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Neostigmine (Neo), pyridostigmine (Pyr), and physostigmine (Phy) at low concentrations inhibited acetylcholine (ACh) esterase, thereby indirectly potentiating ACh enhancement of [3H]perhydrohistrionicotoxin (H12-HTX) binding to the channel sites of the nicotinic ACh receptor of Torpedo membranes. However, at higher concentrations, they inhibited ACh action due to their direct binding to the ACh receptor. They displaced binding of [3H]ACh and 125I-alpha-bungarotoxin (alpha-BGT) to the receptor sites with the following order of decreasing potency: Neo greater than Phy greater than Pyr. Furthermore, Neo and Pyr potentiated [3H] H12-HTX binding to the receptor's channel sites. Preincubation of ACh receptors with any of the three carbamates reduced the rate of binding of 125I-alpha-BGT and increased the potency of carbamylcholine in inhibiting 125I-alpha-BGT binding, suggesting that the three carbamates act as partial agonists and potentiate receptor desensitization. Although none of the three carbamates inhibited [3H]H12-HTX binding to the receptor's closed channel conformation, only Phy was a potent inhibitor of [3H]H12-HTX binding to the carbamylcholine-activated conformation. The potency of Phy was not due to the absence of positive charge since Phy methiodide acted similarly. The data suggest that the major action of the three carbamates at nicotinic cholinergic synapses is inhibition of ACh-esterase. Their interactions with the nicotinic ACh receptor are with its "receptor" as well as allosteric "channel" sites, but they differ in their effects. Neo and Pyr act mainly as partial agonists, while Phy is mostly an inhibitor of the channel in the activated receptor conformation.

The interactions of four tricyclic antidepressants, including two dibenzazepines, imipramine and desipramine, and two dibenzocyclo-heptenes, amitriptyline and nortryptiline, were studied with the ion channel associated with the nicotinic acetylcholine receptor from the electric organs of Torpedo ocellata . These drugs inhibited the binding of tritiated perhydrohistrionicotoxin and phencyclidine to sites on the ion channel. All four compounds interacted with the ion channel with approximately equal affinities, inhibition constants for the two sites ranging from 1.1 to 3.4 μM in the absence and from 0.16 to 0.75 μM in the presence of 1 μM acetylcholine. The affinities of the secondary amines were increased by acetylcholine to a greater degree than were those of the tertiary amine compounds.

The enantiomers of perhydrohistrionicotoxin were studied in their effects on endplate currents recorded at the junctional region of sartorius muscles of Rana pipiens. The two optical antipodes progressively decreased the peak amplitude of the endplate currents and were indistinguishable from each other at all times. The enantiomers shortened equally the time constants for endplate current decay, but did not alter their voltage sensitivities. Although perhydrohistrionicotoxin contains 4 chiral centers, complete steric inversion does not alter its effects on the acetylcholine receptor—ion channel complex. By contrast the recognition site of the AcChR is extremely sensitive to any change in the chirality of agonists.

The attempted suicide by 2 women with a kyushin overdose is reported. Kyushin caused them to produce a significant elevation of a serum digoxin-like immunoreactive substance (2.35 and 1.84 ng/ml) and symptoms of nausea, vomiting and general malaise. Their blood biochemistry and electrolytes were normal. In one patient, an electrocardiogram revealed a second degree Wenckebach atrioventricular block and T-wave change. Toad venom, a kyushin ingredient, is possibly responsible for the development of these clinical features and electrocardiographic changes.

Desensitization of the nicotinic acetylcholine (ACh) receptor of Torpedo electric organs was studied by monitoring activated [3H]perhydrohistrionicotoxin binding to the channel sites of the receptor. Receptor desensitization was produced by incubating Torpedo membranes that had been pretreated with diisopropylfluorophosphate with ACh and was quenched by the addition of AChesterase. Desensitization caused concentration- and time-dependent decrease in the binding of [3H]perhydrohistrionicotoxin to carbamylcholine-activated receptors and an increase in the apparent affinity of carbamylcholine to the receptor. d-Tubocurarine and concanavalin A antagonized the desensitizing effects of ACh. The kinetics of this desensitization showed an extremely rapid temperature- and concentration-dependent component which was complete by the earliest measurement (i.e., 6 sec) and a much slower component (T1/2 = 55 sec) which reached a plateau after 120 sec. The rate of this slow component was temperature- and concentration-independent. Recovery from desensitization was temperature dependent, but independent of the ACh concentration used for desensitization. It also showed biphasic kinetics: half the receptors recovered fast (T1/2 = 36 sec), whereas the other was slow (T1/2 = 180 sec).

