Four novel tarantula toxins as selective modulators of voltage-gated sodium channel subtypes

Four novel peptide toxins that act on voltage-gated sodium channels have been isolated from tarantula venoms: ceratotoxins 1, 2, and 3 (CcoTx1, CcoTx2, and CcoTx3) from Ceratogyrus cornuatus and phrixotoxin 3 (PaurTx3) from Phrixotrichus auratus. The pharmacological profiles of these new toxins were characterized by electrophysiological measurements on six cloned voltage-gated sodium channel subtypes expressed in Xenopus laevis oocytes (Na(v)1.1/beta(1), Na(v)1.2/beta(1), Na(v)1.3/beta(1), Na(v)1.4/beta(1), Na(v)1.5/beta(1), and Na(v)1.8/beta(1)). These novel toxins modulate voltage-gated sodium channels with properties similar to those of typical gating-modifier toxins, both by causing a depolarizing shift in gating kinetics and by blocking the inward component of the sodium current. PaurTx3 is one of the most potent peptide modulators of voltage-gated sodium channels described thus far from spider venom, modulating Na(v)1.2 with an IC(50) value of 0.6 +/- 0.1 nM. CcoTx1 and CcoTx2, differing by only one amino acid, are potent modulators of different voltage-gated sodium channel subtypes from the central nervous system, except for Na(v)1.3, which is only affected by CcoTx2. The potency of CcoTx3 is lower, although this toxin seems to be more selective for the tetrodotoxin-resistant channel subtype Na(v)1.5/beta(1) (IC(50) = 447 +/- 32 nM). In addition to these results, molecular modeling indicates that subtle differences in toxin surfaces may relate to their different pharmacological profiles. Furthermore, an evolutionary trace analysis of these toxins and other structurally related three-disulfide spider toxins provides clues for the exploration of toxin-channel interaction and future structure-function research.     

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.     

Evidence for multiple effects of ProTxII on activation gating in Na(V)1.5

The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na(+) and Ca(2+) channels. ProTxII is distinct from other known Na(+) channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage-dependent activation, and current kinetics in Na(V)1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G(max) appeared at relatively low concentrations (40-100nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (>/=500nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba(2+), known to screen and/or bind to surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba(2+) indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.     

Novel mechanism of blocking axonal Na(+) channels by three macrocyclic polyamine analogues and two spider toxins

1. The mechanism of Na(+) channel block by three macrocyclic polyamine derivatives and two spider toxins was studied with voltage clamp and internal perfusion method in squid axons. 2. All these chemicals specifically block Na(+) channels in the open state only from the internal surface, and do not affect K(+) channels. 3. The blocking effect is enhanced as the depolarizing pulse becomes larger. Blocked channels are unable to shift to the inactivated state. 4. In the case of cyclam and guanidyl-side armed cyclam (G-cyclam), quick release of these chemicals from the binding sites is proven by the increase in the tail current and prolongation of the time course of the off gating current. On the other hand, in the presence of N-4 and the spider toxins, their detachment was delayed significantly. 5. Molecular requirements for the block of Na(+) channels by these molecules are the presence of positive charge and hydrophobicity.     

Tetrahydroacridine inhibits voltage-dependent Na^＋ current in guinea-pig ventricular myocytes

AIM: To study the effects of tetrahydroacridine (tacrine) on voltage-gated Na^ channels in cardiac tissues. METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na^ current was recorded using whole cell voltage-clamp technique. RESULTS: (1) Tacrine reversibly inhibited Na^ current with an IC50 value of 120μmol/L(95% confidence range: 108-133μmol/L). (2) The inhibitory effects of tacrine on Na^ current exhibited both a tonic nature and use-dependence. (3) Tacrine at 100μmol/L caused a negative shift (about 10mV) in the voltage-dependence of steady-state inactivation of Na^ current, and retarded its recovery from inactivation, but did not affect its activation curve. (4) Intracellular application of tacrine significantly inhibited Na^ current. CONCLUSION: In addition to blocking other voltage-gated ion channels,tacrine blocked Na^ channels in guinea-pig ventricular myocytes. Tacrine acted as inactivation stabilizer of Na^ channels in cardiac tissues.     

