State-dependent mibefradil block of Na+ channels

Mibefradil is a T-type Ca2+ channel antagonist with reported cross-reactivity with other classes of ion channels, including K+, Cl-, and Na+ channels. Using whole-cell voltage clamp, we examined mibefradil block of four Na+ channel isoforms expressed in human embryonic kidney cells: Nav1.5 (cardiac), Nav1.4 (skeletal muscle), Nav1.2 (brain), and Nav1.7 (peripheral nerve). Mibefradil blocked Nav1.5 in a use/frequency-dependent manner, indicating preferential binding to states visited during depolarization. Mibefradil blocked currents of all Na+ channel isoforms with similar affinity and a dependence on holding potential, and drug off-rate was slowed at depolarized potentials (k(off) was 0.024/s at -130 mV and 0.007/s at -100 mV for Nav1.5). We further probed the interaction of mibefradil with inactivated Nav1.5 channels. Neither the degree nor the time course of block was dependent on the stimulus duration, which dramatically changed the residency time of channels in the fast-inactivated state. In addition, inhibiting the binding of the fast inactivation lid (Nav1.5 ICM + MTSET) did not alter mibefradil block, confirming that the drug does not preferentially interact with the fast-inactivated state. We also tested whether mibefradil interacted with slow-inactivated state(s). When selectively applied to channels after inducing slow inactivation with a 60-s pulse to -10 mV, mibefradil (1 microM) produced 45% fractional block in Nav1.5 and greater block (88%) in an isoform (Nav1.4) that slow-inactivates more completely. Our results suggest that mibefradil blocks Na+ channels in a state-dependent manner that does not depend on fast inactivation but probably involves interaction with one or more slow-inactivated state(s).     

A mutation in the local anaesthetic binding site abolishes toluene effects in sodium channels

Toluene is a solvent of abuse that inhibits cardiac sodium channels in a manner that resembles the action of local anaesthetics. The purpose of this work was to analyze toluene effects on skeletal muscle sodium channels with and without beta1 subunit (Nav1.4+beta1 and Nav1.4-beta1, respectively) expressed in Xenopus laevis oocytes and to compare them with those produced in the F1579A mutant channel lacking a local anaesthetic binding site. Toluene inhibited Nav1.4 sodium currents (IC50=2.7 mM in Nav1.4+beta1 and 2.2 mM in Nav1.4-beta1 in a concentration dependent way. Toluene (3 mM) blocked sodium currents in Nav1.4 channels proportionally throughout the entire current-voltage relationship producing inactivation at more negative potentials. Minimal inhibition was produced by 3 mM toluene in F1579A mutant channels. Recovery from inactivation was slower both in Nav1.4 and F1579A channels in the presence of 3 mM toluene. The solvent blocked sodium currents in a use-dependent and frequency-dependent manner in Nav1.4 channels. A single mutation in the local anaesthetic binding site of Nav1.4 channels almost abolished toluene effects. These results suggest that this site is important for toluene action.     

Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides

Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel alpha subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na(v)1.2a, Na(v)1.4, Na(v)1.5, and Na(v)1.8 sodium channel alpha subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat beta1 auxiliary subunit on the sensitivity of the Na(v)1.2a and Na(v)1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel alpha subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na(v)1.4 > Na(v)1.2a > Na(v)1.5 > Na(v)1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na(v)1.8 isoform was most sensitive, the Na(v)1.4 isoform was completely insensitive, and the sensitivities of the Na(v)1.5 and Na(v)1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na(v)1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na(v)1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na(v)1.2a or Na(v)1.4 isoforms with the beta1 subunit was the modest reduction in the sensitivity of the Na(v)1.2a isoform to RH 3421. These results demonstrate that mammalian sodium channel isoforms differ in their sensitivities to pyrazoline-type insecticides.     