When SK-N-SH human neuroblastoma cells were exposed to nicotine (NIC) or KCl they showed a dose-dependent transient increase (2- to 4-fold) in intracellular Ca2+ concentration ([Ca2+])i as detected by quin-2 fluorescence, with half maximal effects (EC50) observed at 13 microM and 26 mM, respectively. Tubocurarine and 1-isodihydrohistrionicotoxin potently blocked the NIC-evoked (IC50 congruent to 1 microM and 0.3 microM, respectively), but not the high [K+]o-evoked [Ca2+]i accumulation. The KCl-induced response was inhibited by verapamil and diltiazem (IC50 = 1.4 and 10.9 microM, respectively). Tetrodotoxin (3 microM) and tetraethylammonium (10 microM) had no effect on [Ca2+]i accumulation induced by either agent. Increases in [Ca2+]i could be evoked sequentially by NIC and KCl in the same cells suggesting independent mechanisms of Ca2+ entry. In a Ca2+-free medium, no response to either KCl or NIC was observed. However, when Ca2+ ions were restored, [Ca2+]i accumulation was enhanced to the same extent as cells suspended in a Ca2+-containing buffer. Long-term (18 hr) pretreatment of SK-N-SH cells with pertussis (100 ng/ml) or cholera toxins (10 nM) had no effect on NIC or KCl-induced [Ca2+]i accumulation. Together, these data demonstrate the presence of NIC receptors and voltage-sensitive Ca2+ channels on SK-N-SH neuroblastoma cells, through which [Ca2+]i may be modulated.

The novel tricyclic alkaloid, gephyrotoxin ( GyTX ), found in the skin secretions of the frog Dendrobates histrionicus , potentiates and blocks the indirectly elicited muscle twitch in a concentration-dependent manner. GyTX prolongs the falling phase of the muscle action potential and decreases delayed rectification, supporting the idea that the alkaloid blocks the voltage-sensitive potassium conductance of the electrically excitable membrane. The peak amplitude of the end-plate currents (EPC) and miniature end-plate currents ( MEPC ) were depressed, but no significant deviation from linearity relative to control was seen in the current-voltage relationship. The decay time constant of the EPC (tau EPC) was markedly shortened by GyTX , the effect being greater at 10 degrees than at 22 degrees. The relationship between the log of tau EPC and membrane potential disclosed a linear relationship at all concentrations tested, but a progressive loss of voltage sensitivity of tau EPC was seen when GyTX concentrations were increased. Also, the plot of 1/tau EPC against GyTX concentration revealed a linear relationship. The lack of voltage and time dependence suggests that GyTX has little effect on the ACh receptor-ionic channel complex in the closed conformation. Single-channel conductance studied by means of fluctuation analysis did not change after GyTX application, but the channel lifetime decreased by about 40% at clamp potentials of -105 mV and at a toxin concentration of 7.5 microM. Repetitive nerve stimulation led to a pronounced " rundown " in the EPCs which was frequency-dependent. These findings were taken as evidence that GyTX interacts with the acetylcholine receptor complex, causing a blockade of its channel mainly in the open conformation.

Histrionicotoxin, a spiropiperidine alkaloid, and twenty-two analogs inhibited binding of [3H]perhydrohistrionicotoxin [( 3H]H12-HTX) and of [3H]phencyclidine [( 3H]PCP) to sites on the acetylcholine receptor-ion complex of Torpedo electroplax membranes. Structural alterations to the nitrogen (secondary amine) or oxygen (alcohol) functions or to the five carbon and four carbon side chain of histrionicotoxin altered the potency versus [3H]H12-HTX and [3H]PCP binding measured in the presence or absence of a receptor agonist, carbamylcholine. Histrionicotoxin itself was 3-fold more potent versus [3H]PCP binding than versus [3H]H12-HTX binding. N-Methylation or O-acetylation increased this difference, while alterations to the side chains either slightly decreased or markedly increased this difference. Histrionicotoxin was some 3.5-fold more potent versus [3H]H12-HTX binding in the presence of carbamylcholine than in its absence. O-Acetylation increased this selectivity for the carbamylcholine-activated state of the receptor channel complex, while alterations in the side chains either reduced or increased the selectivity. Histrionicotoxin was some 2.2-fold more potent versus [3H]PCP binding in the presence of carbamylcholine than in its absence. N-Methylation of O-acetyl-histrionicotoxin greatly increased this selectivity, while alterations in the side chains either reduced or had no effect on selectivity.

