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Brightly colored frogs from the Phyllobates genus hop about the Colombian rain forest without fear of being gobbled up by predators, thanks to (-)-batrachotoxin, a potent steroidal toxin in their skin. Natives of the region capture the frogs to use this toxin on the tips of blowdarts, giving the amphibians their more common name: poison dart frogs. Toxic to the heart and nervous system, (-)-batrachotoxin is an agonist that forces open voltage-gated sodium ion channels. This property also makes the compound a useful biochemical tool for studying these channels, which are important for cardiac health and pain perception. But as Phyllobates frogs have become endangered, supplies of (-)-batrachotoxin have become scarce. Now, chemists at Stanford University report at 24-step asymmetric synthesis of (-)-batrachotoxin, giving researchers an opportunity to generate the molecule as well as novel analogs, which could be used as biological probes and, possibly, even as drugs (Science 2016

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 twoelectrode voltage clamp. The V410M mutation caused depolarizing shifts of |9 mV and |5 mV 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 α subunit is located at the interface between sodium channel domains I and II. © 2001 Elsevier Science Ltd. All rights reserved.

Batrachotoxins, including many congeners not previously described, were detected, and relative amounts were measured by using HPLC-mass spectrometry, in five species of New Guinean birds of the genus Pitohui as well as a species of a second toxic bird genus, Ifrita kowaldi . The alkaloids, identified in feathers and skin, were batrachotoxinin-A cis -crotonate (1), an allylically rearranged 16-acetate (2), which can form from 1 by sigmatropic rearrangement under basic conditions, batrachotoxinin-A and an isomer (3 and 3a, respectively), batrachotoxin (4), batrachotoxinin-A 3′-hydroxypentanoate (5), homobatrachotoxin (6), and mono- and dihydroxylated derivatives of homobatrachotoxin. The highest levels of batrachotoxins were generally present in the contour feathers of belly, breast, or legs in Pitohui dichrous , Pitohui kirhocephalus, and Ifrita kowaldi . Lesser amounts are found in head, back, tail, and wing feathers. Batrachotoxin (4) and homobatrachotoxin (6) were found only in feathers and not in skin. The levels of batrachotoxins varied widely for different populations of Pitohui and Ifrita , a result compatible with the hypothesis that these birds are sequestering toxins from a dietary source.

Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.

Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.

Background Chiral local anesthetics, such as ropivacaine and levobupivacaine, have the potential advantage over racemic mixtures in showing reduced toxic side effects. However, these S-(levo, or "-")isomers also have reportedly lower potency than their optical antipode, possibly resulting in no advantage in therapeutic index. Potency for local anesthetics inhibiting Na+ channels or action potentials depends on the pattern of membrane potential and so also does the stereopotency ratio. Here the authors have quantitated the stereopotencies of R-, S-, and racemic bupivacaine, comparing several in vitro assays of neuronal Na+ channels with those from in vivo functional nerve block, to establish relative potencies and to understand better the role of different modes of channel inhibition in overall functional anesthesia. Methods The binding of bupivacaine to Na+ channels was assessed indirectly by its antagonism of [3H]-batrachotoxin binding to rat brain synaptosomes. Inhibition of Na+ currents by bupivacaine was directly assayed in voltage-clamped GH-3 neuroendocrine cells. Neurobehavioral functions were disrupted by bupivacaine percutaneously injected (0.1 ml; 0.0625-1.0%) at the rat sciatic nerve and semiquantitatively assayed. Concentration-dependent actions of R-, S-, and racemic bupivacaine were compared for their magnitude and duration of action. Results Competitive batrachotoxin displacement has a stereopotency ratio of R:S = 3:1. Inhibition of Na+ currents with different prepulse potentials shows that S > R potency when the membrane is hyperpolarized, and R > S potency when it is depolarized from normal resting values. Functional deficits assayed in vivo usually demonstrate no consistent enantioselectivity and only a modest stereopotency (R:S = 1.2-1.3) for peak analgesia achieved at the lowest doses. Other functions display no significant stereopotency in either the degree, the duration, or their product (area under the curve) at any dose. Conclusion Although the in vitro actions of bupivacaine showed stereoselectivity ratios of 1.3-3:1 (R:S), in vivo nerve block at clinically used concentrations showed much smaller ratios for peak effect and no significant enantioselectivity for duration. A primary role for the blockade of resting rather than open or inactivated Na+ channels may explain the modest stereoselectivity in vivo, although stereoselective factors controlling local disposition cannot be ruled out. Levo-(S-)bupivacaine is effectively equipotent to R- or racemic bupivacaine in vivo for rat sciatic nerve block.

Grayanotoxin I (GTX I) is a diterpenoid extracted from the family of Ericaceae that binds to Na(+) channels and causes persistent activation. We investigated the interaction of GTX I with the amino acid residues I1575, F1579 and Y1586 in transmembrane segment D4S6 of micro1. In F1579A, GTX shifted the threshold potential about 50 mV in the hyperpolarizing direction and modified Na(+) channels twice as efficiently as that in wild-type. In contrast, these GTX-effects were eliminated completely in the I1575A mutant and were reduced substantially in mutant Y1586A. Lysine substitution for F1579 significantly reduced and for Y1586 completely eradicated the GTX-effect. Our data suggest that the GTX receptor site shares overlapping but non-identical molecular determinants with BTX in D4S6 and has common molecular determinants in D1S6.

