Comparative analysis of the effects of synthetic derivatives of batrachotoxin on sodium currents in frog node of Ranvier

1. In voltage-clamp experiments on frog myelinated nerve fibers, the effects of nine synthetic derivatives of batrachotoxin (BTX) obtained from 7,8-dihydrobatrachotoxinin A (DBTX-A) on Na+ currents (INa) have been investigated. 2. Both of 20 alpha-esters of DBTX-A with 2,4,5-trimethylpyrrol-3-carboxylic acid (DBTX-P) and benzoic acid (DBTX) at a 10(-5) M concentration caused modification of INa qualitatively similar to that induced by BTX. 3. The quaternary derivative of DBTX (QDBTX) produced such changes in INa only at a 5.10(-4) M concentration, apparently due to its much lower lipid solubility. 4. Replacement of a -CH2- by a -C = O. group in the homomorpholine ring near the tertiary nitrogen atom abolished the DBTX activity, strongly suggesting the necessity of tertiary nitrogen protonation for the toxin interaction with the channel receptor. 5. Transfer of an 11-hydroxygroup from the alpha- to the beta-position in the DBTX molecule did not decrease its activity in spite of the fact that in the beta-position this group is sterically very hindered. The activity of 11 beta-DBTX is at variance with the prediction of Codding's (1983) "oxygen triad" hypothesis. 6. DBTX-A and compounds obtained from DBTX by oxidation of the 11 alpha-hydroxygroup (K-DBTX), acetylation (Ac-DBTX), or reduction of the hemiketal moiety (H2DBTX) even at a concentration as high as 10(-3) M were able to modify only a very small fraction of the Na channels. However, a clear-cut reversible blocking action on both normal and modified Na channels was observed. 7. These results led us to conclude that BTX modifies the Na channels only in the charged form and hemiketal and 20 alpha-ester moieties provide adequate disposition of toxin on the receptor surface. The inability of H2DBTX, DBTX-A, and K-DBTX and Ac-DBTX to modify most of the Na channels can be explained by a low "probability of correct disposition" of these ligands on the receptor surface.     

A study of properties of batrachotoxin modified sodium channels

A further analysis of the effects of the steroidal alkaloid batrachotoxin (BTX) on sodium channels in frog node of Ranvier has been carried out under voltage-clamp conditions. The main properties of modified channels as compared with those of normal ones are as follows: The rate of channel closing is drastically decreased, whereas that of opening is changed slightly if at all; The steady-state voltage dependence of channel activation is shifted towards more negative potentials by 60-70 mV; Currents through modified channels do not show a decay during maintained depolarization as it is typical for normal channels. However modified channels retain the ability to partial inactivation as shown by experiments with depolarizing prepulses; Sodium against potassium selectivity beyond--20 mV suggesting either nonhomogeneity of the modified channels as for their kinetic and selectivity properties or potential-dependence of ionic selectivity for each channel; The selectivity sequence determined from peak current reversal potential measurements is as follows: H: Na :NH4:K = 528:1:0.47: :0.19; The effective pK value of proton block is decreased by about 0.4; 7) The sensitivity of the channels to tetrodotoxin (TTX) block is practically unchanged.     

Interaction of steroidal alkaloid toxins with calcium channels in neuronal cell lines

Depolarization with 50 mM K+ increased 45Ca2+ uptake into neuronal clonal cell lines NG108-15, N1E-115 and NH15-CA2. In each cell line this depolarization-induced uptake was blocked by inorganic and organic blockers of voltage sensitive calcium channels. However, tetrodotoxin (10(-6) M) was ineffective. Moreover, in the presence of tetrodotoxin, neither batrachotoxin nor veratridine inhibited the depolarization-induced uptake. The novel dihydropyridine BAY K8644 enhanced depolarization-induced 45Ca2+ uptake into each cell line in a nitrendipine reversible fashion. In the presence of tetrodotoxin, the BAY K8644/50 mM K+ stimulated uptake could be partially inhibited by batrachotoxin (10(-6) M) and veratridine (5 X 10(-5) M). These effects were not altered by the presence of scorpion venom (1 microgram/ml). The results indicate that both batrachotoxin and veratridine can modulate the effects of dihydropyridines on the gating properties of voltage sensitive calcium channels.     

Saxitoxin blocks batrachotoxin-modified sodium channels in the node of Ranvier in a voltage-dependent manner.

