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Query: UNIPROT:P06889 (Mol)
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The sodium channel activators veratridine and batrachotoxin, the sodium ionophore gramicidin, and the calcium ionophore ionomycin stimulated phosphoinositide breakdown, as indicated by the increased accumulation of [3H]inositol monophosphate in embryonic chick heart cells. The levels of [3H]inositol trisphosphate and [3H]inositol bisphosphate were also increased by veratridine, indicating that there was increased hydrolysis of phosphatidylinositol bisphosphate by phospholipase C. The response to veratridine required both extracellular sodium and calcium, suggesting that calcium entry via Na/Ca exchange might activate phospholipase C. Fluorescence measurements with fura-2 confirmed that the sodium agents greatly increased the cytoplasmic calcium concentration. Veratridine (100 microM) increased cytoplasmic calcium from 94 +/- 4 nM to 862 +/- 103 nM, giving a maximal calcium increase in about 2 min. Batrachotoxin (1 microM) induced an even greater increase in calcium but required a longer time. Gramicidin also induced a large increase in cytoplasmic calcium which was maximal within 0.5 min. To directly test the calcium dependency of phospholipase C, we permeabilized the chick heart cells with saponin and monitored the production of inositol phosphates at different calcium concentrations. Raising the calcium concentration from 3 to 1000 nM increased the accumulation of [3H]inositol phosphates by nearly 4-fold with a half-maximal effect at about 200 nM calcium. The guanine nucleotide guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) also stimulated accumulation of the InsPs and the response to (GTP gamma S) was potentiated by increasing the calcium concentration. The data suggest that the effect of the sodium agents on phosphoinositide hydrolysis results from an elevation of intracellular calcium which increases GTP-dependent phospholipase C activity. Thus, drugs or other conditions that elevate cytoplasmic calcium in heart cells may increase the hydrolysis of membrane phosphoinositides.
Mol Pharmacol 1988 Mar
PMID:Elevation of cytoplasmic calcium concentration stimulates hydrolysis of phosphatidylinositol bisphosphate in chick heart cells: effect of sodium channel activators. 245 Nov 16

The antiarrhythmic action of type I antiarrhythmic drugs may be mediated via binding of the drugs to a receptor associated with the cardiac sodium channel. This suggested that the effects of type I drugs might be stereospecific. We measured the effect of the tocainide stereoisomers (which have stereospecific antiarrhythmic effects) on conduction time and on radioligand binding to the cardiac sodium channel. The concentration-dependent effects of the individual enantiomers of tocainide on interventricular conduction time measured during constant rate ventricular pacing at 350 msec were assessed in 47 isolated perfused rabbit heart preparations. Significant increases (p less than 0.05) in conduction time occurred for both R-(-)-tocainide (75 microM, 10 +/- 5 msec) and S-(+)-tocainide (150 microM, 4 +/- 1 msec). R-(-)-Tocainide was more potent than the S-(+)-tocainide in prolonging conduction time (p less than 0.05). This stereospecific prolongation of conduction time suggested a stereospecific interaction with the sodium channel. The affinities of the enantiomers for the channel were measured with a radioligand binding assay using [3H]batrachotoxinin benzoate and freshly isolated cardiac myocytes. Both enantiomers inhibited [3H]batrachotoxin benzoate binding, but the IC50 (+/- SD) values were different: R-(-)-tocainide 184 +/- 8 microM; S-(+)-tocainide, 546 +/- 37 microM (p less than 0.003). Tocainide isomers are stereospecific in terms of prolonging conduction time and in binding to the sodium channel. The stereospecific electrophysiologic effects of tocainide may result from binding to a receptor associated with the cardiac sodium channel.
Mol Pharmacol 1988 Mar
PMID:Stereospecific interaction of tocainide with the cardiac sodium channel. 245 Nov 17