Voltage-gated Na(+) channels are the primary targets of local anesthetics (LAs). Amino acid residues in domain 4, transmembrane segment 6 (D4-S6) form part of the LA binding site. LAs inhibit binding of the neurotoxin batrachotoxin (BTX). Parts of the BTX binding site are located in D1-S6 and D4-S6. The affinity of BTX-resistant Na(+) channels mutated in D1-S6 (mu1-N434K, mu1-N437K) toward several LAs is significantly decreased. We have studied how residue mu1-N434 influences LA binding. By using site-directed mutagenesis, we created mutations at mu1-N434 that vary the hydrophobicity, aromaticity, polarity, and charge and investigated their influence on state-dependent binding and stereoselectivity of bupivacaine. Wild-type and mutant channels were transiently expressed in human embryonic kidney 293t cells and investigated under whole-cell voltage-clamp. For resting channels, bupivacaine enantiomers showed a higher potency in all mutant channels compared with wild-type channels. These changes were not well correlated with the physical properties of the substituted residues. Stereoselectivity was small and almost unchanged. In inactivated channels, the potency of bupivacaine was increased in mutations containing a quadrupole of an aromatic group (mu1-N434F, mu1-N434W, mu1-N434Y), a polar group (mu1-N434C), or a negative charge (mu1-N434D) and was decreased in a mutation containing a positive charge (mu1-N434K). In mutation mu1-N434R, containing the positively charged arginine, the potency of S(-)-bupivacaine was selectively decreased, resulting in a stereoselectivity (stereopotency ratio) of 3. Similar results were observed with cocaine but not with RAC 109 enantiomers. We propose that in inactivated channels, residue mu1-N434 interacts directly with the positively charged moiety of LAs and that D1-S6 and D4-S6 form a domain-interface site for binding of BTX and LAs in close proximity.

The effect of grayanotoxin (GTX) on site-specific mutants of the alpha-subunit of rat skeletal muscle Na(+) channels (micro1) (micro1-I433K, micro1-N434K and micro1-L437K), which are resistant to batrachotoxin (BTX) (Wang and Wang (1998) Proc Natl Acad Sci USA, 95, 2653-2658) was studied using a whole-cell patch-clamp method. The GTX modification of the Na(+) channels was detected as a characteristic-sustained Na(+) current flow with repetitive pulses. We also studied the GTX action on mutants of the alpha-subunit of rat heart Na(+) channels (RH1) (RH1-V406K and RH1-L410K) which match with micro1-I433 and micro1-L437. All the mutants lost their sensitivity to GTX. This finding indicates that GTX may share a binding site with BTX in transmembrane segment I-S6 of two different Na(+) channel isoforms, micro1 and RH1.

Atracotoxins are novel peptide toxins from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. To analyse their interaction with known sodium channel neurotoxin receptor sites we determined their effect on scorpion toxin, batrachotoxin and saxitoxin binding. Nanomolar concentrations of delta-atracotoxin-Hv1 and delta-atracotoxin-Ar1 completely inhibited the binding of the scorpion alpha-toxin AaH II to rat brain synaptosomes as well as the binding of LqhalphaIT, a scorpion alpha-toxin highly active on insects, to cockroach neuronal membranes. Moreover, delta-atracotoxin-Hv1 cooperatively enhanced batrachotoxin binding to rat brain synaptosomes in an analogous fashion to scorpion alpha-toxins. Thus the delta-atracotoxins represent a new class of toxins which bind to both mammalian and insect sodium channels at sites similar to, or partially overlapping with, the receptor binding sites of scorpion alpha-toxins.

The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-α-benzoate binding affinity, causing a 60-fold increase in K d . Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in K d . In contrast, mutant Y1771A shows no change in BTX binding affinity. For wild-type and mutant Y1771A, BTX shifted the voltage for half-maximal activation ≈40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to ≈60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain–interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on l -type calcium channels.

delta-Atracotoxins from the venom of Australian funnel-web spiders are a unique group of peptide toxins that slow sodium current inactivation in a manner similar to scorpion alpha-toxins. To analyze their interaction with known sodium channel neurotoxin receptor sites, we studied their effect on [3H]batrachotoxin and 125I-Lqh II (where Lqh is alpha-toxin II from the venom of the scorpion Leiurus quinquestriatus hebraeus) binding and on alkaloid toxin-stimulated 22Na+ uptake in rat brain synaptosomes. delta-Atracotoxins significantly increased [3H]batrachotoxin binding yet decreased maximal batrachotoxin-activated 22Na+ uptake by 70-80%, the latter in marked contrast to the effect of scorpion alpha-toxins. Unlike the inhibition of batrachotoxin-activated 22Na+ uptake, delta-atracotoxins increased veratridine-stimulated 22Na+ uptake by converting veratridine from a partial to a full agonist, analogous to scorpion alpha-toxins. Hence, delta-atracotoxins are able to differentiate between the open state of the sodium channel stabilized by batrachotoxin and veratridine and suggest a distinct sub-conductance state stabilized by delta-atracotoxins. Despite these actions, low concentrations of delta-atracotoxins completely inhibited the binding of the scorpion alpha-toxin, 125I-Lqh II, indicating that they bind to similar, or partially overlapping, receptor sites. The apparent uncoupling between the increase in binding but inhibition of the effect of batrachotoxin induced by delta-atracotoxins suggests that the binding and action of certain alkaloid toxins may represent at least two distinguishable steps. These results further contribute to the understanding of the complex dynamic interactions between neurotoxin receptor site areas related to sodium channel gating.

