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.     

Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore.

Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.     

Interactions of neurotoxins with the action potential Na+ ionophore

Four neurotoxins that activate the action potential Na⁺ ionophore of electrically excitable neuroblastoma cells interact with two distinct classes of sites, one specific for the alkaloids veratridine, batrachotoxin, and aconitine, and the second specific for scorpion toxin. Positive heterotropic cooperativity is observed between toxins bound at these two classes of sites. Tetrodotoxin, a specific inhibitor of the action potential Na⁺ current, inhibits activation by each of these toxins in a noncompetitive manner (K_I = 4–8 nM). These results suggest the existence of three functionally separable components of the action potential Na⁺ ionophore: two regulatory components, which bind activating neurotoxins and interact allosterically in controlling the activity of a third ion-transport component, which binds tetrodotoxin. The dissociation constant for scorpion toxin binding is increased 10-fold by depolarization of the cells with K⁺, suggesting that the scorpion toxin binding site is located on a voltage-sensitive regulatory component of the ionophore.     

MEMBRANE POTENTIAL DEPENDENT BINDING OF SCORPION TOXIN TO THE ACTION POTENTIAL SODIUM IONOPHORE. STUDIES WITH A 3-(4-HYDROXY 3-[125I] IODOPHENYL) PROPIONYL DERIVATIVE

Abstract— A polypeptide toxin purified 80-fold from the venom of the scorpion Leiurus quinquestriatus enhances activation of the action potential Na⁺ ionophore by the alkaloid neurotoxins veratridine, batrachotoxin and aconitine in electrically excitable neuroblastoma cells. The purified toxin can be labelled with [¹²⁵I] by reaction with N-succinimidyl 3-(4-hydroxy 3-[¹²⁵I] iodophenyl) propionate. The [¹²⁵I] labelled toxin obtained from carboxymethyl Sephadex ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing. The [¹²⁵I] labelled toxin binds to a single class of saturable binding sites and also activates the action potential Na⁺ ionophore in electrically excitable neuroblastoma cells showing identical concentration dependence for both the binding and the activation effects. The labelled toxin does not show any saturable binding or activation of the action potential Na⁺ ionophore in variant neuroblastoma clones that specifically lack the action potential Na⁺ ionophore. The results indicate that scorpion toxin binds specifically to the action potential Na⁺ ionophore. The binding sites have a mean equilibrium dissociation constant of 3 IIH, a mean binding capacity of 46fmol toxin per mg cell protein and a mean density of 24 sites per μm² of cell surface membrane. A single action potential Na⁺ ionophore transports 1 × 10⁸ ions per min and has a conductance of 3 psiemens at physiologic ion concentrations. Depolarization of cells by elevated K⁺ concentration inhibits the saturable binding. Depolarization of cells by incubation in high Na⁺ medium (130mm -Na⁺, 5mm -K⁺) with gramicidin A or batrachotoxin also inhibits the saturable toxin binding. These results suggest that scorpion toxin binds specifically to a regulatory component (gate) of the Na⁺ ionophore. whose conformation is dependent on membrane potential.     

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.     

The binding of the insect selective neurotoxin (AaIT) from scorpion venom to locust synaptosomal membranes

The binding of the radioiodinated insect selective neurotoxin from the venom of the scorpion Androctonus australis (AaIT), to synaptic plasma membrane vesicles derived from osmotically shocked insect synaptosomes was studied under kinetic and equilibrium conditions. The integrity of these vesicles and the existence of membrane potential and its modifiability were demonstrated by assays of the uptake of the lipophilic cation tetraphenylphosphonium. It has been shown that ¹²⁵I-labeled AaIT binds specifically and reversibly to a single class of noninteracting binding sites of high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 1.2–3}\text{nM}$) and low capacity (1.2–2.0 pmol/mg protein). The values of the rate association and dissociation constants k₁ and k_?1 are, respectively, 1.36 · 10⁶ M^?1 · s^?1 and 1.9 · 10^?3 s^?1, and are in a good accordance with the equilibrium constant. The use of various ionophores and changes in external potassium concentration shown to modify the membrane potential of the present neuronal preparation, did not affect the binding of ¹²⁵I-AaIT, thus indicating its voltage-independence. Veratridine, tetrodotoxin, sea anemone toxin and the α and β scorpion toxins specific for vertebrates did not affect the binding of ¹²⁵I-AaIT. Furthermore, the above scorpion toxins were devoid of specific binding to the present insect neuronal preparation. Two additional insect toxins derived from the venom of the scorpion Buthotus judaicus, BjIT₁ (spastic-excitatory toxin, homologus to the AaIT) and BjIT₂ (flaccidity inducing-depressory toxin), were both shown to displace the ¹²⁵I-AaIT with a high affinity (K_d = 2.2 and 1.3 nM, respectively). These data are compared and discussed in light of the information concerning the interaction of scorpion venom toxins affecting vertebrates with mammalian neuronal tissues.     