The effects of saturating concentrations of DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and the pyrethroid insecticides cismethrin and deltamethrin on alkaloid-dependent activation of the voltage-sensitive sodium channel were studied using measurements of 22Na+ uptake into mouse brain synaptosomes. In survey experiments, these compounds enhanced sodium uptake stimulated by veratridine and batrachotoxin, but inhibited uptake stimulated by aconitine. Concentration response curves for aconitine run in the absence and presence of 10 microM cismethrin demonstrated that the inhibition was noncompetitive. This unanticipated inhibitory effect of insecticides on aconitine-dependent sodium uptake suggests a possible overlap or negative allosteric coupling between the binding sites for insecticides and aconitine and reveals unique characteristics of the action of aconitine that are not shared by veratridine and batrachotoxin. More detailed studies of the effects of insecticides on veratridine- or batrachotoxin-stimulated uptake found small insecticide-dependent increases in the potency of these activators. In addition to this effect, DDT and deltamethrin also enhanced maximal uptake stimulated by veratridine. Possible mechanisms underlying these effects of insecticides on alkaloid-dependent uptake are discussed in light of a qualitative model formulated from these results and previous biochemical and electrophysiological studies. Additional experiments were designed to assess the interactions of insecticides and toxin II of the sea anemone Anemonia sulcata (ATX II) as modifiers of alkaloid-dependent uptake. DDT and ATX II acted synergistically to increase uptake stimulated by veratridine. Moreover, DDT shifted the potency of ATX II for enhancing veratridine-dependent uptake to 5-fold lower concentrations. In contrast, DDT and subsaturating concentrations of ATX II acted independently in their enhancement of sodium channel activation by batrachotoxin. Mutually exclusive effects on veratridine-dependent uptake were observed when cismethrin was co-applied with ATX II. However, independent effects of cismethrin and ATX II were found with aconitine-modified channels, in that cismethrin was able to inhibit ATX II-enhanced aconitine-dependent sodium flux. Thus, the interactions between insecticides and ATX II as modifiers of alkaloid-dependent uptake are complex and depend on the insecticide-activator combination under study.
Mol Pharmacol 1988 May
PMID:Pyrethroid insecticides and DDT modify alkaloid-dependent sodium channel activation and its enhancement by sea anemone toxin. 245 70

Pyrethroid insecticides are synthetic neurotoxins patterned after the naturally occurring pyrethrins. Their mechanism of action is thought to involve effects primarily at the voltage-sensitive sodium channel of both insect and mammalian neurons, although recent studies have raised the possibility that these compounds may also act at the gamma-aminobutyric acid receptor-chloride ionophore complex. Here we show that active pyrethroids of the alpha-cyano-3-phenoxybenzyl class allosterically enhance the binding of [3H]batrachotoxinin-A 20-alpha-benzoate to voltage-sensitive sodium channels of rat brain in a dose-dependent and stereospecific manner. Comparison of the rank order of potency for enhancement of [3H]batrachotoxinin-A 20-alpha-benzoate binding and insecticidal activity in a series of toxic stereoisomers of cypermethrin, representative of the class, reveals a correlation between the two measures. These results support a sodium channel site model for pyrethroid action and suggest a useful and practical method to help evaluate the relationship between effects at the sodium channel and insecticidal potency for members of this class of compounds.
Mol Pharmacol 1988 Jul
PMID:Pyrethroid insecticides: stereospecific allosteric interaction with the batrachotoxinin-A benzoate binding site of mammalian voltage-sensitive sodium channels. 245 60

The state-dependent binding of class I antiarrhythmic drugs to a receptor associated with the cardiac sodium channel was assessed using [3H]batrachotoxinin A 20-alpha-benzoate [( 3H]BTXB) binding. [3H]BTXB binds specifically to and stabilizes activated states of the sodium channel. Quinidine (IC50 = 40 microM) and lidocaine [IC50 = 61 microM) inhibited equilibrium [3H]BTXB binding to sodium channels present on freshly isolated rat cardiac myocytes. Scatchard analysis of [3H]BTXB binding in the presence of quinidine and lidocaine revealed two apparent patterns of inhibition. Quinidine (33 microM) increased the KD but had no significant effect on the Bmax, whereas lidocaine (91 microM) reduced the Bmax but had no significant effect on the KD. To address drug binding to activated and nonactivated states, we exploited the state-specific binding of [3H]BTXB. Drugs that increase the rate of dissociation (k-1) of [3H]BTXB must bind to sodium channels to which [3H]BTXB is already bound (i.e., activated channels). Therefore, drug-mediated increases in k-1 measure drug binding to activated states. Both quinidine and lidocaine increased the k-1 of [3H]BTXB, indicating drug binding to and destablization of activated sodium channels. However, the minimal affinities of quinidine and lidocaine for activated channels (KDact) were estimated to be 433 and 455 microM, respectively, concentrations much higher than the equilibrium IC50 values. Drugs that allosterically decrease the rate of association (k+1) of [3H]BTXB must bind to sodium channels to which [3H]BTXB is not already bound (i.e., nonactivated channels). Therefore, drug-mediated decreases in k+1 measures drug binding to nonactivated states. Quinidine and lidocaine decreased the k+1 of [3H]BTXB, indicating drug binding to and stablization of nonactivated sodium channels. The affinity of quinidine and lidocaine for nonactivated channels (KDnon) was estimated to be 10 and 35 microM, respectively, concentrations close to the equilibrium IC50 values. The markedly different KDact and KDnon values for both quinidine and lidocaine indicate state-dependent binding of quinidine and lidocaine to the class I receptor on the cardiac sodium channel. Both drugs destabilize activated channels and stabilize nonactivated channels. The Scatchard results suggest that quinidine and lidocaine may have different mechanisms of allosteric inhibition of [3H]BTXB binding.
Mol Pharmacol 1989 Jul
PMID:Class I antiarrhythmic drug receptor: biochemical evidence for state-dependent interaction with quinidine and lidocaine. 254 48