Local anesthetics (LAs) are noncompetitive antagonists of batrachotoxin (BTX) in voltage-gated Na+ channels. The putative LA receptor has been delineated within the transmembrane segment S6 in domain IV of voltage-gated Na+ channels, whereas the putative BTX receptor is within segment S6 in domain I. In this study, we created BTX-resistant muscle Na+ channels at segment I-S6 (micro1-N434K, micro1-L437K) to test whether these residues modulate LA binding. These mutant channels were expressed in transiently transfected human embryonic kidney 293T cells, and their sensitivity to lidocaine, QX-314, etidocaine, and benzocaine was assayed under whole-cell, voltage-clamp conditions. Our results show that LA binding in BTX-resistant micro1 Na+ channels was reduced significantly. At -100 mV holding potential, the reduction in LA affinity was maximal for QX-314 (by 17-fold) and much less for neutral benzocaine (by 2-fold). Furthermore, this reduction was residue specific; substitution of positively charged lysine with negatively charged aspartic acid (micro1-N434D) restored or even enhanced the LA affinity. We conclude that micro1-N434K and micro1-L437K residues located near the middle of the I-S6 segment of Na+ channels can reduce the LA binding affinity without BTX. Thus, this reduction of the LA affinity by point mutations at the BTX binding site is not caused by gating changes induced by BTX alone. We surmise that the BTX receptor and the LA receptor within segments I-S6 and IV-S6, respectively, may align near or within the Na+ permeation pathway.

The interactions of a family of aminopyridine derivatives with Site II of the voltage-dependent sodium channel were examined by measuring the ability of these compounds to inhibit [3H]batrachotoxin binding and veratridine-induced increases in [Ca2+]i. Aminopyridines substituted with indole, carbazole, and pyrrole rings were evaluated. All compounds that had an aromatic ring linked to the amine group of 4-aminopyridine showed positive results in both assays. For example, the most potent compound, besipirdine (N-(n-propyl)-N-(4-pyridinyl)-1H-indol-1-amine), had IC50 values of 5 μM and 23.8 μM in the two assays, respectively. Small substitutions on either the aromatic ring or on 4-aminopyridine did not substantially change their potencies. Indoles linked to the amino group of 2- and 3-aminopyridine also showed positive results. These results indicate that aminopyridine derivatives substituted with an aromatic ring on the amino nitrogen are inhibitors of voltage-dependent sodium channels. Drug Dev. Res. 44:8–13, 1998. © 1998 Wiley-Liss, Inc.

Batrachotoxin (BTX) is a steroidal alkaloid that causes Na + channels to open persistently. This toxin has been used widely as a tool for studying Na + channel gating processes and for estimating Na + channel density. In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine → lysine substitution of the rat muscle Na + channel α-subunit, μ1-N434K, renders the channel completely insensitive to 5 μM BTX when expressed in mammalian cells. This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, μ1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified μ1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional. This finding implies that the μ1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the μ1–434 position apparently diminishes the efficacy of BTX on eliminating Na + channel inactivation. Mutants of two adjacent residues, μ1-I433K and μ1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the μ1-I433, μ1-N434, and μ1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor.

This extensive bilayer study of unpurified human brain channels from non-diseased and tumorous human brain involves more than 300 lipid bilayer experiments. Single channel conductances and subconductances, single channel fractional open times, the voltage-dependence of tetrodotoxin (TTX) block and the steady-state activation behavior of four different human brain synaptosomal preparations have been examined. Reproducible values have been obtained for the molecular electrophysiological parameters and their standard deviations, providing a database for future comparisons involving disease or drug-related changes in molecular sodium channel functions. In comparison with sodium channels from other species and under other experimental conditions, the bilayer system proved to be a reliable experimental setting. Despite the very different histology of the tissue probes, there were no significant differences in any of the examined electrophysiological features.

Extracts of the plant Aconitum spec. are used in traditional Chinese medicine predominantly as anti-inflammatory and analgesic agents, the latter allegedly equally potent as morphine but without any habit-forming potential. As the only pharmacologically active compounds, the C19 diterpenoid alkaloid aconitine, and some of its derivatives, have been proven to be antinociceptive in different analgesic assays, but the mode of action is unknown. To elucidate the mode of action, ten aconitine-like derivatives were investigated with respect to their affinity for voltage-dependent Na+ channels, the action on synaptosomal Na+ and Ca2+ homoeostasis and their antinociceptive, arrhythmogenic and acute toxic properties. Since aconitine is known to bind to site II of Na+ channels, we determined the affinity of the aconitine-like derivatives in vitro to synaptosomal membranes by the [3H]-batrachotoxinin-binding assay and their properties on intrasynaptosomal concentrations of free Na+ and Ca2+ ([Na+]i and [Ca2+]i), both the latter determined fluorometrically with SBFI and Fura-2 respectively. Furthermore, the alkaloids’ arrhythmogenic potential was investigated in guinea-pig isolated atria and the antinociceptive action on formalin-induced hyperalgesia and the acute toxic action estimated in mice. The results show that the alkaloids could be divided into at least three groups. The first is characterized by a high affinity to the site II of Na+ channels (K i about 1.2 μM), the ability to enhance [Na+]i and [Ca2+]i (EC50 about 3 μM), a strong arrhythmogenic action that starts at about 30 nM, an antinociceptive effect (ED50 about 0.06 mg/kg) and high acute toxicity (LD50 values about 0.15 mg/kg). To this group belong aconitine, 3-acetylaconitine and hypaconitine. The second group, with lappaconitine as the only member, has an affinity to Na+ channels an order of magnitude lower (K i = 11.5 μM), less acute toxicity (LD50 about 5 mg/kg), and a two orders of magnitude lower antinociceptive action (ED50 about 2.8 mg/kg) and lower cardiotoxicity (bradycardia observed at 3 μM). Additionally, lappaconitine suppresses the increase in [Ca2+]i of aconitine-stimulated synaptosomes and increases the excitation threshold of left atria, indicating an inhibition of Na+ channels. The other derivatives, i.e. delcorine, desoxydelcorine, karakoline, lappaconidine, lappaconine and lycoctonine, belong to the third group, which has hardly any effects. They have a low affinity to Na+ channels with K i values in the millimolar range, show no effect on synaptosomal [Na+]i and [Ca2+]i, no arrhythmogenic potential up to 100 μM, no antinociceptive activity and low toxicity with LD50 values greater than 50 mg/kg. For the investigated alkaloids we suggest two different antinociceptive-like modes of action. Aconitine, hypaconitine and 3-acetylaconitine may induce a block of neuronal conduction by a permanent cell depolarisation, whereas lappaconitine might act like local anaesthetics. However, because of the low LD50/ED30 quotients of 2–6, the antinociceptive-like action of the Aconitum alkaloids seems to reflect severe intoxication rather than a specific antinociceptive action. The structure/activity relationship shows that alkaloids that activate or block Na+ channels have a benzoyl ester side chain in the C-14 or C-4 positions respectively, whereas the other compounds lack this group.