Mechanism of action of a polypeptide neurotoxin from the coral Goniopora on sodium channels in mouse neuroblastoma cells

Goniopora toxin (GPT), a polypeptide toxin of 9700 Da isolated from coral, markedly slows inactivation of sodium currents recorded under voltage clamp in mouse neuroblastoma cells. The voltage dependence of sodium channel activation is shifted to more negative membrane potentials by 9.8 +/- 2.1 mV, and the voltage dependence of channel inactivation is shifted to more positive membrane potential by 6.0 +/- 2.5 mV. These actions of GPT are voltage dependent with an e-fold increase in K0.5 for toxin action for each 48.3-mV depolarization between -80 and +40 mV. GPT requires Na+ or another alkali metal cation in the extracellular medium for its effect on sodium channels. The relative effectiveness of the different cations tested is Na+ greater than K+ greater than Rb+ greater than Li+ greater than Cs+ much greater than choline+. Like other polypeptide neurotoxins that slow inactivation of sodium channels, GPT enhances persistent activation of sodium channels by veratridine. However, GPT does not block the binding of 125I-labeled Leiurus scorpion toxin to neurotoxin receptor site 3 on sodium channels at concentrations which effectively slow channel inactivation. Therefore, our results define a new site on the sodium channel at which specific effects on inactivation can occur.     

A negative charge in transmembrane segment 1 of domain II of the cockroach sodium channel is critical for channel gating and action of pyrethroid insecticides

Voltage-gated sodium channels are the primary target of pyrethroids, an important class of synthetic insecticides. Pyrethroids bind to a distinct receptor site on sodium channels and prolong the open state by inhibiting channel deactivation and inactivation. Recent studies have begun to reveal sodium channel residues important for pyrethroid binding. However, how pyrethroid binding leads to inhibition of sodium channel deactivation and inactivation remains elusive. In this study, we show that a negatively charged aspartic acid residue at position 802 (D802) located in the extracellular end of transmembrane segment 1 of domain II (IIS1) is critical for both the action of pyrethroids and the voltage dependence of channel activation. Charge-reversing or -neutralizing substitutions (K, G, or A) of D802 shifted the voltage dependence of activation in the depolarizing direction and reduced channel sensitivity to deltamethrin, a pyrethroid insecticide. The charge-reversing mutation D802K also accelerated open-state deactivation, which may have counteracted the inhibition of sodium channel deactivation by deltamethrin. In contrast, the D802G substitution slowed open-state deactivation, suggesting an additional mechanism for neutralizing the action of deltamethrin. Importantly, Schild analysis showed that D802 is not involved in pyrethroid binding. Thus, we have identified a sodium channel residue that is critical for regulating the action of pyrethroids on the sodium channel without affecting the receptor site of pyrethroids.     

Identification of amino acid residues in the insect sodium channel critical for pyrethroid binding

The voltage-gated sodium channel is the primary target site of pyrethroids, which constitute a major class of insecticides used worldwide. Pyrethroids prolong the opening of sodium channels by inhibiting deactivation and inactivation. Despite numerous attempts to characterize pyrethroid binding to sodium channels in the past several decades, the molecular determinants of the pyrethroid binding site on the sodium channel remain elusive. Here, we show that an F-to-I substitution at 1519 (F1519I) in segment 6 of domain III (IIIS6) abolished the sensitivity of the cockroach sodium channel expressed in Xenopus laevis oocytes to all eight structurally diverse pyrethroids examined, including permethrin and deltamethrin. In contrast, substitution by tyrosine or tryptophan reduced the channel sensitivity to deltamethrin only by 3- to 10-fold, indicating that an aromatic residue at this position is critical for the interaction of pyrethroids with sodium channels. The F1519I mutation, however, did not alter the action of two other classes of sodium channel toxins, batrachotoxin (a site 2 toxin) and Lqhalpha-IT (a site 3 toxin). Schild analysis using competitive interaction of pyrethroid-stereospecific isomers demonstrated that the F1519W mutation and a previously known pyrethroid-resistance mutation, L993F in IIS6, reduced the binding affinity of 1S-cis-permethrin, an inactive isomer that shares the same binding site with the active isomer 1R-cis-permethrin. Our results provide the first direct proof that Leu993 and Phe1519 are part of the pyrethroid receptor site on an insect sodium channel.     

