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.     

Veratridine-activated release of adenosine-5'-triphosphate from synaptosomes: evidence for calcium dependence and blockade by tetrodotoxin.

Cholinergic synaptosomes from squid brain were found to release almost 50% of their total endogenous ATP when exposed to veratridine, an alkaloid which activates action potential sodium channels in nervous tissue. Veratridine also depolarizes synaptosomes and induces transmitter release by a mechanism which is dependent upon free Ca⁺⁺ in the medium and is inhibited by tetrodotoxin, a specific veratridine inhibitor. ATP release activated by veratridine was also found to be calcium dependent and tetrodotoxin-sensitive. A new filter assay was developed to measure the kinetics of ATP release quantitatively, and veratridine-activated ATP release from synaptosomes was found to be complete in less than 30 seconds. Since ATP is a major component of cholinergic vesicles, this finding supports the concept that transmitter release from synaptosomes may occur from a vesicular rather than from a cytoplasmic pool.     

Constitution and properties of axonal membranes of crustacean nerves.

The purification of axonal membranes of crustaceans was followed by measuring enrichment in [3H]tetrodotoxin binding capacity and in Na+, K+-ATPase activity. A characteristic of these membranes is their high content of lipids and their low content of protein as compared to other types of plasmatic membranes. The axonal membrane contains myosin-like, actin-like, tropomyosin-like, and tubulin-like proteins. It also contains Na+, K+-ATPase and acetylcholinesterase. The molecular weights of these two enzymes after solubilization are 280,000 and 270,000, respectively. The molecular weights of the catalytic subunits are 96,000 for ATPase and 71,000 for acetylcholinesterase. We confirmed the presence of a nicotine binding component in the axonal membrane of the lobster but we have been unable to find [3H]nicotine binding to crab axonal membranes. The binding to axonal membranes og of the sodium channel, has been studied in detail. The dissociation constant for the binding of [3H]tetrodotoxin to the axonal membrane receptor is 2.9 nM at pH 7.4. The concentration of the tetrodotoxin receptor in crustacean membranes is about 10 pmol/mg of membrane protein, 7 times less than the acetylcholinesterase, 30 times less than the Na+, K+-ATPase, and 30 times less than the nicotine binding component in the lobster membrane. A reasonable estimate indicates that approximately only one peptide chain in 1000 constitutes the tetrodotoxin binding part of the sodium channel in the axonal membrane. Veratridine, which acts selectively on the resting sodium permeability, binds to the phospholipid part of the axonal membrane. [3H]Veratridine binding to membranes parallels the electrophysiological effect. Veratridine and tetrodotoxin have different receptor sites. Although tetrodotoxin can repolarize the excitable membrane of a giant axon depolarized by veratridine, veratridine does not affect the binding of [3H]tetrodotoxin to purified axonal membranes. Similarly, tetrodotoxin does not affect the binding of [3H]veratridine to axonal membranes. Scorpion neurotoxin I, a presynaptic toxin which affects both the Na+ and the K+ channels, does not interfere with the binding of [3H]tetrodotoxin or [3H]veratridine to axonal membranes. Tetrodotoxin, veratridine, and scorpion neurotoxin I, which have in common the perturbation of the normal functioning of the sodium channel, act upon three different types of receptor sites.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

Regulation of the (Na+ + K+)-ATPase in cultured chick skeletal muscle. Modulation of expression by the demand for ion transport

The levels of (Na+ + K+)-ATPase expression during muscle development and in response to modulation of demand for ion transport were studied in chick skeletal muscle cells in culture. The number of (Na+ + K+)-ATPase molecules on the myogenic cell surface, quantified with 125I-labeled monoclonal antibodies, increased 20-fold during muscle differentiation, with a substantial increase in (Na+ + K+)-ATPase molecules/unit area of membrane. The demand for sodium ion transport by the (Na+ + K+)-ATPase was modulated by activating voltage-sensitive sodium channels with veratridine or exposing cultures to low [K+]o (0.5 mM). Exposure to veratridine (10 microM) resulted in a 60-100% increase in cell surface and a smaller increase in intracellular (Na+ + K+)-ATPase over a 24-36-h period. Neither high [K+]o (50 mM) nor Ca2+ ionophore A23187 (1 microM) produced any such change, suggesting that neither membrane depolarization nor elevated cytosolic calcium was mediating the effect of veratridine. Veratridine stimulated up-regulation was specific for the (Na+ + K+)-ATPase, blocked by tetrodotoxin, and completely reversible. The kinetics of the reversal (down-regulation) process were much faster (t1/2 = 3 h) than those of up-regulation (t1/2 = 18 h). Up-regulation of the (Na+ + K+)-ATPase by veratridine occurred by a combination of two mechanisms: the first an early phase involving a stimulated biosynthesis of the (Na+ + K+)-ATPase and a later phase in which the biosynthetic rate returned to approximately control levels while the degradation rate slowed (t1/2 control = 31 h, t1/2 veratridine = 64 h).     