Human central nervous system (CNS) sodium channels modified by batrachotoxin and incorporated inter voltage-clamped lipid bilayers, were exposed to various concentrations of the opioid alfentanil (0.2-8.0 mM). Alfentanil caused a concentration-dependent and membrane potential independent reduction of the single channel amplitude and the fractional channel open-time. The weighted computer fit of the dose-response curve yielded a maximal conductance block of 50% with an EC50 of 1.3 mM. These effects occurred at levels beyond clinically relevant human serum/brain levels (0.003 mM) but within the predicted concentration range using the Meyer-Overton (lipid solubility/anaesthetic potency) correlation. Thus, human CNS sodium channels are probably not a main target site for the clinical effects of alfentanil but they provide a model system to estimate the proportion of the lipophilic interactions contributing to its overall effect.

Previous studies have shown that chronic administration of class I antiarrhythmic drugs, which have definite inhibitory action on the fast Na + channel, result in up-regulation of cardiac Na + channel expression, and suggest that this effect may contribute to their deleterious effects during chronic administration. Recent studies have shown that the antiarrhythmic effects of free n − 3 polyunsaturated fatty acids (PUFA) are associated with an inhibition of the Na + channel. Whether the PUFA when used chronically will mimic the effect of the class I drugs on the expression of the Na + channel is not known. To answer this question, we determined the level of mRNA encoding cardiac Na + channels and the number of the Na + channels per cell in cultured neonatal rat cardiac myocytes after supplementation of the cells with the n − 3 PUFA eicosapentaenoic acid (EPA), the class I drug mexiletine, or both EPA and mexiletine for 3–4 days. The number of sodium channels was assessed with a radioligand binding assay using the sodium channel-specific toxin [ 3 H]batrachotoxinin benzoate ([ 3 H]BTXB). The supplementation of myocytes with mexiletine (20 μM) induced a 4-fold increase in [ 3 H]BTXB specific binding to the cells. In contrast, chronic treatment with EPA (20 μM) alone did not significantly affect [ 3 H]BTXB binding. However, the combination of EPA with mexiletine produced a 40–50% reduction in the [ 3 H]BTXB binding, compared with that seen with mexiletine alone. RNA isolated from cardiac myocytes was probed with a 2.5-kb cRNA transcribed with T7 RNA polymerase from the clone Na-8.4, which encodes nucleotides 3361–5868 of the α-subunit of the R IIA sodium channel subtype. The changes in the level of mRNA encoding sodium channel α-subunit were correlated with comparable changes in sodium channel number in the cultured myocytes, indicating that regulation of transcription of mRNA or its processing and stability is primarily responsible for the regulation of sodium channel number. These data demonstrate that chronic EPA treatment not only does not up-regulate the cardiac sodium channel expression but also reduces the mexiletine-induced increase in the cardiac sodium channel expression.

Human CNS sodium channels provide a protein model system for our continuing study of anaesthetic drug interactions at the molecular level. The impact of ethanol, an alcohol with general anaesthetic properties, on sodium channel function and their significance for the overall anaesthetic effect was quantified. Sodium channels from human brain cortex tissue were incorporated into voltage-clamped planar lipid bilayers in the presence of batrachotoxin and studied at various ethanol concentrations (0.085 – 0.84 M). Ethanol caused a concentration-dependent and membrane potential independent reduction of the single channel amplitude (major effect) and of the fractional channel open-time (minor effect) with no effect on channel steady-state activation. Severe membrane perturbing effects at the highest ethanol levels terminated the measurements. The weighted computer fit of the concentration-response curve with an estimate of a maximal conductance block of 40% yielded an EC50 of 1.03 M. The EC50 for the 100% maximal theoretical block was calculated to be 3.3 M. These effects occurred at levels far beyond toxic human serum levels (0.1 M; 0.5%). Thus, the human CNS sodium channel is not a main target site for the clinical effects of ethanol and other, more sensitive central receptors are involved in ethanol's mechanism of action. © 1997 John Wiley & Sons, Ltd.

The preparation and SAR of a series of batrachotoxin partial structures that antagonize binding and activity of batrachotoxin are reported in this communication.

Pentacyclic analogues of the potent voltage-gated sodium ion channel agonist batrachotoxin can be accessed through an intermediate furan by exploiting Diels–Alder cycloaddition reactions with ring-strained dienophiles. The use of 3-bromofuran as a 1,2-dianion equivalent, the application of carbamate reductive N-alkylation for homomorpholine ring assembly, and the demonstration of CsF as an effective reagent for generating benzyne, cyclohexyne, and related dienophiles underscore this work.