Spiders of medical importance in the Asia-Pacific: atracotoxin,latrotoxin and related spider neurotoxins

1. The spiders of medical importance in the Asia-Pacific region include widow (family Theridiidae) and Australian funnel-web spiders (subfamily Atracinae). In addition, cupboard (family Theridiidae) and Australian mouse spiders (family Actinopodidae) may contain neurotoxins responsible for serious systemic envenomation. Fortunately, there appears to be extensive cross-reactivity of species-specific widow spider antivenom within the family Theridiidae. Moreover, Sydney funnel-web antivenom has been shown to be effective in the treatment of mouse spider envenomation. 2. alpha-Latrotoxin (alpha-LTx) appears to be the main neurotoxin responsible for the envenomation syndrome known as "latrodectism" following bites from widow spiders. This 120 kDa protein binds to distinct receptors (latrophilin 1 and neurexins) to induce neurotransmitter vesicle exocytosis via both Ca2+-dependent and -independent mechanisms, resulting in vesicle depletion. This appears to involve disruption to a process that normally inhibits vesicle fusion in the absence of Ca2+. Precise elucidation of the mechanism of action of alpha-LTx will lead to a major advancement in our understanding of vesicle exocytosis. 3. delta-Atracotoxins (delta-ACTX) are responsible for the primate-specific envenomation syndrome seen following funnel-web spider envenomation. These peptides induce spontaneous repetitive firing and prolongation of action potentials in excitable cells. This results from a hyperpolarizing shift of the voltage-dependence of activation and a slowing of voltage-gated Na+ channel inactivation. This action is due to voltage-dependent binding to neurotoxin receptor site-3 on insect and mammalian voltage-gated Na+ channels in a manner similar, but not identical, to scorpion alpha-toxins and sea anemone toxins. delta-Atracotoxins provide us with highly specific tools to study Na+ channel structure and function 4. omega- and Janus-faced ACTX, from funnel-web spider venom, are novel neurotoxins that show selective toxicity to insects. In particular omega-ACTX define a new insecticide target due to a specific action to block insect voltage-gated Ca2+ channels. Both these ACTX show promise for the development of baculoviral recombinant biopesticides expressing these toxins for the control of insecticide-resistant agricultural pests. In addition, they should provide valuable tools for the pharmacological and structural characterization of insecticide targets.     

Isoform-specific effects of a novel BmK 11(2) peptide toxin on Na channels.

BmK 11(2) is a 7216Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch. Nanomolar concentrations of the toxin prolong amphibian nerve action potentials without attenuation of the amplitude. The pharmacological action of the toxin and its sequence similarity to other alpha-scorpion toxins suggest that BmK 11(2) selectively alters voltage-gated Na channels. In order to test whether BmK 11(2) preferentially modulates the gating or kinetics of certain channel isoforms, we applied BmK 11(2) to muscle, heart and neuronal Na channels. 100nM BmK 11(2) increased the peak current amplitude of skeletal muscle (micro1) and neuronal (N1E-115) Na currents by 40 and 20%, respectively, and reduced the cardiac Na (hH1) current by 15%. The toxin slowed current decay of all isoforms, most prominently in N1E-115 (tau(BmK)/tau(Control)=12), micro1 (11), and less so for hH1 (1.3). BmK 11(2) shifted the voltage dependence of activation of micro1 and N1E-115 currents. BmK 11(2) had no effect on steady-state inactivation, use-dependent availability, and the kinetics of entry into slowly recovering inactivated states.     

Differential blockade of neuronal voltage-gated Na(+) and K(+) channels by antidepressant drugs

The effects of a range of antidepressants were investigated on neuronal voltage-gated Na(+) and K(+) channels. With the exception of phenelzine, all antidepressants inhibited batrachotoxin-stimulated 22Na(+) uptake, most likely via negative allosteric inhibition of batrachotoxin binding to neurotoxin receptor site-2 on the Na(+) channel. Imipramine also produced a differential action on macroscopic Na(+) and K(+) channel currents in acutely dissociated rat dorsal root ganglion neurons. Imipramine produced a use-dependent block of Na(+) channels. In addition, there was a hyperpolarizing shift in the voltage-dependence of steady-state Na(+) channel inactivation and slowed repriming kinetics consistent with imipramine having a higher affinity for the inactivated state of the Na(+) channel. At higher concentrations, imipramine also blocked delayed-rectifier and transient outward K(+) currents in the absence of alterations to the voltage-dependence of activation or the kinetics of inactivation. These actions on voltage-gated ion channels may underlie the therapeutic and toxic effects of these drugs.     

Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channels.

1. Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. 2. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials. 3. The effect on Na+ currents in rat cardiomyocytes was studied using the patch-clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively. 4. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 microM), shift the steady-state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. 5. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. 6. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels.     

Purification and characterization of Hainantoxin-V, a tetrodotoxin-sensitive sodium channel inhibitor from the venom of the spider Selenocosmia hainana.

A neurotoxic peptide, named Hainantoxin-V (HNTX-V), was isolated from the venom of the Chinese bird spider Selenocosmia hainana. The complete amino acid sequence of HNTX-V has been determined by Edman degradation and found to contain 35 amino acid residues with three disulfide bonds. Under whole-cell patch-clamp mode, HNTX-V was proved to inhibit the tetrodotoxin-sensitive (TTX-S) sodium currents while it had no any effects on tetrodotoxin-resistant (TTX-R) sodium currents on adult rat dorsal root ganglion neurons. The inhibition of TTX-S sodium currents by HNTX-V was tested to be concentrate-dependent with the IC(50) value of 42.3nM. It did not affect the activation and inactivation kinetics of currents and did not have the effect on the active threshold of sodium channels and the voltage of peak inward currents. However, 100nM HNTX-V caused a 7.7mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation. The results indicated that HNTX-V inhibited mammalian voltage-gated sodium channels through a novel mechanism distinct from other spider toxins such as delta-ACTXs, micro -agatoxins I-VI which bind to receptor site three to slow the inactivation kinetics of sodium currents.     

Site-3 toxins and cardiac sodium channels.

Site-3 toxins are small polypeptide venoms from scorpions, sea anemones, and spiders that bind with a high specificity to the extracellular surface of voltage-gated Na channels. After binding to a site near the S4 segment in domain IV the toxin causes disruption of the normal fast inactivation transition resulting in a marked prolongation of the action potentials of excitable tissues including those of cardiac and skeletal muscle and nerve. In this review we discuss the specific binding interactions between residues of the toxin and those of the Na channel, and the specific modification of Na channel kinetic behavior leading to a change in fast inactivation focusing on interactions deduced primarily from the study of sea anemone toxins and the cardiac Na channel (Na(V)1.5). We also illustrate the usefulness of site-3 toxins in the study of altered Na channel behavior by drug-modification.     

Effects of a Lasiodora spider venom on Ca2+ and Na+ channels.

The venom of a Brazilian spider, Lasiodora sp (Mygalomorphae, Theraphosidae), was screened for activity against ion channels using Ca2+ imaging and whole-cell patch clamp in GH3 cells. When tetrodotoxin (TTX) was present to block Na+ channels, the venom abolished the Ca2+ oscillations that are normally present in these cells and reduced the basal level of intracellular Ca2+. Under patch clamp, the venom reduced the L-type Ca2+ channel conductance and caused a positive shift in its voltage dependence of activation. In addition to these effects, when applied without TTX, the venom also caused a slow and noisy increase in intracellular Ca2+. The sensitivity of this second effect to TTX suggested an effect on Na+ channels, which was tested using patch clamp. Control Na+ currents inactivated completely as a single exponential. Treatment with the venom did not affect the amplitude of I(Na), but caused it to divide in two slower exponential components plus a sustained component, all of which were suppressed by TTX. The venom also caused a negative shift in the voltage dependence of activation and steady-state inactivation of I(Na). The observed effects of this venom on whole-cell currents explain the changes it causes in intracellular Ca2+ in GH3 cells and demonstrate that the venom of this spider is a source of toxins active against ion channels.     

Spider Neurotoxins 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 sodium channel gating and/or kinetics. A number of polypeptide toxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized that interact with several of these sites. Certain huwentoxins and hainantoxins appear to target site 1 to block Na_v channel conductance. The δ -atracotoxins and Magi 4 slow Na_v-channel inactivation via an interaction with neurotoxin site 3. The δ -palutoxins, and most likely μ -agatoxins and curtatoxins, target site 4. However, their action is complex with the μ -agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion β -toxins, but with both δ -palutoxins and μ -agatoxins slowing Na_v channel inactivation, a site 3-like action. Many spider toxins target undefined sites, while others are likely to cross-react with other ion channels due to conserved structures within domains of voltage-gated ion channels. It is already clear, however, that many spider toxins represent highly potent and specific molecular tools to define novel links between sites modulating channel activation and inactivation. Other spider toxins show phyla specificity and are being considered as lead compounds for the development of biopesticides. Others display tissue specificity via interactions with specific Na_v channel subtypes and should provide useful tools to delineate the molecular determinants to target ligands to these channel subtypes. 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 mentioned areas remains underdeveloped.     

