Dual receptor-sites reveal the structural basis for hyperactivation of sodium channels by poison-dart toxin batrachotoxin.

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin's potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.

Screening an In-House Isoquinoline Alkaloids Library for New Blockers of Voltage-Gated Na+ Channels Using Voltage Sensor Fluorescent Probes: Hits and Biases.

Voltage-gated Na+ (NaV) channels are significant therapeutic targets for the treatment of cardiac and neurological disorders, thus promoting the search for novel NaV channel ligands. With the objective of discovering new blockers of NaV channel ligands, we screened an In-House vegetal alkaloid library using fluorescence cell-based assays. We screened 62 isoquinoline alkaloids (IA) for their ability to decrease the FRET signal of voltage sensor probes (VSP), which were induced by the activation of NaV channels with batrachotoxin (BTX) in GH3b6 cells. This led to the selection of five IA: liriodenine, oxostephanine, thalmiculine, protopine, and bebeerine, inhibiting the BTX-induced VSP signal with micromolar IC50. These five alkaloids were then assayed using the Na+ fluorescent probe ANG-2 and the patch-clamp technique. Only oxostephanine and liriodenine were able to inhibit the BTX-induced ANG-2 signal in HEK293-hNaV1.3 cells. Indeed, liriodenine and oxostephanine decreased the effects of BTX on Na+ currents elicited by the hNaV1.3 channel, suggesting that conformation change induced by BTX binding could induce a bias in fluorescent assays. However, among the five IA selected in the VSP assay, only bebeerine exhibited strong inhibitory effects against Na+ currents elicited by the hNav1.2 and hNav1.6 channels, with IC50 values below 10 µM. So far, bebeerine is the first BBIQ to have been reported to block NaV channels, with promising therapeutical applications.

Does batrachotoxin autoresistance coevolve with toxicity in Phyllobates poison-dart frogs?

Toxicity is widespread among living organisms, and evolves as a multimodal phenotype. Part of this phenotype is the ability to avoid self-intoxication (autoresistance). Evolving toxin resistance can involve fitness tradeoffs, so autoresistance is often expected to evolve gradually and in tandem with toxicity, resulting in a correlation between the degrees of toxicity and autoresistance among toxic populations. We investigate this correlation in Phyllobates poison frogs, notorious for secreting batrachotoxin (BTX), a potent neurotoxin that targets sodium channels, using ancestral sequence reconstructions of BTX-sensing areas of the muscular voltage-gated sodium channel. Reconstructions suggest that BTX resistance arose at the root of Phyllobates, coinciding with the evolution of BTX secretion. After this event, little or no further evolution of autoresistance seems to have occurred, despite large increases in toxicity throughout the history of these frogs. Our results, therefore, provide no evidence in favor of an evolutionary correlation between toxicity and autoresistance, which conflicts with previous work. Future research on the functional costs and benefits of mutations putatively involved in BTX resistance, as well as their prevalence in natural populations, should shed light on the evolutionary mechanisms driving the relationship between toxicity and autoresistance in Phyllobates frogs.

Computational Structural Pharmacology and Toxicology of Voltage-Gated Sodium Channels.

Voltage-gated sodium channels are targets for many toxins and medically important drugs. Despite decades of intensive studies in industry and academia, atomic mechanisms of action are still not completely understood. The major cause is a lack of high-resolution structures of eukaryotic channels and their complexes with ligands. In these circumstances a useful approach is homology modeling that employs as templates X-ray structures of potassium channels and prokaryotic sodium channels. On one hand, due to inherent limitations of this approach, results should be treated with caution. In particular, models should be tested against relevant experimental data. On the other hand, docking of drugs and toxins in homology models provides a unique possibility to integrate diverse experimental data provided by mutational analysis, electrophysiology, and studies of structure-activity relations. Here we describe how homology modeling advanced our understanding of mechanisms of several classes of ligands. These include tetrodotoxins and mu-conotoxins that block the outer pore, local anesthetics that block of the inner pore, batrachotoxin that binds in the inner pore but, paradoxically, activates the channel, pyrethroid insecticides that activate the channel by binding at lipid-exposed repeat interfaces, and scorpion alpha and beta-toxins, which bind between the pore and voltage-sensing domains and modify the channel gating. We emphasize importance of experimental data for elaborating the models.

Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype-phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the first to demonstrate the genetic basis of autoresistance in frogs with alkaloid defenses.

Selectivity problems with drugs acting on cardiac Na⁺ and Ca²âº channels.

With the increase of our knowledge on cardioactive agents it comes more and more clear that practically none of the currently used compounds shows absolute selectivity to one or another ion channel type. This is particularly true for Na(+) and Ca(2+) channel modulators, which are widely applied in the clinical practice and biomedical research. The best example might be probably the marine guanidine poison tetrodotoxin, which has long been considered as a selective Na(+) channel blocker, while recently it turned out to effectively inhibit cardiac Ca(2+) currents as well. In the present study the cross actions observed between the effects of various blockers of Na(+) channels (such as toxin inhibitors, class I antiarrhythmics and local anesthetics) and Ca(2+) channels (like phenylalkylamines, dihydropyridine compounds, diltiazem and mibefradil) are overviewed in light of the known details of the respective channel structures. Similarly, activators of Na(+) channels, including veratridine and batrachotoxin, are also compared. The binding of tetrodotoxin and saxitoxin to Cav1.2 and Nav1.5 channel proteins is presented by construction of theoretical models to reveal common structures in their pore forming regions to explain cross reactions. Since these four domain channels can be traced back to a common ancestor, a close similarity in their structure can well be demonstrated. Thus, the poor selectivity of agents acting on cardiac Na(+) and Ca(2+) channels is a consequence of evolution. As a conclusion, since the limited selectivity is an intrinsic property of drug receptors, it has to be taken into account when designing new cardioactive compounds for either medical therapy or experimental research in the future.

Batrachotoxin, pyrethroids, and BTG 502 share overlapping binding sites on insect sodium channels.

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.

Monte Carlo simulation for statistical mechanics model of ion-channel cooperativity in cell membranes.

Voltage-gated ion channels are key molecules for the generation and propagation of electrical signals in excitable cell membranes. The voltage-dependent switching of these channels between conducting and nonconducting states is a major factor in controlling the transmembrane voltage. In this study, a statistical mechanics model of these molecules has been discussed on the basis of a two-dimensional spin model. A new Hamiltonian and a new Monte Carlo simulation algorithm are introduced to simulate such a model. It was shown that the results well match the experimental data obtained from batrachotoxin-modified sodium channels in the squid giant axon using the cut-open axon technique.

Accurate quantitative determination of monoaromatic compounds for the monitoring of bioremediation processes.

This work demonstrated that the protocol for sample treatment, necessary to remove the microbial biomass prior to an analysis, is a critical issue for obtaining accurate results when volatile compounds are present. Two phenomena were observed, solute adsorption and stripping in the gas phase in contact with the liquid. It was demonstrated that the best protocol involved centrifugation using poly tetra fluoro ethylene (PTFE) capped tubes completely filled with the liquid suspension, i.e. without any gas phase inside it. This approach allowed a solute loss lower than 1%. The results also indicated that the optimum centrifugation conditions were 10000g at 10 degrees C for 10 min. Alternatively, it was found that the centrifugation technique developed could be used for the experimental determination of the activity coefficient of solubilized volatile compounds. This study additionally highlighted the fact that polyvinylidene fluoride micro filters (PVDF) and propylene GH polypro membranes (GHP) with a pore size of 0.45 microm could be used for biomass separation, although 10-12% monoaromatic adsorption by membrane was still present. In addition, a simple and sensitive method using high performance liquid chromatography (HPLC) with a UV detector set at the optimum point of 208 nm was developed for assessing the concentrations of BTX in samples taken from bioremediation processes. Minimum detection limits of 5, 4 and 10 microg L(-1) were obtained for benzene, toluene and mixed xylenes, respectively.