A facile and general strategy is developed for the stereoselective synthesis of substituted 1,4-oxazepane derivatives through an intramolecular reductive etherification reaction. The effects of the substituents present on the oxonium ion and the position of this ion with respect to the nitrogen atom on the yield and selectivity of the reaction are also studied.

The effects of three vinca derivatives on [3H]batrachotoxin binding in rat cortical synaptosomes, on the inhibition of whole-cell Na+ currents evoked in voltage-clamped cortical neurones of the rat, on the protection against veratridine-induced cell death in cortical cultures and on the maximal electroshock-induced seizures in mice were compared. Vinpocetine, vincamine and vincanol reduced [3H]batrachotoxin binding with IC50 values of 0.34, 1.9 and 10.7 microM, blocked Na+ currents with IC50 values of 44.72 and 40 microM, and protected cortical against veratridine-induced cell death with IC50 values of 0.49, 26 and 33 microM, respectively. Upon i.p. administration, vinpocetine, vincamine and vincanol attenuated maximal electric shock-induced convulsions in a dose-dependent manner with ED50 values of 27, 15.4 and 14.6 mg/kg, respectively. The present findings indicate that the three vinca derivatives are potent blockers of voltage-gated Na+ channels, a mechanism that may contribute at least in part to the pharmacological/therapeutic benefit of these drugs.

The binding site for batrachotoxin, a lipid-soluble neurotoxin acting at Na+ channel receptor site 2, was localized using a photoreactive radiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na+ channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allosteric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit was specifically covalently labeled. The region of the alpha subunit specifically photolabeled by the photoreactive batrachotoxin derivative was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, and anti-peptide antibody recognizing an amino acid sequence adjacent to Na+ channel transmembrane segment IS6 was able to immunoprecipitate up to 70% of the labeled peptides. Analysis of a more complete digestion with trypsin or V8 protease indicated that the batrachotoxin receptor site is formed in part by a portion of domain I. The identification of a specifically immunoprecipitated photolabeled 7.3-kDa peptide containing transmembrane segment S6 from domain I restricted the site of labeling to residues Asn-388 to Glu-429 if V8 protease digestion was complete or Leu-380 to Glu-429 if digestion was incomplete. These results implicate the S6 transmembrane region of domain I of the Na+ channel alpha subunit as an important component of the batrachotoxin receptor site.

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.

Lidocaine effects were studied at the single channel level on batrachotoxin-activated eel electroplax (muscle-derived) and on rat brain sodium channels in planar lipid bilayers to investigate whether these effects were the same on structurally different sodium channels. Lidocaine blocked the open state of brain channels with the same voltage dependence, but with 15-times as high a potency as muscle-derived channels. In brain channels, but not muscle-derived ones, the level of the open channel block showed periods of relief. Lidocaine at microM concentrations stabilized the highest conductance state in both channel types and at mM concentrations stabilized subconductance-like states in electroplax, but not in brain channels. In both channel types, lidocaine increased the lifetime and rate of entry to a long-nonconducting state. Since both channel types were studied under identical lipid and ionic conditions, the observed functional differences in the lidocaine action (effects, potency) must reflect channel structural differences.

1. Besipirdine (HP 749) is a compound undergoing clinical trials for efficacy in treating Alzheimer's disease. Among other pharmacological effects, besipirdine inhibits voltage-dependent sodium and potassium channels. This paper presents a pharmacological study of the interaction of besipirdine with voltage-dependent sodium channels. 2. Besipirdine inhibited [3H]-batrachotoxin binding (IC50 = 5.5 +/- 0.2 microM) in a rat brain vesicular preparation and concentration-dependently inhibited veratridine (25 microM)-stimulated increases in intracellular free sodium ([Na+]i) and calcium ([Ca2+]i) in primary cultured cortical neurones of rat. 3. Besipirdine (30-100 microM) concentration-dependently inhibited (up to 100%) veratridine-stimulated release of [3H]-noradrenaline (NA) from rat cortical slices. 4. When examined in greater detail, besipirdine was found to inhibit [3H]-batrachotoxin binding in vesicular membranes competitively. However, when examined in rat brain synaptosomes, we found that the antagonism by besipirdine was not competitive; that is, the maximal stimulation of [Ca2+]i induced by veratridine decreased with increasing concentrations of besipirdine. 5. These results show that besipirdine is an inhibitor of voltage-sensitive sodium channels and appears to bind to a site close to the batrachotoxin/veratridine binding site.

The extracellular side of single batrachotoxin-activated voltage-dependent Na channels isolated from rat skeletal muscle membranes incorporated into neutral planar lipid bilayers were treated in situ with the carboxyl methylating reagent, trimethyloxonium (TMO). These experiments were designed to determine whether TMO alters Na channel function by a general through-space electrostatic mechanism or by methylating specific carboxyl groups essential to channel function. TMO modification reduced single-channel conductance by decreasing the maximal turnover rate. Modification increased channel selectivity for sodium ions relative to potassium ions as measured under biionic conditions. TMO modification increased the mu-conotoxin (muCTX) off-rate by three orders of magnitude. Modification did not alter the muCTX on-rate at low ionic strength or Na channel voltage-dependent gating characteristics. These data demonstrate that TMO does not act via a general electrostatic mechanism. Instead, TMO targets protein residues specifically involved in ion conduction, ion selectivity, and muCTX binding. These data support the hypothesis that muCTX blocks open-channel current by physically obstructing the ion channel pore.

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.