The inhibition by saxitoxin (STX) of single Na channels incorporated into planar lipid bilayers and modified by batrachotoxin (BTX) previously has been shown to be voltage dependent (Krueger, B.K.,J.F. Worley, and R. J. French, 1983, Nature [Lond.], 303:172-175; Moczydlowski, E., S. Hall, S. S. Garber, G. S. Strichartz, and C. Miller, 1984, J. Gen. Physiol., 84:687-704). We tested for such a voltage dependence of STX block of the Na current in voltage-clamped frog nodes of Ranvier. The block by STX of normal Na channels showed no modulation in response to maintained (20 s) changes of the membrane potential or to a train of brief pulses to potentials more positive than the holding potential. However, when the nodal channels were modified by BTX, the train of pulses produced a modulation of the block of the Na current by STX. The modulation of STX block depended on the voltage of the conditioning pulses and this voltage dependence agreed well with that predicted from the single channel studies over the membrane potential range used in those studies. In addition, we found that the voltage dependence of STX block was manifest only at potentials equal to or more positive than required to activate the channels. Most of the apparent differences among data from single channels in bilayers, equilibrium binding studies of STX, and the experiments described here are resolved by the hypotheses that (a) STX binding to open channels is voltage dependent, and (b) the affinities of STX for closed and inactivated channels are independent of voltage, equal, and less than the open channel affinity at potentials less than 0 mV. Whether these hypotheses apply to the STX block of all Na channels or just of BTX-modified channels remains to be determined.     

Synthesis and biological activity of 7,8-dihydro derivatives of batrachotoxinin interacting with fast sodium channels

7,8-Dihydrobatrachotoxinin (A) (I) was synthesized from 11 alpha-hydroxyprogesterone (III) by a 37-stage procedure. Trimethylpyrrolcarboxylate, benzoate as well as 2-azido-benzoate derivatives of (I) were obtained by mixed anhydride technique, the latter two derivatives being prepared also with tritium atoms in aromatic rings (sp. radioactivity about 28 Cu/mmol). Upon interaction with rat brain synaptosomes the apparent Kd of 7,8-dihydrobatrachotoxinin A 20 alpha-[4-3H]benzoate (Iv) was about 2,5 x 10(-6) M. The (Iv) specific binding was inhibited by aconitine with K0,5 = 1,3 x 10(4) M. Anemonia sulcata toxin II (ATX II) enhanced (Iv) affinity for the receptor up to 7 x 10(-7) M, the maximum binding capacity being 2,5 pmol/mg of protein. Benzocaine and tetracaine competitively displaced specifically bound toxin with K0,5 = 3,1 x 10(-4) M and 5,7 x 10(-7) M, respectively, in the presence of 10(-5) M ATX II. 2-Azido[5-3H]benzoate derivative (Id) was shown to be an effective probe for covalent labeling of the alkaloid toxin receptor of the sodium channel.     

Differences in the blocking action of benzocaine and amines on batrachotoxin-modified sodium channels of the Ranvier node

Experiments by the voltage clamp method on Ranvier nodes of the frog nerve fiber showed that batrachotoxin sharply reduces the sensitivity of sodium channels to the blocking action of various tertiary (trimecaine, procaine, ajmaline, strychnine) and quaternary (QX-572, N-propylajmaline) amines but has no appreciable effect on blocking of sodium channels by neutral benzocaine. Inhibition of batrachotoxin-modified sodium currents by trimecaine is distinctly time-and potential-dependent in character. None of the amines tested gives rise to frequency-dependent (cumulative) blocking of the modified channels. Unblocking of these channels during rinsing of the node takes place much faster than unblocking of normal channels. The time course of recovery of the normal and modified currents after blocking by benzocaine is about the same. Relations between batrachotoxin "receptors" and the various blocking agents in the sodium channel are discussed.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 636–643, November–December, 1982.     

The V410M mutation associated with pyrethroid resistance in Heliothis virescens reduces the pyrethroid sensitivity of house fly sodium channels expressed in Xenopus oocytes