The cannabinoid receptor agonist CP-55,940 and ethyl arachidonate interfere with [(3)H]batrachotoxinin A 20 alpha-benzoate binding to sodium channels and inhibit sodium channel function.

Recent investigations in our laboratory showed that voltage-gated sodium channels (VGSCs) in brain are sensitive to inhibition by various synthetic cannabinoids and endocannabinoids. The present experiments examined the effects of the cannabinoid-1 (CB1) receptor agonist CP-55,940 and ethyl arachidonate on [(3)H]batrachotoxinin A 20 alpha-benzoate ([(3)H]BTX-B]) binding and VGSC-dependent depolarization of the nerve membrane in synaptoneurosomes isolated from mouse whole brain. CP-55,940 acted as a full inhibitor of [(3)H]BTX-B binding and its IC(50) was established at 22.3 microM. At its maximum effect concentration, ethyl arachidonate achieved partial (approximately 70%) inhibition and was less effective than CP-55,940 as an inhibitor of binding (IC(50)=262.7 microM). The potent CB1 receptor antagonist AM251 (2 microM) had no significant effect on the displacement of [(3)H]BTX-B by either compound (P>0.05). Scatchard analyses showed that CP-55,940 and ethyl arachidonate reduce the binding of [(3)H]BTX-B by lowering its B(max) but ethyl arachidonate also increased the K(d) of radioligand binding. In kinetic experiments, CP-55,940 and ethyl arachidonate were found to boost the dissociation of [(3)H]BTX-B from VGSCs to rates that exceed the maximum velocity achievable by veratridine, indicating they operate as allosteric inhibitors of [(3)H]BTX-B binding. Neither compound was effective at changing the initial rate of association of [(3)H]BTX-B with sodium channels. CP-55,940 and ethyl arachidonate inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes with IC(50)s of 3.2 and 50.1 microM respectively. These inhibitory effects were again not influenced by 2 microM AM251. Our data demonstrate that the potent cannabinoid receptor agonist CP-55,940 and the ethyl ester of arachidonic acid have the ability to associate with VGSCs and inhibit their function independently of effects on CB1 receptors. Binding data comparisons using mouse brain preparations indicate CP-55,940 is approximately 10,000 times more potent as a CB1 receptor ligand than a sodium channel ligand while ethyl arachidonate shows a much smaller differential. Ethyl arachidonate has been shown previously to be the principal metabolite of ethanol in the brains of intoxicated individuals and effects of this ester on VGSCs and CB1 receptors may contribute to the depressant effects of alcohol.

Mechanisms of action of ligands of potential-dependent sodium channels.

Potential-dependent sodium channels play a leading role in generating action potentials in excitable cells. Sodium channels are the site of action of a variety of modulator ligands. Despite numerous studies, the mechanisms of action of many modulators remain incompletely understood. The main reason that many important questions cannot be resolved is that there is a lack of precise data on the structures of the channels themselves. Structurally, potential-dependent sodium channels are members of the P-loop channel superfamily, which also include potassium and calcium channels and glutamate receptor channels. Crystallization of a series of potassium channels showed that it was possible to analyze the structures of different members of the superfamily using the "homologous modeling" method. The present study addresses model investigations of the actions of ligands of sodium channels, including tetrodotoxin and batrachotoxin, as well as local anesthetics. Comparison of experimental data on sodium channel ligands with x-ray analysis data allowed us to reach a new level of understanding of the mechanisms of channel modulation and to propose a series of experimentally verifiable hypotheses.

Trans-channel interactions in batrachotoxin-modified skeletal muscle sodium channels: voltage-dependent block by cytoplasmic amines, and the influence of mu-conotoxin GIIIA derivatives and permeant ions.

External mu-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two mu-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the mu-conotoxin and the DEA-binding site of approximately 15 A. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by approximately 4-fold; and 2), increasing external [Na(+)] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions.

Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers.