Batrachotoxin (BTX) is an excitatory component in the skin secretions of dendrobatid frogs, which advertise their armament with the gaudy colors of their lethal skin. BTX is a hydrophobic alkaloid, which crosses cell membranes and activates voltage-gated sodium (Nav) channels of muscle, nerve and heart. BTX binding causes a shift of the activation curve toward more negative voltages, and suppresses fast and slow inactivation resulting in a massive increase in excitability of the tissue and ultimately causing paralysis. BTX is thought to act by binding in the cytoplasmic cavity of the ion-conducting pore, of pseudo-symmetric eukaryotic Nav channels. This binding site is near, or overlapping, receptor sites for amphiphylic inhibitor drugs including local anesthetics, anticonvulsants and anti-arrhythmics.The four homologous domains of eukaryotic Navs parallel the homo-tetrameric arrangement of BacNav channels, which have yielded high-resolution crystal structures. Despite the absence of a “ball-and chain” or “hinged-lid” fast inactivation mechanism, BacNavs display a “pore based” inactivation, that in some cases is quite rapid. Thus, they offer an opportunity to explore BTX actions on a molecular system of gating that is amenable to structural verification.The BacNav channels NaChBac and NavSp1 both show dramatic modification by BTX, with negative shifts of activation and inhibition of inactivation. These actions are enhanced by phenylalanine substitutions of pore domain (S6) residues, at positions which align with Phe residues that are important for BTX activation in eukaryotic Nav channels.

Voltage gated sodium channels initiate signaling in neurons and other cells which go on to become sensations of pleasure and pain, as well as thoughts and feelings. Sodium channel malfunction has been linked with cardiac arrhythmia, epilepsy, and neuropathic pain. We performed large-scale molecular dynamics simulations of the prokaryotic voltage gated sodium channel to probe dynamics and conformational change of channel activation. In collaboration with experimentalists, we propose a model for binding of batrachotoxin.

Ion channels are targets for many naturally occurring toxins and small-molecule drugs. Despite great progress in the X-ray crystallography of ion channels, we still do not have a complete understanding of the atomistic mechanisms of channel modulation by ligands. In particular, the importance of the simultaneous interaction of permeant ions with the ligand and the channel protein has not been the focus of much attention. Considering these interactions often allows one to rationalize the highly diverse experimental data within the framework of relatively simple structural models. This has been illustrated in earlier studies on the action of local anesthetics, sodium channel activators, as well as blockers of potassium and calcium channels. Here, we discuss the available data with a view to understanding the use-, voltage-, and current carrying cation-dependence of the ligand action, paradoxes in structure--activity relationships, and effects of mutations in these ion channels.

A stereoselective intramolecular Diels-Alder reaction has been developed using annelated furans to produce functionalized steroidal nuclei, which can serve as advanced intermediates in the synthesis of batrachotoxin.

The oxazepane ring system present in batrachotoxin has been synthesized within the framework of an advanced intermediate via a novel Michael reaction.

Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel.

The effects of batrachotoxin (BTX) on cloned α-subunit Na+ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na+ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at −100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11×106 mol−1·s−1 at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The KD value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (KD=14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between −20 mV to +50 mV. Below −30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH3 cells. Neutral local anesthetic benzocaine, however, drastically enhanced the steady-state inactivation of BTX-modified NaIIA channels, with its maximal effect around −60 mV. We conclude that BTX can bind and modify the NaIIA α-subunit. However, a specific subtype of α-subunits and/or an unidentified modulating process may be required for the optimal steady-state inactivation of BTX-modified Na+ channels.

Despite a widespread approach to explain the molecular mechanisms of anaesthetic agents, the complex state of "general anaesthesia" is still not completely understood. Voltage-activated sodium channels from human brain cortex served as a model membrane protein to investigate anaesthetic drug-protein interactions using a novel electrophysiological voltage-clamp technique. Sodium channels are already well-characterized important integral membrane proteins responsible for the generation of the fast-propagated action potential and thus are vital components for neuronal signal integration and cell communication. In order to elucidate the molecular interactions of intravenous anaesthetics with single human brain sodium channels, representative compounds of four different clinical intravenous anaesthetic groups were used to correlate different types of clinical anaesthesia with differential anaesthetic effects on the molecular level. Methods. Single sodium channels from human brain cortex were incorporated into artificial phospholipid bilayers and studied under our standard experimental conditions (Electrolyte solution: 500 mM NaCl, 100 mM HEPES, pH 7.4, Temp. 22 – 25 °C) with an electrophysiological voltage-clamp technique. In the presence of a channel activator (1 µM batrachotoxin) single-channel characteristics (fractional open time, single-channel conductance and amplitude, steady-state activation behaviour) were characterized for control conditions and in the presence of various doses of four different anaesthetic agents (pentobarbital, propofal, ketamine, midazolam). Results. During control measurements the investigated human brain sodium channels showed stable and reproducible characteristics on the range expected for batrachotoxin-modified sodium channels in bilayers. After completion of the control measurement the effects of the four different general anaesthetics pentobarbital, propofol, ketamine and midazolam were investigated on the same control sodium channels. All four substances demonstrated a blocking effect of sodium channel conductance (pentobarbital: K50: concentration for 50% block of the maximal conductance block: 0.60 mM; blockmax: maximal conductance block (%): 100%; propofol: K50: 0.02 mM, blockmax: 28%; ketamine: K50: 1.1 mM, blockmax: 71%; midazolam: K50: 0.52 mM, blockmax: 100%). Furthermore, a destabilization of the steady-state activation process could be demonstrated. These effects were dose dependent, but only pentobarbital and propofol demonstrated these effects at or near clinically relevant serum concentrations. Discussion. At the clinical level, "general anaesthesia" is a highly complex phenomenon. Similarly, anaesthetics may demonstrate a multimechanistic mode of action also at the molecular level. In this study all four investigated anaesthetic compounds interacted with at least two primary sodium channel functions, leading to a voltage-independent reduction of the fractional channel open time and an interaction with the steady-state activation behaviour, respectively. The effects of pentobarbital and propofol were detectable at concentrations within the range of serum concentrations achieved during clinical anaesthesia, whereas ketamine and midazolam demonstrated qualitatively similar effects exceeding this range 10- to 50-fold. Thus, the human brain sodium channel might serve as a molecular target only for pentobarbital and propofol. This suggests that different types of clinical anaesthesia may correlate with differential actions of anaesthetics on the molecular level.