A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms

The specific, acute interaction of tolperisone, an agent used as a muscle relaxant and for the treatment of chronic pain conditions, with the Na(v1.2), Na(v1.3), Na(v1.4), Na(v1.5), Na(v1.6), Na(v1.7), and Na(v1.8) isoforms of voltage dependent sodium channels was investigated and compared to that of lidocaine. Voltage dependent sodium channels were expressed in the Xenopus laevis oocyte expression system and sodium currents were recorded with the two electrode voltage clamp technique. Cumulative dose response relations revealed marked differences in IC(50) values between the two drugs on identical isoforms, as well as between isoforms. A detailed kinetic analysis uncovered that tolperisone as well as lidocaine exhibited their blocking action not only via state dependent association/dissociation with voltage dependent sodium channels, but a considerable fraction of inhibition is tonic, i.e. permanent and basic in nature. Voltage dependent activation was affected to a minor extent only. A shift in steady-state inactivation to more negative potentials could be observed for most drug/isoform combinations. The contribution of this shift to overall block was, however, small at drug concentrations resulting in considerable overall block. Recovery from inactivation was affected notably by both drugs. Lidocaine application led to a pronounced prolongation of the time constant of the fast recovery process for the Na(v1.3), Na(v1.5), and Na(v1.7) isoforms, indicating common structural properties in the local anesthetic receptor site of these three proteins. Interestingly, this characteristic drug action was not observed for tolperisone.     

Insect-selective spider toxins targeting voltage-gated sodium channels.

The voltage-gated sodium (Na(v)) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Na(v) channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Na(v) channel conductance. Magi 2 and Tx4(6-1) slow Na(v) channel inactivation via an interaction with site-3. The delta-palutoxins, and most likely mu-agatoxins and curtatoxins, target site-4. However, their action is complex with the mu-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion beta-toxins, but with both delta-palutoxins and mu-agatoxins slowing Na(v) channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as delta-atracotoxins, are known to target both insect and vertebrate Na(v) channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped.     

Inhibition of neuronal voltage-gated sodium channels by brilliant blue G

Brilliant blue G (BBG), best known as an antagonist of P2X7 receptors, was found to inhibit voltage-gated sodium currents in N1E-115 neuroblastoma cells. Sodium currents elicited from a holding potential of -60 mV were blocked with an IC(50) of 2 μM. Block was enhanced in a use-dependent manner at higher stimulation rates. The voltage-dependence of inactivation was shifted in the hyperpolarizing direction, and recovery from inactivation was slowed by BBG. The most dramatic effect of BBG was to slow recovery from inactivation after long depolarizations, with 3 μM BBG increasing half-time for recovery (measured at -120 mV) from 24 to 854 ms after a 10-s step to 0 mV. These results were mimicked by a kinetic model in which BBG binds weakly to resting channels (K(d) = 170 μM) but tightly to fast-inactivated channels (K(d) = 5 μM) and even more tightly (K(d) = 0.2 μM) to slow-inactivated channels. In contrast to BBG, the structurally related food-coloring dye Brilliant Blue FCF had very little effect at concentrations up to 30 μM. These results show that BBG inhibits voltage-gated sodium channels at micromolar concentrations. Although BBG inhibition of sodium channels is less potent than inhibition of P2X7 receptors, there may be significant inhibition of sodium channels at BBG concentrations achieved in spinal cord or brain during experimental treatment of spinal cord injury or Huntington's disease. Considered as a sodium channel blocker, BBG is remarkably potent, acting with more than 10-fold greater potency than lacosamide, another blocker thought to bind to slow-inactivated channels.     

An inner pore residue (Asn406) in the Nav1.5 channel controls slow inactivation and enhances mibefradil block to T-type Ca2+ channel levels

Mibefradil is a tetralol derivative once marketed to treat hyper-tension. Its primary target is the T-type Ca(2+) channel (IC(50), approximately 0.1-0.2 microM), but it also blocks Na(+),K(+),Cl(-), and other Ca(2+) channels at higher concentrations. We have recently reported state-dependent mibefradil block of Na(+) channels in which apparent affinity was enhanced when channels were recruited to slow-inactivated conformations. The structural determinants controlling mibefradil block have not been identified, although evidence suggests involvement of regions near or within the inner pore. We tested whether mibefradil interacts with the local anesthetic (LA) binding site, which includes residues in the S6 segments of domains (D) I, III, and IV. Mutagenesis of DIII S6 and DIVS6 did not reveal critical binding determinants. Substitution of Asn406 in DI S6 of cardiac Na(v)1.5, however, altered affinity in a manner dependent on the identity of the substituting residue. Replacing Asn406 with a phenylalanine or a cysteine increased affinity by 4- and 7-fold, respectively, thus conferring T-type Ca(2+) channel-like mibefradil sensitivity to the Na(+) channel. A series of other substitutions that varied in size, charge, and hydrophobicity had minimal effects on mibefradil block, but all mutations dramatically altered the magnitude and voltage-dependence of slow inactivation, consistent with data in other isoforms. Channels did not slow-inactivate, however, at the voltages used to assay mibefradil block, supporting the idea that Asn406 lies within or near the mibefradil binding site.     