Donepezil blocks voltage-gated ion channels in rat dissociated hippocampal neurons

Donepezil (E2020) is a novel cholinesterase inhibitor for the treatment of Alzheimer's disease. Recent studies show that it may act on targets other than acetylcholinesterase in the brain. In the present study, the actions of donepezil on voltage-gated Na+ and K+ channels were investigated in rat dissociated hippocampal neurons. Donepezil reversibly inhibited voltage-activated Na+ current (I(Na)), delayed rectifier K+ current (I(K)) and fast transient K+ current (I(A)). The inhibition of donepezil on I(Na) was dependent on the holding potential. When neurons were held at -100, -80 and -60 mV, the IC50 value was 436+/-19, 291+/-26 and 3.8+/-0.3 microM, respectively. The drug did not affect the activation, fast inactivation of I(Na) and its recovery from fast inactivation. The inhibition of donepezil on I(K) (IC50=78+/-5 microM) was voltage-dependent, whereas that on I(A) (IC50=249+/-25 microM) was voltage-independent. Donepezil caused a significant hyperpolarizing shift of the voltage-dependence of the activation and steady-state inactivation of I(K), without affecting the kinetic properties of I(A). Due to the high concentrations used, the blocking effects of donepezil on the voltage-gated ion channels are unlikely to contribute to the clinical benefits in patients with Alzheimer's disease.     

Electrophysiological effect of l-cis-diltiazem, the stereoisomer of d-cis-diltiazem, on isolated guinea-pig left ventricular myocytes

l-cis-Diltiazem, the stereoisomer of the L-type Ca(2+) channel blocker d-cis-diltiazem, protects cardiac myocytes from ischemia and reperfusion injury in the perfused heart and from veratridine-induced Ca(2+) overload. We determined the effect of l-cis-diltiazem on the voltage-dependent Na(+) current (I(Na)) and lysophosphatidylcholine-induced currents in isolated guinea-pig left ventricular myocytes by a whole-cell patch-clamp technique. l-cis-Diltiazem inhibited I(Na) in a dose-dependent manner without altering the current-voltage relationship for I(Na) (K(d) values : 729 and 9 microM at holding potentials of -140 and -80 mV, respectively). A use-dependent block of I(Na), the leftward shift of the steady-state inactivation curve and the delay of recovery from inactivation suggest that l-cis-diltiazem has a higher affinity for the inactivated state of Na(+) channels. In addition to I(Na), the lysophosphatidylcholine-induced currents were inhibited by l-cis-diltiazem in a similar concentration range. It is suggested that inhibition of both Na(+) channels and lysophosphatidylcholine-activated non-selective cation channels contributes to the cardioprotective effect of l-cis-diltiazem.     

Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels.

Sea anemones produce a myriad of toxic peptides and proteins of which a large group acts on voltage-gated Na+ channels. However, in comparison to other organisms, their venoms and toxins are poorly studied. Most of the known voltage-gated Na+ channel toxins isolated from sea anemone venoms act on neurotoxin receptor site 3 and inhibit the inactivation of these channels. Furthermore, it seems that most of these toxins have a distinct preference for crustaceans. Given the close evolutionary relationship between crustaceans and insects, it is not surprising that sea anemone toxins also profoundly affect insect voltage-gated Na+ channels, which constitutes the scope of this review. For this reason, these peptides can be considered as insecticidal lead compounds in the development of insecticides.     

Inhibition of voltage-gated potassium currents by gambierol in mouse taste cells.