Calcium ions, applied internally, externally, or symmetrically, have been used in conjunction with rate-theory modeling to explore the energy profile of the ion-conducting pore of sodium channels. The block, by extracellular and/or intracellular calcium, of sodium ion conduction through single, batrachotoxin-activated sodium channels from rat brain was studied in planar lipid bilayers. Extracellular calcium caused a reduction of inward current that was enhanced by hyperpolarization and a weaker block of outward current. Intracellular calcium reduced both outward and inward sodium current, with the block being weakly dependent on voltage and enhanced by depolarization. These results, together with the dependence of single-channel conductance on sodium concentration, and the effects of symmetrically applied calcium, were described using single- or double-occupancy, three-barrier, two- site (3B2S), or single-occupancy, 4B3S rate-theory models. There appear to be distinct outer and inner regions of the channel, easily accessed by external or internal calcium respectively, separated by a rate- limiting barrier to calcium permeation. Most of the data could be well fit by each of the models. Reducing the ion interaction energies sufficiently to allow a small but significant probability of two-ion occupancy in the 3B2S model yielded better overall fits than for either 3B2S or 4B3S models constrained to single occupancy. The outer ion- binding site of the model may represent a section of the pore in which sodium, calcium, and guanidinium toxins, such as saxitoxin or tetrodotoxin, compete. Under physiological conditions, with millimolar calcium externally, and high potassium internally, the model channels are occupied by calcium or potassium much of the time, causing a significant reduction in single-channel conductance from the value measured with sodium as the only cation species present. Sodium conductance and degree of block by external calcium are reduced by modification of single channels with the carboxyl reagent, trimethyloxonium (TMO) (Worley et al., 1986) Journal of General Physiology. 87:327-349). Elevations of only the outermost parts of the energy profiles for sodium and calcium were sufficient to account for the reductions in conductance and in efficacy of calcium block produced by TMO modification.

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.

Batrachotoxin (BTX), a steroidal alkaloid, and pyrethroid insecticides bind to distinct but allosterically coupled receptor sites on voltage-gated sodium channels and cause persistent channel activation. BTX presumably binds in the inner pore, whereas pyrethroids are predicted to bind at the lipid-exposed cavity formed by the short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6. The alkylamide insecticide (2E,4E)-N-(1,2-dimethylpropyl)-6-(5-bromo-2-naphthalenyl)-2,4-hexadienamide (BTG 502) reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. However, a pyrethroid-sensing residue, Phe(3i17) in IIIS6, which does not face the pore, is essential for the activity of BTG 502 but not for BTX. In this study, we found that three additional deltamethrin-sensing residues in IIIS6, Ile(3i12), Gly(3i14), and Phe(3i16) (the latter two are also BTX-sensing), and three BTX-sensing residues, Ser(3i15) and Leu(3i19) in IIIS6 and Phe(4i15) in IVS6, are all critical for BTG 502 action on cockroach sodium channels. Using these data as constraints, we constructed a BTG 502 binding model in which BTG 502 wraps around IIIS6, probably making direct contacts with all of the above residues on the opposite faces of the IIIS6 helix, except for the putative gating hinge Gly(3i14). BTG 502 and its inactive analog DAP 1855 antagonize the action of deltamethrin. The antagonism was eliminated by mutations of Ser(3i15), Phe(3i17), Leu(3i19), and Phe(4i15) but not by mutations of Ile(3i12), Gly(3i14), and Phe(3i16). Our analysis revealed a unique mode of action of BTG 502, its receptor site overlapping with those of both BTX and deltamethrin.

O. Vital Brazil and M. D. Fontana . Toxins as tools in the study of sodium channel distribution in the muscle fibre membrane. Toxicon31, 1085–1098, 1993.—The number of tetrodotoxin molecules bound to the membrane of the fibres of muscles in normal conditions and after detubulation produced by glycerol-induced osmotic shock pointed to a higher sodium channel density at the surface membrane than at the membrane in the transverse tubules. Study of the maximum rate of rise of the action potential at the junctional and nonjunctional regions of the muscle fibre membrane suggested that the Na+ channel density is also not the same along the muscle fibre membrane, being higher at the junctional region. Further studies on the distribution of the Na+ channel along the muscle fibre membrane were carried out with the use of (1) the loose patch voltage-clamp technique, (2) labelling the Na+ channels with fluorescently labelled scorpion toxins, (3) autoradiography of localized Na+ channels with 125I-labelled scorpion toxins, and (4) toxins that induce persistent activation of the Na+ channel. The studies referred to in (1), (2) and (3) demonstrate that the density of the Na+ channel is much higher at the junctional region than elsewhere in the membrane of the muscle fibre. On the other hand, in experiments carried out on curarized rat diaphragms several sodium channel activating toxins (crotamine, Phoneutria nigriventer venom, its toxin PhTx2, veratrine) were found to produce a much greater depolarization of the membrane at the junctional region than at nonjunctional regions. However, it was also found that some toxins (veratridine, batrachotoxin) depolarized equally well the junctional and nonjunctional regions. Two alternative hypotheses to explain the uniform depolarization of the muscle fibre membrane induced by these toxins are suggested.