1. [3H]H12-HTX binding to Torpedo electric organ membranes is saturable and inhibited by ligands that modulate EPCs. There is a small proportion of nonspecific binding to the membrane. 2. The validity of utilizing [3H]H12-HTX as a specific label for the ionic channel of the nicotinic receptor is extablished by the good correlation between the potency of HTX analogues in reducing EPC amplitudes and their inhibition of [3H]H12-HTX binding. 3. Receptor drugs and toxins inhibit [3H]ACh binding to the ACh receptor, but do not inhibit significantly [3h]h12-htx binding to the ionic channel at similar concentrations. 4. Various drugs and toxins with different modes of action modulate EPCs and inhibit [3H]H12-HTX binding to the ionic channel of the nicotinic receptor. Most such drugs and toxins, at similar concentrations, do not inhibit [3H]ACh binding to the ACh receptor, but some do, such as quinacrine and TEA. 5. The ionic channel of the nicotinic receptor is a protein which is solubilized by cholate and retains its affinity for H12-HTX as well as its drug sensitivity. 6. The ACh receptor is separated from its ionic channel, and different conditions affect the proportion of separated molecules.

Fractionation of the poisonous secretion of the toad Bufo marinus paracnemis Lutz, by dialysis and chromatography on QAE-Sephadex, led to the isolation of a fraction which was adsorbed to the ion exchanger. This fraction, when incubated with human serum, yielded an anticomplementary effect that was evaluated by measuring the kinetics of lytic activity on sensitized sheep red cells (classical pathway) and unsensitized rabbit cells (alternative pathway).

Several microtubule-active drugs block cholinergically mediated catecholamine secretion from adrenal chromaffin cells without affecting secretion induced by other secretagogues. Interactions of these agents with nicotinic acetylcholine receptor-ion channel complexes from Torpedo californica electric organs were studied using radiolabeled probes for receptor and associated ion channel-binding sites. Colchicine, taxol, and the Vinca alkaloids had minimal affinity for cholinergic receptor-binding sites (nicotinic or muscarinic). The Vinca alkaloids (vinblastine, vincristine, vindesine) and colchicine inhibited [3H]perhydrohistrionicotoxin ([3H]H12-HTX) binding to the receptor-gated ion channel with IC50 values of 2-32 microM and 6 mM, respectively. The ability of the microtubule-active drugs to inhibit [3H]H12-HTX binding was increased by up to 5-fold in the presence of 1 microM carbamylcholine. The IC50 values for inhibition of [3H]H12-HTX binding by colchicine and three Vinca alkaloids were closely correlated with their abilities to inhibit acetylcholine-induced catecholamine secretion from cultured bovine adrenal chromaffin cells. As a consequence of its interaction (direct or indirect) with the ion channel, at least one Vinca alkaloid (vinblastine) stabilized a high agonist affinity conformation of the nicotinic receptor complex. beta-Lumicolchicine, an analog of colchicine devoid of microtubule activity, also blocked ion channel binding. On the other hand, taxol, a microtubule-stabilizing agent which also selectively blocks cholinergically mediated secretion, did not affect receptor or ion channel binding. The present results indicate that interactions with the nicotinic receptor-ion channel complex may underlie the actions of certain microtubule-active agents on catecholamine secretion by adrenal chromaffin cells.

The actions of phencyclidine [1-(1-phenylcyclohexyl)piperidine, PCP] and its morpholine analog [1-(1-phenylcyclohexyl)morpholine, PCM] on ionic currents of nicotinic acetylcholine receptors were studied at the neuromuscular junction of frog skeletal muscle and on embryonic rat muscle cells in tissue culture. PCP and PCM reduced the peak amplitude and the decay time constant of the endplate current (EPC). PCP produced a voltage-dependent curvature and a time-dependent hysteresis loop at negative potentials (at potentials from -50 to -150 mV). In contrast, PCM caused a depression of EPC peak amplitude, but the current-voltage relationship (+60 to -150 mV) remained linear. When PCP-modified EPCs were elicited in trains at hyperpolarized potentials the amplitudes of successive events were progressively decreased and the magnitude of the decrease was dependent on the level of hyperpolarization. At positive potentials the process was reversed; the amplitude increased with successive stimulations. The EPC decayed exponentially in the presence of PCP and PCM, with a shortened time constant of decay that was less dependent on membrane potential than control. PCP and PCM caused only a 20% decrease of the amplitude of the iontophoretically evoked acetylcholine potential, which was significantly different from that induced by the desensitizing alkaloid perhydrohistrionicotoxin. Both PCP and PCM reduced by 50% the mean channel open time obtained from rat myoballs, giving a potency ratio for PCP to PCM of 2.5. This relative potency was correlated with that obtained for the reduction in the decay time constant of the EPC (ratio = 2.2). The effects of PCP on the peak amplitude of the EPC seem to be related to a conformational change of the acetylcholine receptor occurring before channel activation and not to a receptor desensitization.