Structure-activity relationships for the action of 11 pyrethroid insecticides on rat Na v 1.8 sodium channels expressed in Xenopus oocytes

Pyrethroid insecticides bind to voltage-sensitive sodium channels and modify their gating kinetics, thereby disrupting nerve function. This paper describes the action of 11 structurally diverse commercial pyrethroid insecticides on the rat Na v 1.8 sodium channel isoform, the principal carrier of the tetrodotoxin-resistant, pyrethroid-sensitive sodium current of sensory neurons, expressed in Xenopus laevis oocytes. All 11 compounds produced characteristic sodium tail currents following a depolarizing pulse that ranged from rapidly-decaying monoexponential currents (allethrin, cismethrin and permethrin) to persistent biexponential currents (cyfluthrin, cyhalothrin, cypermethrin and deltamethrin). Tail currents for the remaining compounds (bifenthrin, fenpropathrin, fenvalerate and tefluthrin) were monoexponential and decayed with kinetics intermediate between these extremes. Reconstruction of currents carried solely by the pyrethroid-modified subpopulation of channels revealed two types of pyrethroid-modified currents. The first type, found with cismethrin, allethrin, permethrin and tefluthrin, activated relatively rapidly and inactivated partially during a 40-ms depolarization. The second type, found with cypermethrin, cyfluthrin, cyhalothrin, deltamethrin, fenpropathrin and fenvalerate, activated more slowly and did not detectably inactivate during a 40-ms depolarization. Only bifenthrin did not produce modified currents that fit clearly into either of these categories. In all cases, the rate of activation of modified channels was strongly correlated with the rate of tail current decay following repolarization. Modification of Na v 1.8 sodium channels by cyfluthrin, cyhalothrin, cypermethrin and deltamethrin was enhanced 2.3- to 3.4-fold by repetitive stimulation; this effect appeared to result from the accumulation of persistently open channels rather than preferential binding to open channel states. Fenpropathrin was the most effective compound against Na v 1.8 sodium channels from the perspective of either resting or use-dependent modification. When use dependence is taken into account, cypermethrin, deltamethrin and tefluthrin approached the effectiveness of fenpropathrin. The selective expression of Na v 1.8 sodium channels in nociceptive neurons suggests that these channels may be important targets for pyrethroids in the production of paresthesia following dermal exposure.     

Critical molecular determinants of voltage-gated sodium channel sensitivity to mu-conotoxins GIIIA/B

GIIIA/B mu-conotoxins block the rat skeletal muscle sodium channel (rNa(v)1.4) with high affinity by binding to specific residues in the pore. However, human Na(v)1.4 (hNa(v)1.4) channels, which are resistant to block by GIIIA/B, have these same pore residues. We used chimera constructs, site-directed mutagenesis, and electrophysiological techniques to investigate which residues determine GIIIA/B selectivity. Exchange of serine 729 in the D2/S5-S6 linker of rat Na(v)1.4 with leucine (S729L), the corresponding residue in hNa(v)1.4, reduces the sensitivity of rNa(v)1.4 by approximately 20-fold and largely accounts for the differential sensitivity of rNa(v)1.4 and hNa(v)1.4 to both GIIIA and GIIIB. To determine whether D2/S5-S6 linker residues might contribute to the resistance of neuronal channels to GIIIA/B, we exchanged residues in this linker that differed between rNa(v)1.4 and neuronal channels. Substitution of aspargine 732 with lysine (N732K), the corresponding residue in rNa(v)1.1a and rNa(v)1.7, reduced the GIIIB sensitivity of rNa(v)1.4 by approximately 20-fold. The N732K substitution, however, only reduced GIIIA sensitivity of rNa(v)1.4 by approximately 4-fold, demonstrating that GIIIA and GIIIB have distinct interactions with the D2/S5-S6 linker. Our data indicate that naturally occurring variants in the extra-pore region of the D2/S5-S6 linker contribute to the isoform-specific sensitivity of sodium channels to GIIIA/B. Because S729 and N732 are not part of the high-affinity binding site for mu-conotoxins, these extra-pore residues probably influence the accessibility of the toxin to the binding site within the pore and/or the stability of the toxin-channel complex. Our results should aid the development of toxins that block specific neuronal sodium channel isoforms.     

Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel

After opening, Na(+) channels may enter several kinetically distinct inactivated states. Whereas fast inactivation occurs by occlusion of the inner channel pore by the fast inactivation gate, the mechanistic basis of slower inactivated states is much less clear. We have recently suggested that the inner pore of the voltage-gated Na(+) channel may be involved in the process of ultra-slow inactivation (I(US)). The local anesthetic drug lidocaine is known to bind to the inner vestibule of the channel and to interact with slow inactivated states. We therefore sought to explore the effect of lidocaine binding on I(US). rNa(V) 1.4 channels carrying the mutation K1237E in the selectivity filter were driven into I(US) by long depolarizing pulses (-20 mV, 300 s). After repolarization to -120 mV, 53 +/- 5% of the channels recovered with a very slow time constant (tau(rec) = 171 +/- 19 s), typical for recovery from I(US). After exposure to 300 microM lidocaine, the fraction of channels recovering from I(US) was reduced to 13 +/- 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on I(US), indicating that lidocaine has to bind to the inner vestibule of the channel to modulate I(US). We propose that I(US) involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.     

Molecular modeling of local anesthetic drug binding by voltage-gated sodium channels

Voltage-gated sodium (Na+) channels are targets for local anesthetic (LA) drugs that bind in the inner pore of the channel with affinities related to the channel gating states. Our core model of the sodium channel (P loops and S5 and S6 segments from each of the four domains) was closed because it was developed using coordinates from the KcsA channel crystallographic structure. We developed a model of the activated, open channel based on the structure of the open MthK channel, which was characterized by bends at the S6 glycine or serine residues. This created a conformation that allowed energetically appropriate docking of the LA drugs. The alkylamino head of ionizable LA molecules was docked closer to the selectivity filter and in association with Phe-1579 of IVS6 and Leu-1280 of IIIS6 (Nav1.4), and the aromatic ring interacted with Tyr-1586 of IVS6 and Asn-434 of IS6. Comparison of multiple LA drugs showed relative binding affinities in the model consistent with experimental studies. The ionizable LA alkylamino heads interact primarily by van der Waals forces that position the charge so as to create a positive electrostatic barrier for cation permeation. Permanently uncharged benzocaine could be docked in the closed conformation as well, stabilizing the closed conformation. The structurally different anticonvulsant lamotrigine and one of its derivatives have a binding site that fully overlaps with that of the LA drugs. The open, activated channel creates the high-affinity binding site for these sodium channel blocker drugs, and block may be mainly electrostatic.     

A quantitative and comparative study of the effects of a synthetic ciguatoxin CTX3C on the kinetic properties of voltage-dependent sodium channels