Scorpion neurotoxin - a presynaptic toxin which affects both Na+ and K+ channels in axons.

A neurotoxin from the venom of the scorpion, $\text{Androctonus australis Hector}$, affects the closing of the Na⁺ channel and the opening of the K⁺ channel in giant axons of crayfish and lobster nerves. It blocks both Na⁺ and K⁺ conductances in $\text{Sepia}$ giant axons. Dose-response curves are markedly cooperative with all types of axons. Apparent dissociation constants for the receptor-toxin complexes are 0.25 μM, 0.7 μM and 2–4 μM for the crayfish, lobster and $\text{Sepia}$ axons, respectively. This toxin will be probably a useful tool for biochemical investigation of Na⁺ and K⁺ channels.     

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.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniprotiens of scorpion venom, stimulated the passive uptake of Na⁺ and Ca²⁺ in chick ermbryo heart cells. Half-maximum stimulation was obtained for 20–30 nM Na⁺ and 40–50 nM Ca²⁺. Scorpion toxin-activated Na⁺ and Ca²⁺ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na⁺ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nm) for both Na⁺ and Ca²⁺. Scorpion toxin-stimulated Ca²⁺ uptake was dependent on extracellular Na⁺ concentration and was not inhibited by Ca²⁺ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca²⁺ transport, which is coupled to passive Na⁺ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin — sensitive fast channels.     

Induction of recA protein in Escherichia coli by three platinum (II) compounds

The Na⁺ channels of Chinese Hamster lung fibroblasts have receptor sites for tetrodotoxin, batrachotoxin, veratridine, dihydrograyanotoxin, scorpion and sea anemone toxins. The binding properties of these toxic compounds were determined and shown to be very similar to those found in a variety of excitable cells. Electrophysiological experiments indicate that these Na⁺ channels cannot be electrically activated unless previously treated by veratridine.     

Reconstitution of High-Affinity Binding of a β-Scorpion Toxin to Neurotoxin Receptor Site 4 on Purified Sodium Channels

Abstract: Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1–3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a β-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). ¹²⁵I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min^?1 and 0.015 min^?1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with K_D of ～0.1 nM and a major class with a K_D of ～5 nM. Approximately 0.8 mol ¹²⁵I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1–3 or 5, consistent with the characteristics of binding of β-scorpion toxins to sodium channels in cells and membrane preparations. Our results show that specific, high-affinity binding at neurotoxin receptor site 4 on purified sodium channels can be restored by reconstitution into phospholipid vesicles and provide an experimental approach to analysis of the peptide components of the toxin receptor site.     

Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1)

Studies of the localization of the Na⁺-dependent sugar transport in monolayers of LLC PK₁ cells show that the uptake of a methyl α-d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [³H]phlorizin binding to monolayers of LLC PK₁ cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na⁺-dependent process. Binding at the low affinity sites on the contrary is Na⁺-independent. The mode of action of Na⁺ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na⁺ dependence and the matching of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for high affinity binding sites with the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of phlorizin for the inhibition of methyl α-d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α-d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na⁺ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na⁺-dependent sugar transport system in the apical membrane of LLC PK₁ cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Eosin,a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

(1) Eosin bound to the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of K⁺ has practically the same fluorescence as eosin without enzyme while in the presence of Na⁺ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K⁺ concentration but with a low affinity. With equal concentrations of Na⁺ and K⁺ more is bound in the presence of Na⁺, and the difference between 150 mM Na⁺ and 150 mM K⁺ shows one high-affinity eosin binding site per ³²P-labelling site ($\text{K}{}_{\mathrm{D}}$ 0.45 μM). With lower concentrations of the cations there are between one and two Na⁺-dependent high-affinity eosin binding sites per ³²P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na⁺-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na⁺ (+Mg²⁺). (4) Eosin, like ATP, increases the Na⁺ relative to K⁺ affinity $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{= 150}\text{mM}$) for Na⁺ activation of hydrolysis and for Na⁺ protection against inactivation by $\text{N-}\text{ethylmaleimide}$. (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na⁺ opens a high-affinity site for ATP while K⁺ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na⁺ relative to K⁺ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$ the system reacts with two different ATP molecules.     