Extracts of the skin of Pseudophryne coriacea displayed a powerful stimulant action on the leech helical muscle, both in vitro and in vivo. In the isolated dorsal muscle, the extract caused the appearance of vigorous phasic movements, accompanied by rapid increase in tonus, up to intense spasm. Hyoscine, physostigmine, hexamethonium, tubocurarine and alpha-bungarotoxin did not affect the response to the extract; tetrodotoxin, nifedipine and 5-hydroxytryptamine (5-HT) produced a partial blockade. In the intact animal, the extract at first potently stimulated the musculature, evoking the appearance of a succession of incoordinated, spastic movements, with twisting and rolling of the body of the animal. Stimulation was followed by paralysis and death. It is suggested that the pumiliotoxin-like alkaloid of Pseudophryne coriacea, responsible for these effects, acts directly on the helical muscle.

Ten pyrethroids affected binding of [3H]perhydrohistrionicotoxin ([ 3H]H12-HTX) to the channel sites of the nicotinic acetylcholine (ACh) receptor/channel of the electric organ of the electric ray, Torpedo ocellata, and inhibited 45Ca2+ flux through the receptor's ionic channel. Most pyrethroids stimulated binding of [3H]H12-HTX to the channel sites in 30 s, in absence of carbamylcholine, with little or no effect on binding of [3H]ACh to the receptor sites, which suggests that the pyrethroids are binding to a third kind of site. However, in presence of carbamylcholine, all pyrethroids inhibited binding of [3H]H12-HTX, with esters of cyclopentenolone more potent, and generally more rapid in doing so, than esters of alpha-cyano-3-phenoxybenzyl alcohol. Changes in the acidic moiety of the pyrethroid had little effect. Kadethrin, whose alcohol moiety is 5-benzyl-3-furylmethyl, was the most potent pyrethroid in stimulating [3H]H12-HTX binding (in absence of carbamylcholine) in 30 s (18-fold) and in inhibiting it in 120 min. The pyrethroids were more potent in their modulation of [3H]H12-HTX binding at lower temperatures. Inhibition of receptor binding and receptor-regulated ion transport by concentrations of pyrethroids similar to those at which they affect nerve conduction suggests that the nicotinic ACh receptor may be an additional target for the toxic action of pyrethroids.

Histrionicotoxin, a toxin isolated from skin secretions of a Colombian arrow poison frog, Dendrobates histrionicus, decreased the amplitude and time-course of the endplate current, and altered the voltage dependence of the half-decay time. In addition, the toxin produced a characteristic nonlinearity in the current-voltage relationship of the endplate current when 3-s voltage conditioning steps were used. Reduction in time of the conditioning steps to 10 ms made the current-voltage relationship linear. The decrease in peak amplitude of the endplate current (epc) produced by histrionicotoxin measured during long hyperpolarizing conditioning steps was fitted by a single exponential function. The calculated rate constants ranged from 0.03 to 0.14 s-1 and varied with membrane potential at hyperpolarizing levels. The voltage- and time-dependent action of histrionicotoxin does not require an initial activation of receptors by acetylcholine (ACh). The characteristic of the current-voltage relationship can be accounted for by the observed voltage and time dependency of the attenuation of the endplate current amplitude in the presence of histrionicotoxin during long conditioning steps. These effects of histrionicotoxin on the peak amplitude, and on the voltage and time dependence of the epc were concentration-dependent and slowly reversible upon washing out the toxin. Thus, the voltage- and time-dependent action of histrionicotoxin at the endplate is related to an increase in the affinity between the toxin and the ACh receptor-ionic channel complex. This increase in affinity is postulated to be due to a conformational change of the macromolecule in the presence of histrionicotoxin which is demonstrated to be relatively slow, i.e., on the order of tens of seconds.