Some strains of Heliothis virescens carry a novel sodium channel mutation, corresponding to the replacement of Val410 by Met (designated V410M) in the house fly Vssc1 sodium channel, that is genetically and physiologically associated with pyrethroid resistance. To test the functional significance of this mutation, we created a house fly Vssc1 sodium channel containing the V410M mutation by site-directed mutagenesis, expressed wildtype and specifically mutated sodium channels in Xenopus laevis oocytes, and evaluated the effects of the V410M mutation on the functional and pharmacological properties of the expressed channels by two-electrode voltage clamp. The V410M mutation caused depolarizing shifts of approximately 9mV and approximately 5mV in the voltage dependence of activation and steady-state inactivation, respectively, of Vssc1 sodium channels. The V410M mutation also reduced the sensitivity of Vssc1 sodium channels to the pyrethroid cismethrin at least 10-fold and accelerated the decay of cismethrin-induced sodium tail currents. The degree of resistance conferred by the V410M mutation in the present study is sufficient to account for the degree of pyrethroid resistance in H. virescens that is associated with this mutation. Although Val410 is located in a sodium channel segment identified as part of the binding site for batrachotoxin, the V410M mutation did not alter the sensitivity of house fly sodium channels to batrachotoxin. The effects of the V410M mutation on the voltage dependence and cismethrin sensitivity of Vssc1 sodium channels were indistinguishable from those caused by another sodium channel point mutation, replacement of Leu1014 by Phe (L1014F), that is the cause of knockdown resistance to pyrethroids in the house fly. The positions of the V410M and L1014F mutations in models of the tertiary structure of sodium channels suggest that the pyrethroid binding site on the sodium channel alpha subunit is located at the interface between sodium channel domains I and II.     

Ionic current through batrachotoxin-modified sodium channels of the ranvier node membrane at high positive and negative potentials

Ionic current through batrachotoxin (BTX)-modified sodium channels within a wide range of membrane potentials were measured by the voltage clamp method on the membrane of a myelinated frog nerve fiber. At high positive voltages (above +80 mV) the current decreased with time; with an increase in voltage the steady-state level of the currents fell. The results of measurement of "instant" currents showed that this phenomenon is connected with a decrease in overall conductivity of the modified channels. Scorpion toxin had no significant effect on the kinetics of decline of the currents. This indicates that they are due to processes which differ from ordinary inactivation. In the presence of procaine, at high positive voltages slow (tens of milliseconds) potential-dependent blocking of BTX-modified channels was observed. An increase in negative potentials above ?100 mV caused a decrease in "instant" currents, connected with rapid potential-dependent blocking of BTX-modified sodium channels by calcium ions.     

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers.

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.     

Potential-dependent changes in ionic selectivity of batrachotoxin-modified sodium channels of frog nerve fiber

Ionic currents through sodium channels modified by batrachotoxin were measured by the voltage clamp method on a myelinated frog nerve fiber membrane. The reversal potential (E_rev) of steady-state currents was shown to be on the average 5 mV less positive than E_rev corresponding to the initial (peak) values of the currents. The results of control experiments using procaine and tetrodotoxin showed that the change in E_rev observed during a depolarizing pulse is not connected with the presence of unmodified sodium channels or unblocked potassium channels, with nonlinearity of leakage, or with a change in transmembrane gradients of current-carrving cations. In experiments with measurement of "instant" currents it was shown that E_rev becomes less positive as the amplitude and duration of preliminary depolarization increase. The results support the view that sodium-potassium selectivity of batrachotoxin-modified sodium channels depends on potential.     

Voltage dependence of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier

Sodium current and sodium channel intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). BTX caused an approximately threefold increase in steepness of the Q vs. voltage relationship and a 50-mV negative shift in its midpoint. The maximum amount of intramembrane charge was virtually identical before and after BTX treatment. BTX treatment eliminated the charge immobilization observed in untreated nodes after relatively long depolarizing pulses and slowed the rate of OFF charge movement after a pulse. After BTX treatment, the voltage dependence of charge movement was the same as the steady-state voltage dependence of sodium conductance activation. The observations are consistent with the hypothesis that BTX induces an aggregation of the charged gating particles associated with each channel and causes them to move as a unit having approximately three times the average valence of the individual particles. Movement of this single aggregated unit would open the BTX-modified sodium channel.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins

Regulation of Na+ channels by neurotoxins has been studied in pinched- off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.     

BTX modification of Na channels in squid axons. I. State dependence of BTX action

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.     

Selectivity and sensitivity to hydrogen ion blocking of batrachotoxin-modified sodium channels in nerve fiber membrane

Currents through batrachotoxin-modified sodium channels were measured by the voltage clamp method on the Ranvier node membrane. In experiments with replacement of Na⁺ in the external solution by K⁺ or NH ₄ ⁺ the following series of permeabilities, determined as reversal potentials according to the equation of a static field, was obtained — P_Na: \(P_{NH_4 }\) :P_K=1:0.47:0.19. The relative permeability for H⁺ was determined by measuring currents after replacement of Na⁺ in the external solution by nonpenetrating choline ions and lowering pH to 3.7–3.8. The ratio p_H/p_Na for sodium channels modified by batrachotoxin averaged 528±46. Modified channels were less sensitive to the blocking action of H⁺ than normal sodium channels. The difference in the effective values of pK of the acid group of normal and modified channels was 0.40–0.45.     