Effects of cholera toxin on cellular and paracellular sodium fluxes in rabbit ileum

The diarrhea observed in patients which cholera is known to be related to secretion of water and electrolytes into the intestinal lumen. However, the exact mechanisms involved in these secretory processes have remained unclear. Although it is clear that purified toxin acts on epithelial cell metabolism, its activity on Na⁺ transport across intestinal mucosa is equivocal: reported either to prevent net Na⁺ absorption or to cause net secretion of Na⁺ from serosa to mucosa. Since total transmural Na⁺ fluxes across “leaky” epithelia involve very significant movement via a paracellular shunt pathway, we studied the effects of cholera toxin on the cellular and paracellular pathways of Na⁺ movement. Unidirectional Na⁺ fluxes were examined as functions of applied potential in control tissues and in tissues from the same animal treated with purified cholera toxin. Treatment of rabbit ileum in vitro with toxin stimulated the cellular component of serosa-to-mucosa Na⁺ flux (from $\text{2.41 ± 0.49 μ}\text{equiv./h}$ per cm² under control conditions to $\text{4.71 ± 0.43 μ}\text{equiv./h}$ per cm² after treatment with toxin, $\text{P < 0.01}$). The effect of cholera toxin on Na⁺ movement through the cells from mucosa to serosa appeared to be insignificant. Finally, a marked decrease in the Na⁺ permeability ($\text{P < 0.01}$) and no detectable significant changes in transference number for Na⁺ of the paracellular shunt pathway were observed following treatment with cholera toxin. These results provide direct evidence for the hypothesis that purified cholera toxin stimulates active sodium secretion but has minimal effect on sodium absorption.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Electrophysiological studies on embryonic heart cells in culture. Scorpion toxin as a tool to reveal latent fast sodium channel

Trypsin-dispersed heart cells were obtained from 11-day-old chick embryos. After culture as unstirred suspensions in dimethylsulfoxide-containing medium, spherical aggregates of cells beating spontaneously and apparently synchronously for months were obtained. Two kinds of cell were characterized by electrophysiological recordings: (1) cells with a slow rate of depolarizing phase showing tetrodotoxin-resistant action potential and blocked by D 600 (‘slow’ cells); (2) cells with high value of rising phase which was strongly decreased by tetrodotoxin and in which D 600 provoked uncoupling of excitation-contraction (‘fast’ cells).Toxin II from Androctonus australis scorpion venom increased the duration of action potential, which was ascribed to a slowing down of Na⁺ current inactivation and enhance the maximum rate of depolarization, especially in slow cells. Effects were antagonized by tetrodotoxin in both fast and slow cells. Washing experiments confirmed the results of previous studies, namely that tetrodotoxin and scorpion toxin bind to different receptors. It is concluded that slow cells with tetrodotoxin-resistant action potential contain latent fast Na⁺ channels that are revealed (activated) by toxin binding to the membrane.     

Pulsed NMR studies on Na + binding to simple carbohydrates

Pulsed NMR spectroscopy has been used to study Na⁺ binding to several simple carbohydrates in aqueous solution. Changes in the ²³Na spin-lattice relaxation time (T₁) were monitored to indicate complex formation between sodium ions and a ligand. It was found that Na⁺ interacts with these hydroxy-compounds in a manner similar to other metal cations, but very weakly. Among the sugars investigated, $\text{c i s}$-inositol forms the strongest complexes with the stability constant about 1.2 M^?1 (if 1:1 complexes are assumed). A qualitative study of competition between Na⁺ and Ca²⁺ was done, indicating that both cations have the same binding sites.     

Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium channel

Four new toxins have been isolated from the sea anemone Radianthus paumotensis: RpI, RpII, RpIII, and RpIV. They are polypeptides comprised of 48 or 49 amino acids; the sequence of RpII has been determined. Toxicities of these toxins in mice and crabs are similar to those of the other known sea anemone toxins, but they fall into a different immunochemically defined class. The sequence of RpII shows close similarities with the N-terminal end (up to residue 20) of the previously sequenced long sea anemone toxins, but most of the remaining part of the molecule is completely different. Like the other sea anemone toxins, Radianthus toxins are active on sodium channels; they slow down the inactivation process. Through their Na+ channel action, Radianthus toxins stimulate Na+ influx into tetrodotoxin-sensitive neuroblastoma cells and tetrodotoxin-resistant rat skeletal myoblasts. The efficiency of the toxins is similar in the two cellular systems. In that respect, Radianthus toxins behave much more like scorpion neurotoxins than sea anemone toxins from Anemonia sulcata or Anthopleura xanthogrammica. In binding experiments to synaptosomal Na+ channels, Radianthus toxins compete with toxin II from the scorpion Androctonus australis but not with toxins II and V from Anemonia sulcata.