Batrachotoxin (BTX) is one of a group of potent lipid-soluble neurotoxins which binds voltage-sensitive sodium channels. Here we show that [3H]batrachotoxinin-A-ortho-azidobenzoate ([3H]BTX-OAB), a photolabile derivative of BTX, binds covalently upon irradiation to the BTX sodium channel site of rat cerebral cortical synaptoneurosomes. Another ligand specific for the BTX sodium channel receptor, batrachotoxinin-A 20-alpha-benzoate (BTX-B), competitively inhibited the specific binding of [3H]BTX-OAB. The specific binding of [3H]BTX-OAB was increased by the addition of Leiurus quinquestriatus quinquestriatus scorpion venom (ScTx) and inhibited by veratridine, a member of the same class of sodium channel activators. Examination of the [3H]BTX-OAB-labeled components revealed that over 90% of the specifically incorporated [3H]BTX-OAB was recovered in lipid extracts of photolabeled synaptoneurosomes. Addition of tetrodotoxin (TTX) to the binding mixture increased the specific incorporation of [3H]BTX-OAB into protein components as much as 15-fold. Increasing the incubation temperature from 25 degree C to 37 degrees C had a similar but less marked effect. We conclude that the BTX binding site lies at a lipid-protein interface and that treatments which induce conformational changes in the sodium channel protein (i.e. addition of TTX) can result in a reorientation of BTX at its binding site relative to the protein and lipid domains of voltage-sensitive sodium channels.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.

1. Amitraz stimulated [ 3 H]batrachotoxin in A 20-α-benzoate ([ 3 H]BTX-B) binding to neural membranes from pyrethroid susceptible (S) and resistant (R) tobacco budworm moths, but N ′-(2,4-xylyl)- N -methylformamidine (SN 49844) stimulated binding only with S moths. 2. Chlordimeform stimulated [ 3 H]BTX-B binding only with R moths, and N -(4-chloro- o -tolyl)- N -methylformamidine (demethylchlordimeform) yielded no significant stimulation with either strain. 3. A mixture of amitraz and deltamethrin, a pyrethroid that previously had been shown to enhance [ 3 H]BTX-B binding with tobacco budworm moths, also gave significant stimulation of radioligand binding with S moths. 4. When membranes were prepared from S moths at various intervals following topical application of amitraz, deltamethrin, or a mixture of amitraz and deltamethrin, biphasic stimulation of [ 3 H]BTX-B binding was observed, with maximum enhancement occurring at 2 and 6 hr. 5. These results provided a basis for suggesting that a formamidine binding site is located on or closely associated with the sodium channel protein. 6. Whether this site is the same as the pyrethroid/DDT binding domain remains to be demonstrated; however, similarities in responses elicited by amitraz and deltamethrin alone and in combination indicate that some relationship may exist.

Synaptosomal binding of [ 3 H]batrachotoxinin A-20-α-benzoate ([ 3 H]BTX-B), a potent sodium channel toxin, was measured in kdr -type resistant (Ectiban-R) and susceptible (CSMA) German cockroaches. Previous studies indicated that the Ectiban-R strain was 8.7-fold cross-resistant to batrachotoxin. Scatchard analysis of [ 3 H]BTX-B binding demonstrated that the resistant and susceptible strains have similar binding affinities ( K D values 230 and 252 n M ) and binding site densities ( B max values, 6.0 and 7.6 pmol/mg protein). Batrachotoxin, aconitine, and veratridine displaced [ 3 H]BTX-B binding in both strains with IC 50 values of 1.0-1.2, 18-19, and 73-77 μ M , respectively. The dihydropyrazole insecticide RH 3421 displaced [ 3 H]BTX-B binding by 13% only at 10 μ M , the highest concentration examined. The pyrethroid insecticide deltamethrin had no effect on [ 3 H]BTX-B binding in either strain. Our results indicate that the binding of site 2 neurotoxins to cockroach sodium channels is not altered in the kdr -type resistant strain. Possible molecular mechanisms of kdr -type resistance are discussed.

Repeated administration of a subconvulsant dose of a local anesthetic will eventually induce seizures, a phenomenon similar to electrical kindling. We have investigated the effect of repeated lidocaine and cocaine administration on the phosphoinositide (PI) hydrolysis induced by batrachotoxin (BTX), a specific Na channel activator. Rats were injected with cocaine or saline daily for 6 days and PI hydrolysis was assayed in sliced frontal cortex. Cocaine treatment had no effect on BTX-induced PI hydrolysis while in vitro cocaine blocked the BTX effect. In a second experiment, rats received daily injections of lidocaine or saline. After a rat developed at least two seizures, it was sacrificed together with a rat receiving lidocaine injections which had never seized and a rat receiving saline injections. Basal, BTX and ibotenic acid (IBO; a glutamate receptor agonist)-stimulated PI hydrolysis did not differ among the three groups in slices of either hippocampus (HC) or piriform cortex (PC) though IBO-stimulated PI hydrolysis was mucg greater in the HC than in the PC. Neither in vitro nor in vivo carbamazepine altered the effect of cocaine on BTX-induced PI hydrolysis. These results demonstrate that local anesthetic kindling does not alter PI hydrolysis coupled to Na channel or IBO activation.

A chiral synthesis of the AB ring system of Batrachotoxin (1) is reported. The synthesis utilises a carbene mediated ring expansion of a chiral hydrindane.