Effects of veratridine on Ca fluxes and the release of oxytocin and vasopressin from the isolated rat neurohypophysis

Uptake of radioactive calcium, 45Ca efflux, and hormone release from the isolated rat neurohypophysis were monitored in vitro after the addition of veratridine to the incubation medium. Veratridine dramatically increased hormone release, but the release was not sustained and had declined by about 90% after 2 h. Removal of external Na+ prevented hormone release as did addition to the incubation medium of tetrodotoxin or the calcium antagonists D600 and Mn2+ ions. Veratridine increased 45Ca uptake into the isolated neurohypophysis and the increase could be prevented by addition of tetrodotoxin or D600 to the medium. Efflux of 45Ca was not changed by addition of veratridine. The results underline the importance of both Na+ and Ca+2 channels in the regulation of secretion of neurosecretory products.     

Depolarization Differentially Affects Allosteric Modulation by Neurotoxins of Scorpion α-Toxin Binding on Voltage-Gated Sodium Channels

Abstract: Voltage-gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K⁺ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion α-toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 m M K⁺). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 m M K⁺). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two-state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state-dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.     

Ion selectivity of the nerve membrane sodium channel incorporated into liposomes

Tetrodotoxin-sensitive sodium channels of lobster nerve membranes were incorporated into soybean liposomes by the freeze-thaw-sonication procedure and their ionic selectivity was studied. Veratridine and grayanotoxin-I were used to activate the sodium channels and the increment of the ionic flux through them was specifically abolished by tetrodotoxin. The drug-sensitive 22Na+, 42K+, 86Rb+ and 137Cs+ influxes were measured. The permeability ratios calculated directly from ion fluxes showed that the channels preferably allow the passage of Na+. No anion influx ([32P]phosphate, [35S]sulfate, 36Cl) sensitive to the drugs was observed. The data reveal that the sodium channels incorporated into liposomes remain cation-selective and discriminate among different cations.     

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the inhibitory effect of veratridine (a Na⁺ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 M veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P₄ formation from Ins 1,4,5-P₃.Abbreviations used Ins 1,4,5-P₃ inositol 1,4,5 trisphosphate - Ins 1,3,4,5-P₄ inositol 1,3,4,5-tetrakisphosphate - PMA phorbol 12-myristate 13-acetate     

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 demonstarted 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.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Sodium channels in axons and glial cells of the optic nerve of Necturus maculosa

Experiments investigating both the binding of radioactively labelled saxitoxin (STX) and the electrophysiological response to drugs that increase the sodium permeability of excitable membranes were conducted in an effort to detect sodium channels in glial cells of the optic nerve of Necturus maculosa, the mudpuppy. Glial cells in nerves from chronically enucleated animals, which lack optic nerve axons, show no saturable uptake of STX whereas a saturable uptake is clearly present in normal optic nerves. The normal nerve is depolarized by aconitine, batrachotoxin, and veratridine (10(-6)-10(-5) M), whereas the all-glial preparation is only depolarized by veratridine and at concentrations greater than 10(-3) M. Unlike the depolarization caused by veratridine in normal nerves, the response in the all-glial tissue is not blocked by tetrodotoxin nor enhanced by scorpion venom (Leiurus quinquestriatus). In glial cells of the normal nerve, where axons are also present, the addition of 10(-5) M veratridine does lead to a transient depolarization; however, it is much briefer than the axonal response to veratridine in this same tissue. This glial response to veratridine could be caused by the efflux of K+ from the drug-depolarized axons, and is similar to the glial response to extracellular K+ accumulation resulting from action potentials in the axon.     

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.     

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.     

Effects of neurotoxins on pancreatic islets: Elucidation of a possible role for sodium channels in the secretory response

The neurotoxins veratridine and Leiurus toxin were used to characterize the nature of the sodium channel in the pancreatic β-cell membrane in relation to metabolc and secretory events. Insulin release and glycolytic flux were measured on batch-incubated rat islets. Veratridine, 200 μM, but not 10 μM, elicited a secretory response in the presence of 5.6 mM (basal) glucose, but did not influence the response to 15.3 mM glucose. Leiurus toxin, 20 nM, together with basal glucose and 10 μM veratridine induced insulin release, although Leiurus toxin, alone, was not effective. The secretory responses to the neurotoxins, but not 15.3 mM glucose, were blocked by tetrodotoxin. Glucose utilization was enhanced by 200 μM veratridine in the presence of basal glucose. Leiurus toxin at 20 nM increased the glycolytic rate which was further enhanced by the addition of 10 μM veratridine. The increments in glycolytic flux were partially or completely blocked by tetrodotoxin. Ouabain, 1.0 mM, had no effect on the secretory response to veratridine, but completely blocked the veratridine-induced increase in glycolytic flux. These observations indicate that the sodium channels in the β-cell membrane are pharmacologically similar to those in neuronal plasma membranes. Furthermore, the secretory response elicited by neurotoxins may occur independently of an increase in glycolytic flux. The major role of glycolytic flux may be to provide energy for extrusion of sodium from the β-cell.     