Ciguatera is a food poisoning caused by toxins of Gambierdiscus toxicus, a marine dinoflagellate. The neurological features of this intoxication include sensory abnormalities, such as paraesthesia, heightened nociperception, and also taste alterations. Here, we have evaluated the effect of gambierol, one of the possible ciguatera toxins, on the voltage-gated ion currents in taste cells. Taste cells are excitable cells endowed with voltage-gated Na+, K+, and Cl- currents (I(Na), I(K), and I(Cl), respectively). By applying the patch-clamp technique to single cells in isolated taste buds obtained from the mouse vallate papilla, we have recorded such currents and determined the effect of bath-applied gambierol. We found that this toxin markedly inhibited I(K) in the nanomolar range (IC50 of 1.8 nM), whereas it showed no significant effect on I(Na) or I(Cl) even at high concentration (1 microM). The block of I(K) was irreversible even after a 50-min wash. In addition to affecting the current amplitude, we found that gambierol significantly altered both the activation and inactivation processes of I(K). In conclusion, unlike other toxins involved in ciguatera, such as ciguatoxins, which affect the functioning of voltage-gated sodium channels, the preferred molecular target of gambierol is the voltage-gated potassium channel, at least in taste cells. Voltage-gated potassium currents play an important role in the generation of the firing pattern during chemotransduction. Thus, gambierol may alter action potential discharge in taste cells and this could be associated with the taste alterations reported in the clinical literature.     

Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels.

The membrane actions of three recently isolated polypeptide neurotoxins from the sea anemones Stichodactyla helianthus (toxin ShI), Condylactis gigantea (toxin CgII) and Calliactis parasitica (toxin CpI) were investigated on action potentials and voltage-clamp membrane currents of the giant axon of the crayfish Procambarus clarkii. The first two toxins were also tested on the cockroach (Periplaneta americana) giant axon. All three toxins were particularly lethal to crustaceans, moderately toxic to an insect (cockroach), and essentially non-toxic to a mammal (mouse). ShI and CgII were 50- to 100-fold more potent on crayfish than on cockroach axons; this difference in activity was correlated with the relative reversibility of their effects on these arthropod axons. The crustacean selectivity of these toxins is therefore due largely to their greater affinity for crustacean sodium channels. All three toxins prolonged crayfish giant axon action potentials by selectively slowing Na channel inactivation without greatly affecting activation. Before toxin treatment, inactivation was nearly exponential, with a time constant less than 1 msec. After treatment, the inactivation time course could be described as the sum of two exponentially decaying components, plus a large steady-state component. The major component possessed the slower (10-20 msec) time constant. The steady-state component increased with depolarization, causing the sodium channel steady-state inactivation curve to reach a minimum between -60 and -20 mV and then increase at more positive potentials. All three toxins shifted the peak sodium current-voltage relation to the left. This voltage shift was greater at 20 degrees C than at 10 degrees C. Maintained membrane depolarization during toxin wash-in delayed the appearance of modified Na channels. Also, prolonged depolarization of toxin-treated axons converted modified sodium channels back to normal ones. The toxins did not affect potassium and leakage currents. Our results indicate that the three crustacean-active sea anemone toxins share a common electrophysiological action on arthropod sodium channels, at least at the macroscopic level.     

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.     

Isoflurane and propofol inhibit voltage-gated sodium channels in isolated rat neurohypophysial nerve terminals

Mounting electrophysiological evidence indicates that certain general anesthetics, volatile anesthetics in particular, depress excitatory synaptic transmission by presynaptic mechanisms. We studied the effects of representative general anesthetics on voltage-gated Na+ currents (INa) in nerve terminals isolated from rat neurohypophysis using patch-clamp electrophysiological analysis. Both isoflurane and propofol inhibited INa in a dose-dependent and reversible manner. At holding potentials of -70 or -90 mV, isoflurane inhibited peak INa with IC50 values of 0.45 and 0.56 mM, and propofol inhibited peak INa with IC50 values of 4.1 and 6.0 microM, respectively. Isoflurane (0.8 mM) did not significantly alter the V1/2 of activation; propofol caused a small positive shift. Isoflurane (0.8 mM) or propofol (5 microM) produced a negative shift in the voltage dependence of inactivation. Recovery of INa from inactivation was slower from a holding potential of -70 mV than from -90 mV; isoflurane and propofol further delayed recovery from inactivation. In conclusion, the volatile anesthetic isoflurane and the intravenous anesthetic propofol inhibit voltage-gated Na+ currents in isolated neurohypophysial nerve terminals in a concentration- and voltage-dependent manner. Marked effects on the voltage dependence and kinetics of inactivation and minimal effects on activation support preferential anesthetic interactions with the fast inactivated state of the Na+ channel. These results are consistent with direct inhibition of oxytocin and vasopressin release from the neurohypophysis by isoflurane and propofol. Inhibition of voltage-gated Na+ channels may contribute to the presynaptic effects of general anesthetics on nerve terminal excitability and neurotransmitter release.