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [125I]alpha-bungarotoxin ([125I]BGT) as a probe for the acetylcholine binding site and [3H]perhydrohistrionicotoxin ([3H]H12-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [3H]H12-HTX binding sites (Ki=0.08–0.24 μM), but had no affect on receptor binding. MUP affinity for [3H]H12-HTX binding sites was approximately doubled in the presence of 1 μM carbamylcholine. Introduction of a 2′-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [3H]H12-HTX binding (K i-8.8 μM). 2-Methylpiperidine was considerably less active (K i=600 μM), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [3H]H12-HTX binding sites were decreased in the presence of 1 μM carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 μM MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, “desensitized” conformations.

A protein is purified by differential centrifugation from membrane fragments rich in acetylcholine receptor prepared from Torpedo marmorata electric organ after dissolution by a mixture of non denaturing detergents. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and Coomassie blue staining the purified protein yields a single band of apparent molecular weight 43,000. Spectroscopic experiments carried out in the absence of Ca++ and detergents reveal that the 43 K protein interacts with the fluorescent local anesthetic quinacrine and with the frog toxin histrionicotoxin (apparent KD : 7 X 10(-7) M) but not with carbamylcholine and the alpha toxin from N. nigricollis.

1. Four synthetic histrionicotoxin derivatives (H 8 − , H 12 − , C 4 H 10 − and C 5 H 10 − HTX) were applied to bullfrog (Rana catesbeiana) sympathetic ganglia and their effects were compared electrophysiologically. 2. The derivatives (60–100μM) blocked both acetylcholine receptor-channel complex (ACh RC complex) and Na + channel to cause a transmission failure. They also blocked K + and Ca 2+ channels. 3. Although all 4 derivatives exhibited similar effects, their potencies on respective ionic channels differed from one another. 4. Two types of (presumably subsynaptic and extrasynaptic) ACh RC complexes in ganglion cells were distinguished based on their differential sensitivities to HTX derivatives.

The interactions of naltrexone with the nicotinic acetylcholine receptor were studied electrophysiologically using the frog sciatic nerve-sartorius muscle and biochemically using membranes from the electric organ of Torpedo ocellata. At nanomolar concentrations naltrexone increased the peak amplitude of endplate currents with little change in the decay time constant. At micromolar concentrations there was a concentration-dependent depression of endplate current and miniature endplate current amplitudes and decay time constants. Decay time constant depression was enhanced with hyperpolarization. Only marginal curvature was induced in peak endplate current amplitude versus membrane voltage plots by naltrexone. Naltrexone had no effect on single channel conductance but decreased open channel lifetime, according to fluctuation analysis. Naltrexone alone (less than or equal to 3 microM) did not impair binding of [125I]alpha-bungarotoxin to the receptor in a fast pre-equilibration assay, but increased the ability of acetylcholine to displace [125I]alpha-bungarotoxin. The drug displaced the agonist-stimulated binding of [3H]perhydrohistrionicotoxin to the channel site. Biphasic functional changes in neuromuscular transmission can be attributed to an allosteric mechanism with increased agonist binding to the nicotinic receptor at nanomolar concentrations and caused a non-competitive blockade of the ionic channel at micromolar concentrations.

Chiriquitoxin is a new natural analog of tetrodotoxin in which the -CH2OH group on C6 has been replaced with a yet unidentified group consisting of 104 mass units. It is unique in being the only known stable analog to be equally potent as tetrodotoxin in blocking the sodium channel. It additionally interferes with the delayed rectifier (potassium) channel. In frog skeletal muscle, it significantly reduced the outward current while abolishing the inward current. It also slows the fast repolarization of the action potential and obliterates the voltage response characteristic of delayed rectification to large outward currents. It completes the tetrodotoxin for the same membrane binding site, thereby suggesting that the same molecule interferes with both the sodium and the potassium channels. A new working hypothesis is proposed in which tetrodotoxin and chiriquitoxin are postulated to bind to a membrane receptor located in the outside surface of the muscle fiber membrane. From the structure of tetrodotoxin and a presumed structure of chiriquitoxin, the Na+ and K+ channels have been estimated to be separated from each other by not less than 5 A nor much more than 15 A.