R13X derivatives of mu-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean "blocked" durations of several seconds. We studied interactions between two classes of blockers (mu-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, approximately 100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude ("fast" block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X mu-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA ( approximately 4 mM) and DEA ( approximately 30 mM) increase by approximately 20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the mu-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.

Inhibition of [3H]batrachotoxinin A-20alpha-benzoate binding to sodium channels and sodium channel function by endocannabinoids.

A number of putative endocannabinoids were found to modify the binding of [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]BTX-B) to site 2 on voltage-gated sodium channels of mouse brain and achieve functional inhibition of sodium channels in vitro. 2-Arachidonoyl-glycerol (2-AG), arachidonoyl glycerol ether (AGE), N-arachidonoyl-dopamine (NADA) gave almost complete inhibition of [(3)H]BTX-B binding with IC(50) values of 90.4, 51.2 and 20.7 microM, respectively. The CB1 receptor antagonist AM251 (2 microM) had no effect on the displacement of radioligand by these endocanabinoids. Arachidonoyl-glycine (A-Gly) and arachidonoyl-GABA (A-GABA) were apparently less effective inhibitors of [(3)H]BTX-B binding giving 14.8+/-2.2 and 23.9+/-4.8% inhibition at 100 microM. Phenylmethanesulphonylfluoride (PMSF) did not alter the inhibitory effects of 2-AG, AGE, NADA and A-Gly on binding, but the efficacy of 100 microM A-GABA was increased by 60.3+/-6.3% (P<0.05). Scatchard analyses showed that 2-AG, AGE and NADA reduce the binding of [(3)H]BTX-B by lowering B(max) although increases in K(D) were also evident for AGE and NADA. Our kinetic experiments found that 2-AG, AGE and NADA increase the dissociation velocity of radioligand from site 2 on sodium channels demonstrating that these endocannabinoids operate as allosteric inhibitors of [(3)H]BTX-B binding. 2-AG, AGE and NADA inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes at low micromolar concentrations and again the capacities of A-Gly and A-GABA to inhibit this response were less pronounced. The three most effective endocannabinoids (2-AG, AGE and NADA) were then examined in a synaptosomal transmitter release assay where they were observed to inhibit sodium channel- (veratridine-dependent) release of l-glutamate and GABA in the low micromolar range. These effects also occurred through a mechanism that was not influenced by 2 microM AM251. It is concluded that direct inhibition of sodium channel function leading to reduced neuronal excitation and depression of presynaptic release of amino acid transmitters is a property shared by several endocannabinoids.

Serine-401 as a batrachotoxin- and local anesthetic-sensing residue in the human cardiac Na+ channel.

Sequence alignment of four S6 segments in the human cardiac Na+ channel suggests that serine-401 (hNav1.5-S401) at D1S6 along with asparagine-927 (N927) at D2S6, serine-1458 (S1458) at D3S6, and phenylalanine-1760 (F1760) at D4S6 may jointly form a pore-facing S(401)N(927)S(1458)F(1760) ring. Importantly, this pore-facing structure is adjacent to the putative gating-hinge (G(400)G(926)G(1457)S(1759)) and close to the selectivity filter. Within this SNSF ring, only S401 has not yet been identified as a batrachotoxin (BTX) sensing residue. We therefore created S401 mutants with 12 substitutions (S401C,W,P,A,K,F,R,E,L,N,D,G) and assayed their BTX sensitivity. All S401 mutants expressed Na+ currents but often with altered gating characteristics. Ten mutants were found sensitive to 5 muM BTX, which eliminated Na+ channel fast inactivation after repetitive pulses. However, S401K and S401R became BTX resistant. In addition, the block of open and inactivated hNav1.5-S401K Na+ channels by local anesthetic bupivacaine was reduced by approximately 8-10-fold, but not the block of resting Na+ channels. Qualitatively, these ligand-sensing phenotypes of hNav1.5-S401K channels resemble those of S1458K and F1760K channels reported earlier. Together, our results support that residue hNav1.5-S401 at D1S6 is facing the inner cavity and is in close proximity to the receptor sites for BTX and for local anesthetics.

