indicating that this effect was not mediated by the opening of Na⁺-channels in synaptosomal membranes. Veratridine (50 μM) caused a graded release of ATP which was larger and more prolonged than that caused by elevated K⁺. The release of ATP by veratridine was blocked by TTX indicating that the opening of Na⁺-channels was involved. Neither veratridine nor elevated K⁺ released ATP from microsomal or mitochondrial fractions, showing that the release of ATP probably did not originate from microsomal, vesicular or mitochondrial contaminants of the synaptosomal preparation. Release of ATP by elevated K⁺ was diminished in a medium lacking CaCl₊ or when EGTA was added to chelate Ca²⁺. In contrast, release by veratridine appeared to be augmented in Ca²⁺-free media or in the presence of EGTA. The K⁺-induced release of ATP, which is Ca²⁺ dependent, closely resembles the exocytotic release of putative neurotransmitters from presynaptic nerve-terminals. On the other hand, the apparent lack of a Ca²⁺ requirement for veratridine's action suggests that this process could originate from other sites, or involve mechanisms other than conventional neurotransmitter release processes.     

Persistent human cardiac Na+ currents in stably transfected mammalian cells

Miniature persistent late Na⁺ currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na⁺ currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na⁺ channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na⁺ currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na⁺ currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na⁺ currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na⁺ channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na⁺ currents were blocked by tetrodotoxin with an IC₅₀ value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na⁺ currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na⁺ currents are suitable for studying as well as screening potent open-channel blockers.     

Modification of electroplax excitability by veratridine

Veratridine influences membrane-potential changes arising both from the action potential and from the application of external cholinergic agonists in the isolated monocellular electroplax preparation. The action potential shows a long depolarizing after-potential in the presence of veratridine. The effects of various pharmacological agents and of external ion changes on this after-potential are similar to those reported for other nerve and muscle fibers and are consistent with the view that veratridine acts chiefly to increase the Na⁺ conductance.Membrane depolarizations by cholinergic agonists are inhibited by veratridine at pH 7 but strikingly amplified at pH 9. The former effect appears to involve interaction with the cholinergic receptor at the surface of the membrane, while the latter potentiation parallels the increase in the spike after-potential at pH 9 and presumably arises from a Na⁺ conductance increase.Veratridine appears to interact with the component involved in the Na⁺ conductance in the interior membrane phase. The possible localization of this component in both the conducting and synaptic membrane is discussed.