Ciguatoxins (CTXs) are known to bind to receptor site 5 of the voltage-dependent Na channel, but the toxin's physiological effects are poorly understood. In this study, we investigated the effects of a ciguatoxin congener (CTX3C) on three different Na-channel isoforms, rNa(v)1.2, rNa(v)1.4, and rNa(v)1.5, which were transiently expressed in HEK293 cells. The toxin (1.0 micromol l(-1)) shifted the activation potential (V(1/2) of activation curve) in the negative direction by 4-9 mV and increased the slope factor (k) from 8 mV to between 9 and 12 mV (indicative of decreased steepness of the activation curve), thereby resulting in a hyperpolarizing shift of the threshold potential by 30 mV for all Na channel isoforms. The toxin (1.0 micromol l(-1)) significantly accelerated the time-to-peak current from 0.62 to 0.52 ms in isoform rNa(v)1.2. Higher doses of the toxin (3-10 micromol l(-1)) additionally decreased time-to-peak current in rNa(v)1.4 and rNa(v)1.5. A toxin effect on decay of I(Na) at -20 mV was either absent or marginal even at relatively high doses of CTX3C. The toxin (1 micromol l(-1)) shifted the inactivation potential (V(1/2) of inactivation curve) in the negative direction by 15-18 mV in all isoforms. I(Na) maxima of the I-V curve (at -20 mV) were suppressed by application of 1.0 micromol l(-1) CTX3C to a similar extent (80-85% of the control) in all the three isoforms. Higher doses of CTX3C up to 10 micromol l(-1) further suppressed I(Na) to 61-72% of the control. Recovery from slow inactivation induced by a depolarizing prepulse of intermediate duration (500 ms) was dramatically delayed in the presence of 1.0 micromol l(-1) CTX3C, as time constants describing the monoexponential recovery were increased from 38+/-8 to 588+/-151 ms (n=5), 53+/-6 to 338+/-85 ms (n=4), and 23+/-3 to 232+/-117 ms (n=3) in rNa(v)1.2, rNa(v)1.4, and rNa(v)1.5, respectively. CTX3C exerted multimodal effects on sodium channels, with simultaneous stimulatory and inhibitory aspects, probably due to the large molecular size (3 nm in length) and lipophilicity of this membrane-spanning toxin.     

Mammalian skeletal muscle voltage-gated sodium channels are affected by scorpion depressant "insect-selective" toxins when preconditioned

Among scorpion beta- and alpha-toxins that modify the activation and inactivation of voltage-gated sodium channels (Na(v)s), depressant beta-toxins have traditionally been classified as anti-insect selective on the basis of toxicity assays and lack of binding and effect on mammalian Na(v)s. Here we show that the depressant beta-toxins LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus (Lqh) bind with nanomolar affinity to receptor site 4 on rat skeletal muscle Na(v)s, but their effect on the gating properties can be viewed only after channel preconditioning, such as that rendered by a long depolarizing prepulse. This observation explains the lack of toxicity when depressant toxins are injected in mice. However, when the muscle channel rNa(v)1.4, expressed in Xenopus laevis oocytes, was modulated by the site 3 alpha-toxin LqhalphaIT, LqhIT2 was capable of inducing a negative shift in the voltage-dependence of activation after a short prepulse, as was shown for other beta-toxins. These unprecedented results suggest that depressant toxins may have a toxic impact on mammals in the context of the complete scorpion venom. To assess whether LqhIT2 and Lqh-dprIT3 interact with the insect and rat muscle channels in a similar manner, we examined the role of Glu24, a conserved "hot spot" at the bioactive surface of beta-toxins. Whereas substitutions E24A/N abolished the activity of both LqhIT2 and Lqh-dprIT3 at insect Na(v)s, they increased the affinity of the toxins for rat skeletal muscle channels. This result implies that depressant toxins interact differently with the two channel types and that substitution of Glu24 is essential for converting toxin selectivity.     

Biphasic effects of haloperidol on sodium currents in guinea pig ventricular myocytes

Aim： To study the effects of haloperidol on sodium currents （INa） in guinea pig ventricular myocytes. Method： Whole-cell patch clamp technique was employed to evaluate the effects of haloperidol on INa in individual ventricular myocytes. Results： Haloperidol （0.1-3 wnol/L） inhibited INa in a concentration-dependent manner with an IC50 of 0.253±0.015 larnol/L. The inhibition rate of haloperidol （0.3 μmol/L） on INa was 22.14%±0.02%, and the maximum conductance was reduced. Haloperidol significantly reduced the midpoints for the activation and inactivation of INa by 2.09 and 4.09 mV, respectively. The time constant of recovery was increased. The increase in time intervals could only recover by 90.14%±1.4% （n=6）; however, haloperidol at 0.03 μmol/L enhanced INa conductance. The midpoints for the activation and inactivation Of INa were shifted by 1.38 and 5.69 mV, respectively, at this concentration of haloperidol. Conclusion： Haloperidol displayed a biphasic effect on INa in guinea pig cardiac myocytes. High concentrations of haloperidol inhibited INa, while lower concentrations of haloperidol shifted the activation and inactivation curve to the left. Full recovery of recovery curve was not achieved after 0.3 μmol/L haloperidol administration, indicating that the drug affects the inactivated state of sodium channels.     