Release of d-[3H]aspartic acid from the rat substantia nigra: effect of veratridine-evoked depolarization and cortical ablation

The spontaneous and veratridine-evoked release of radioactive d-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 μM, 1 min) markedly produced a 14-fold increase in d-[³H]aspartic acid release from nigral slices. Omission of Ca²⁺ and increasing Mg²⁺ concentration to 12 mM in the superfusion medium did substantially block d-[³H]aspartate release induced by veratridine depolarization. Nevertheless, veratridine was able to evoke [³H]amino acid release which seemed to be, at least, 30% Ca²⁺-independent. Additional experiments showed that tetrodotoxin (0.01–0.1 μM), a blocker of voltage-dependent Na⁺ channels, totally abolished veratridine-evoked release of d-[³H]aspartate from nigral slices.Lesion studies were performed in order to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce d-[³H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral d-[³H]aspartate and by a large loss from ipsilateral nigral slices in d-[³H]aspartate release evoked by veratridine. In contrast, both the accumulation and veratridine-evoked release of [³H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion.The findings reported suggest that d-[³H]aspartic acid may be taken up and then released, in a Ca²⁺-dependent manner, by nerve terminals located in the substantia nigra. In addition, the results shown provide support to the view that l-glutamate and/or l-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.     

Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system

The electrical properties of the clonal muscle cell line L6 can be revealed by the measurement of ion fluxes. Under many circumstances, this technique provides a useful alternative to electro-physiology. In myoblasts, sodium uptake through voltage-dependent ionophores can be stimulated by veratridine and inhibited by tetrodotoxin. In myotubes which result from fusion of myoblasts, these voltage-dependent sodium channels appear to increase in number, paralleling the development of the action potential. Furthermore, in myotubes (but not myoblasts) carbamylcholine is able to stimulate a sodium influx through ionophores which are inhibitable by curare (dTC) but not tetrodotoxin (TTX). This demonstrates the presence of acetylcholine receptors on the fused cells. The cells also have a manganese-inhibitable calcium channel which appears to be voltage dependent and may be responsible for the calcium-dependent component of the action potential. Depolarizing concentrations of potassium in the medium stimulate calcium uptake both in the presence and absence of sodium. Veratridine and carbamylcholine also stimulate calcium influx, but both require the presence of sodium. This indicates that the depolarization necessary for opening the calcium channel is dependent upon sodium influx in these latter cases. Myoblasts and myotubes appear to have these channels in about equal numbers.     

Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles

1. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplaneta americana) was investigated. 2. Batrachotoxin (K0.5, 0.2 microM) was full agonist as for most effective activator of Na+ uptake; veratridine (K0.5, 2.5 microM) and aconitine (K0.5, 7.6 microM) produced a maximal stimulation of 22Na+ uptake that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I0.5, 11 nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins--stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect synaptosomes than those previously described for vertebrate brain synaptosomes, cultured neuronal cell, nerve membrane vesicles and neuroblastoma cells.     

Electrical properties of a clonal cell line as determined by measurement of ion fluxes

The electrical properties of the clonal muscle cell line L6 can be revealed by the measurement of ion fluxes. Under many circumstances, this technique provides a useful alternative to electro-physiology. In myoblasts, sodium uptake through voltage-dependent ionophores can be stimulated by veratridine and inhibited by tetrodotoxin. In myotubes which result from fusion of myoblasts, these voltage-dependent sodium channels appear to increase in number, paralleling the development of the action potential. Furthermore, in myotubes (but not myoblasts) carbamylcholine is able to stimulate a sodium influx through ionophores which are inhibitable by curare (dTC) but not tetrodotoxin (TTX). This demonstrates the presence of acetylcholine receptors on the fused cells. The cells also have a manganese-inhibitable calcium channel which appears to be voltage dependent and may be responsible for the calcium-dependent component of the action potential. Depolarizing concentrations of potassium in the medium stimulate calcium uptake both in the presence and absence of sodium. Veratridine and carbamylcholine also stimulate calcium influx, but both require the presence of sodium. This indicates that the depolarization necessary for opening the calcium channel is dependent upon sodium influx in these latter cases. Myoblasts and myotubes appear to have these channels in about equal numbers.