Anatoxin-a (AnTX) was shown to be a highly potent and stereospecific agonist at nicotinic synapses in frog skeletal muscle and Torpedo electric organs. AnTX binds to the nicotinic-acetylcholine receptor with a higher affinity than for acetylcholine (ACh) but does not bind to sites in the receptor-gated ionic channel. (+)AnTX caused receptor desensitization, i.e., the loss of agonist-stimulated binding of histrionicotoxin to an allosteric site with time, at a rate significantly slower than that of ACh. Single channel patch clamp recordings indicated that the conductance of channels activated by (+)AnTX (28 pS) and ACh (27 pS) were similar. The (+)AnTX-activated channels contained rapid closing events, the burst times caused by the toxin were shorter than those caused by ACh but had similar voltage dependencies, and the number of short closures per burst was constant at all potentials with both agonists. The bursts of rapid openings and rapid closures (tau = 0.4 msec) appear to result from repetitive opening and closing of the (+)AnTX-bound receptor-ion channel. It is concluded that the semirigid molecule and secondary amine (+)AnTX is a more potent agonist than ACh or carbamylcholine because of a higher affinity for the receptor. At various concentrations the toxin activates the appearance of channels with the same conductances as ACh-induced channels but with a shorter channel lifetime.

The effects of pure philanthotoxin (PhTX), a component of the venom of the wasp Philanthus triangulum, were studied on nicotinic acetylcholine receptors (nAChRs) of vertebrates and insects so as to compare their sensitivities and the mechanism of action of PhTX. Electrophysiological techniques were used on frog muscles and cockroach thoracic ganglia and biochemical techniques were applied to membranes from Torpedo electric organ and honeybee brain. PhTX (1-20 microM) inhibited reversibly the indirectly elicited muscle twitch and reduced the endplate current peak amplitude and its decay time constant in a concentration-dependent manner. In patch clamp studies, PhTX (1-5 microM) when combined with acetylcholine, induced a concentration-dependent decrease in frequency of channel openings and in channel open and burst times. The cockroach fast coxal depressor neuron was inhibited by PhTX in a time- and voltage-dependent manner. The initial rate of binding of [3H]perhydrohistrionicotoxin to Torpedo nAChR in the presence of carbamylcholine was inhibited competitively by PhTX. Binding of alpha-[125I] bungarotoxin to electric organ and honeybee brain membranes was inhibited by PhTX. Binding of [3H]acetylcholine to the electric organ receptor was potentiated by low concentrations of PhTX but inhibited by high concentrations. PhTX, therefore, inhibits both vertebrate and insect nAChRs, which may be important molecular targets for its toxicity. It is suggested that PhTX at high concentration may have some competitive action on nAChR, but it acts mainly as a blocker of the ion channel of the nAChR in its open conformation.

1 The action of C5-decahydrohistrionicotoxin (C5-HTX) has been investigated on the extrajunctional acetylcholine (ACh) receptors of denervated rat muscle. 2 C5-HTX causes both a rapid and slow reduction in amplitude of iontophoretic ACh potentials evoked at all frequencies from the extrajunctional receptors. 3 C5-HTX also causes a time-dependent inhibition of the iontophoretic potentials evoked at frequencies greater than 0.02 Hz. This inhibition was observed either alone or superimposed upon desensitization, and may be caused by a similar mechanism to desensitization.

Tetraethylammonium (Et 4 N + ) ions depressed the amplitude and accelerated the decay rate of spontaneously occurring and nerve-evoked endplate currents (EPCs) in frog sartorius muscle. The relationship between peak EPC amplitude and membrane potential became nonlinear in the presence of 100 μM Et 4 N + , and with drug concentrations of 250 μM or greater the current-voltage relationship exhibited negative conductance in the hyperpolarized region. Et 4 N + modified the exponential dependence of the EPC decay on membrane potential such that the decays between -150 and -50 mV were abbreviated and voltage independent but remained near control levels at more positive membrane potentials. The minimal effective concentration of Et 4 N + for altering the EPC time course was 10, and maximal effects were attained with 100 μM. Little additional shortening in the EPC decay phase was detected on raising the drug concentration to 1000 μM. Acetylcholine noise analysis revealed a voltage-dependent reduction in the mean channel open time, which was comparable in magnitude to the shortening in the EPC decay, and a depression of single-channel conductance. In concomitant biochemical studies, Et 4 N + was found to inhibit the binding of both [ 3 H]acetylcholine and [ 3 H]perhydrohistrionicotoxin to receptor-rich membranes from the electric organ of Torpedo ocellata with K i values of 200 μM and 280 μM, respectively. These results suggest that Et 4 N + interacts with both the acetylcholine receptor and its associated ionic channel. The voltage-dependent actions of Et 4 N + are attributed to blockade of the ionic channel in closed as well as open conformation.

A new protein, of molecular weight 160,000, was isolated from the skinvenom of the crested newt and partially characterized by bioassays and biochemical methods. This protein shows the same functional properrties as the crude venom, which produces convulsions in mice and is cytotoxic.