State-dependent block underlies the tissue specificity of lidocaine action on batrachotoxin-activated cardiac sodium channels.

We have identified two kinetically distinct modes of block, by lidocaine, of cardiac sodium channels, activated by batrachotoxin and incorporated into planar lipid bilayers. Here, we analyze the slow blocking mode which appears as a series of nonconducting events that increase in frequency and duration with increasing lidocaine concentrations. This type of block occurred rarely, if at all, for the skeletal muscle sodium channel subtype. Kinetic analysis showed that a linear open-closed-blocked model is sufficient to account for the major features of our data. Slow block occurs from a long closed state that is a distinguishing characteristic of cardiac channels under these conditions. Slow block showed no significant voltage dependence in the range of -60 to -20 mV for which the detailed kinetic analysis was performed, and was not elicited by application of the permanently charged lidocaine derivative QX-314. By contrast, the fast block, described in the companion paper, results from drug binding to the open state, and is similar for cardiac and skeletal muscle sodium channels. Application of trypsin to the cytoplasmic end of the channel eliminates both the spontaneous, long, gating closures and slow block. Thus, the lidocaine-sensitive closed state of batrachotoxin-activated cardiac sodium channels exhibits a protease susceptibility resembling that of the inactivated state of unmodified sodium channels. It is the slow block caused by lidocaine binding to this closed state that underlies the channel-subtype specificity of lidocaine action in our experiments.     

Fusion of native or reconstituted membranes to liposomes, optimized for single channel recording.

We here describe a protocol for fusing vesicles into large structures suitable for patch clamp recording. The method may be used with native membrane vesicles or with liposomes containing reconstituted/purified ion channels. The resulting unilamellar membranes exhibit high channel surface abundance, yielding multiple channels in the average excised patch. The procedure has been used to record voltage-sensitive Na channels from three native membrane preparations (eel electroplax, rat skeletal muscle, squid optic nerve), and from reconstituted protein purified from eel electroplax. Channels treated with batrachotoxin (BTX) displayed characteristic activation voltage dependence, conductances, selectivity, and sensitivity to saxitoxin (STX).     

20(S)-protopanaxadiol and the ginsenoside Rh2 inhibit Na+ channel-activated depolarization and Na+ channel-dependent amino acid neurotransmitter release in synaptic fractions isolated from mammalian brain

The ginsenoside Rh(2) and its aglycone 20(S)-protopanaxadiol are known to inhibit the binding of [(3)H]batrachotoxinin 20alpha-benzoate to site 2 on voltage-gated sodium channels and electrophysiological investigations conducted by others have shown that ginsenosides cause voltage-dependent inhibition of reconstituted forms of the sodium channel. Here we describe the actions of Rh(2) and 20(S)-protopanaxadiol on sodium channel function and release of neurotransmitters resulting from activation of native sodium channels in synaptic preparations isolated from whole mouse brain. Rh(2) and 20(S)-protopanaxadiol inhibited veratridine-dependent (tetrodotoxin-suppressible) depolarization of synaptoneurosomes as determined using the rhodamine 6G method although 20(S)-protopanaxadiol was more potent as an inhibitor than Rh(2). Veratridine- (sodium channel-) dependent release of the neurotransmitters L-glutamate and GABA was almost fully inhibited by 20(S)-protopanaxadiol, however, less complete inhibition was observed with Rh(2). At its maximum inhibitory concentration, Rh(2) also produced release of l-glutamate and GABA from synaptosomes, in contrast to 20(S)-protopanaxadiol. We conclude that low to moderate micromolar concentrations of Rh(2) and 20(S)-protopanaxadiol inhibit sodium channel function and sodium channel-activated release of neurotransmitters. Apparently the ginsenoside Rh(2) cannot achieve complete inhibition of sodium channel-activated transmitter release because at high concentrations it also stimulates release.     

Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements

We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of both open-state dwell times and closed-state dwell times could be fit by single exponentials. For more depolarized potentials, although the open-state histograms could still be fit by single exponentials, the closed-state histograms required two exponentials. This data together with macroscopic voltage clamp data on the same system could be accounted for by a three-state closed-closed-open model with transition rates between these states that are exponential functions of membrane potential. One of the implications of this model, in agreement with experiment, is that there are always some closed BTX-modified sodium channels, regardless of membrane potential.     

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.