Vanilloid (subtype 1) receptor-modulatory drugs inhibit [3H]batrachotoxinin-A 20-alpha-benzoate binding to Na+ channels.

This investigation was conducted to provide further insight into the effects of vanilloid (subtype 1) receptor (VR1) drugs at voltage-gated sodium channels and examine the potential of this interaction to influence release of neurotransmitters from synaptosomes prepared from mammalian brain. The VR1 modulatory drugs capsaicin, olvanil and capsazepine inhibited the binding of batrachotoxinin-A 20-alpha-benzoate ([(3)H]BTX-B) to receptor site 2 of voltage-gated sodium channels. All drugs reduced the affinity of radioligand for sodium channels, and capsazepine also decreased the number of [(3)H]BTX-B binding sites. In kinetic experiments, no reduction in radioligand association rate was found, but capsaicin, olvanil and capsazepine all enhanced the dissociation rate of [(3)H]BTX-B. All drugs inhibited veratridine-evoked release of L-glutamic acid, gamma-amino butyric acid and L-aspartic acid from synaptosomes; however, their inhibitory effects on transmitter release were much weaker when 35 mM potassium chloride was used to depolarize synaptosomes. The study compounds, in common with other central nervous system depressants, interact with a region on the voltage-gated sodium channel that permits negative allosteric coupling with receptor site 2 and this mechanism likely accounts for blockade of sodium channel-activated transmitter release.

Irreversible block of cardiac mutant Na+ channels by batrachotoxin.

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.

Natural products from ginseng inhibit [3H]batrachotoxinin A 20-alpha-benzoate binding to Na+ channels in mammalian brain.

A [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]BTX-B) binding assay was used to investigate the interaction of two ginseng aglycones (20(S)protopanaxadiol and 20(S)protopanaxatriol) and Rh(2) (a monoglucoside of 20(S)protopanaxadiol) with voltage-gated sodium channels in mouse brain. All compounds inhibited the binding of [(3)H]BTX-B and IC(50)s were established at 42 microM (20(S)protopanaxadiol), 79 microM (20(S)protopanaxatriol) and 162 microM (Rh(2)). Scatchard analysis confirmed that 20(S)protopanaxadiol and Rh-2 reduced the B(max) of [(3)H]BTX-B binding while Rh(2) also increased the K(d). At IC(50) concentrations and above, 20(S)protopanaxadiol and Rh(2) increased the dissociation of the [(3)H]BTX-B:sodium channel complex above that produced by a saturating concentration of veratridine, but failed to reduce the rate of association of [(3)H]BTX-B with sodium channels. Reversal of the inhibition of [(3)H]BTX-B binding by 20(S)protopanaxadiol and Rh(2) occurred slowly. We conclude that the 20(S)protopanaxadiol and the less potent inhibitor Rh(2) destabilize BTX-B-activated sodium channels through non-covalent allosteric modification of neurotoxin binding site 2.

Sodium channel activators: model of binding inside the pore and a possible mechanism of action.

Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state.

Inactivation-deficient human skeletal muscle Na+ channels (hNav1.4-L443C/A444W) in stably transfected HEK-293 cells.

After transient transfection of an hNav1.4-L443C/A444W mutant clone, HEK-293 cells exhibited large inactivation-deficient Na+currents. We subsequently established a stable cell line expressing robust inactivation-deficient Na+currents. Persistent late Na+currents were far more sensitive to block by class 1 anti-arrhythmic flecainide, mexiletine, propafenone, and amiodarone at 10 microM than peak Na+currents. Such results support a hypothesis that persistent late Na+currents are in vivo targets for class 1 anti-arrhythmic drugs at their therapeutic plasma concentrations. Stably transfected HEK-293 cells expressing robust inactivation-deficient Na+currents will likely be suitable for screening novel drugs that target persistent late Na+currents selectively.