High-affinity blockade of voltage-operated skeletal muscle and neuronal sodium channels by halogenated propofol analogues

Background and purpose:Voltage-operated sodium channels constitute major target sites for local anaesthetic-like action. The clinical use of local anaesthetics is still limited by severe side effects, in particular, arrhythmias and convulsions. These side effects render the search for new local anaesthetics a matter of high interest.Experimental approach:We have investigated the effects of three halogenated structural analogues of propofol on voltage-operated human skeletal muscle sodium channels (Na(V)1.4) and the effect of one compound (4-chloropropofol) on neuronal sodium channels (Na(V)1.2) heterologously expressed in human embryonic kidney cell line 293.Key results:4-Iodo-, 4-bromo- and 4-chloropropofol reversibly suppressed depolarization-induced whole-cell sodium inward currents with high potency. The IC(50) for block of resting channels at -150 mV was 2.3, 3.9 and 11.3 muM in Na(V)1.4, respectively, and 29.2 muM for 4-chloropropofol in Na(V)1.2. Membrane depolarization inducing inactivation strongly increased the blocking potency of all compounds. Estimated affinities for the fast-inactivated channel state were 81 nM, 312 nM and 227 nM for 4-iodopropofol, 4-bromopropofol and 4-chloropropofol in Na(V)1.4, and 450 nM for 4-chloropropofol in Na(V)1.2. Recovery from fast inactivation was prolonged in the presence of drug leading to an accumulation of block during repetitive stimulation at high frequencies (100 Hz).Conclusions and implications:Halogenated propofol analogues constitute a novel class of sodium channel-blocking drugs possessing almost 100-fold higher potency compared with the local anaesthetic and anti-arrhythmic drug lidocaine. Preferential drug binding to inactivated channel states suggests that halogenated propofol analogues might be especially effective in suppressing ectopic discharges in a variety of pathological conditions.British Journal of Pharmacology (2008) 155, 265-275; doi:10.1038/bjp.2008.255; published online 23 June 2008.     

Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels.

Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltage-gated sodium channels. Sodium channels, a family of at least 10 structurally different proteins, are responsible for the rising phase of the action potential in membranes of neuronal, cardiac and muscular excitable cells. This work is a comparative study of the binding properties and the cytotoxic effects of ciguatoxins and brevetoxins on human embryonic cells (HEK) stably expressing either the skeletal muscle (Na(v)1.4), or the cardiac (Na(v)1.5) sodium channel alpha-subunit isoforms. We report that type A (PbTx-1) and type B (PbTx-3 and PbTx-2) brevetoxins as well as ciguatoxins target both cardiac and muscle channels; type B brevetoxins show isoform selectivity, presenting a lower affinity for the heart than the skeletal muscle channel. The lower selectivity of type B brevetoxins for heart sodium channels may result from a more rigid backbone structure than is found in type A brevetoxins and ciguatoxins.     

Common mechanisms of drug interactions with sodium and T-type calcium channels

Voltage-gated sodium (Na(v)) and calcium (Ca(v)) channels play important roles in physiological processes, including neuronal and cardiac pacemaker activity, vascular smooth muscle contraction, and nociception. They are thought to share a common ancestry, and, in particular, T-type calcium (T-type) channels share structural similarities with Na(v) channels, both with regard to membrane topology and with regard to gating kinetics, including rapid inactivation. We thus reasoned that certain drugs acting on Na(v) channels may also modulate the activities of T-type channels. Here we show that the specific Na(v)1.8 blocker 5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide (A803467) tonically blocks T-type channels in the low micromolar range. Similarly to Na(v)1.8, this compound causes a significant hyperpolarizing shift in the voltage dependence of inactivation and seems to promote a slow inactivation-like phenotype. We further hypothesized that the structural similarity between T-type and Na(v) channels may extend to structurally similar drug-binding sites. Sequence alignment revealed several highly conserved regions between T-type and Na(v) channels that corresponded to drug-binding sites known to alter voltage-dependent gating kinetics. Mutation of amino acid residues in this regions within human Ca(v)3.2 T-type channels altered A803467 blocking affinity severalfold, suggesting that these sites may be exploited for the design of mixed T-type and Na(v) channel blockers that could potentially act synergistically to normalize aberrant neuronal activity.