BTG 502 [(2E,4E)-N-(1,2-dimethyl)-propyl-6-(5-bromonaphth-2-yl)-hexa -2,4- dienamide], a synthetic analog of insecticidal amides isolated from Piper species, stimulated 22Na+ uptake into mouse brain synaptoneurosomes in the presence of saturating concentrations of Leiurus quinquestriatus venom but had no effect on sodium uptake in the absence of venom. In the presence of Leiurus venom, half-maximal stimulation was achieved at a BTG 502 concentration of 1.7 microM, whereas maximal stimulation (2.3-fold greater than nonspecific uptake) was observed at 50 microM. In the absence of other modifiers, BTG 502 inhibited batrachotoxin (BTX)-dependent sodium uptake, producing 50% inhibition at 2 microM. In the presence of Leiurus venom, BTG 502 was a partial inhibitor of BTX-dependent 22Na+ uptake, producing half-maximal inhibition at 1.5 microM. The levels of residual BTX-dependent sodium uptake and maximal BTG 502-dependent sodium uptake measured in the presence of Leiurus venom were identical. BTG 502 inhibited the specific binding of [3H]batrachotoxinin A-20-alpha-benzoate (BTX-B) to the activator recognition site (site 2) of sodium channels in these preparations, producing half-maximal inhibition at 2 microM and maximal inhibition at 30 microM. Equilibrium analysis showed that BTG 502 was an apparent competitive inhibitor of [3H]BTX-B binding, producing a concentration-dependent decrease in the affinity of sodium channels for this ligand without affecting binding capacity. Kinetic analysis demonstrated that BTG 502 slowed the rate of formation of the ligand-receptor complex but did not alter the rate of dissociation of this complex. The effects of BTG 502 on 22Na+ uptake and [3H]BTX-B binding are consistent with the action of this compound as an antagonist at the activator recognition site of the voltage-sensitive sodium channel in the absence of Leiurus venom and as a partial agonist at this site in the presence of Leiurus venom. These results suggest that the N-alkylamides represent a novel chemical class of neurotoxins that act at site 2 of the sodium channel.
Mol Pharmacol 1989 Aug
PMID:Activation of sodium channels and inhibition of [3H]batrachotoxinin A-20-alpha-benzoate binding by an N-alkylamide neurotoxin. 254 84

The actions of diphenylhydantoin (DPH) and carbamazepine (CBZ) on sodium channels in mouse neuroblastoma cells (clone N18) were analyzed using the patch voltage clamp procedure in the whole cell configuration. DPH and CBZ reduced sodium currents without effect on the voltage dependence of sodium channel activation. Half-maximal inhibition was observed with approximately 30 microM of each drug. Depolarization increased and hyperpolarization reversed channel block by these two drugs in the voltage range from -90 to -45 mV. Repetitive stimulation at 2 Hz or greater enhanced inhibition of sodium channels. The half-time for recovery from voltage-dependent inhibition was greater for DPH (1.36 sec) than for CBZ (0.38 sec). A combination of prolonged depolarizing pulses of 15 mV with superimposed brief maximal depolarizations designed to mimic the electrical activity in an epileptic focus gave additive effects of voltage-dependent and frequency-dependent inhibition. The results support the previous proposal that DPH and CBZ are sodium channel-selective anticonvulsants and provide a potential basis for specific inhibition of neurons in epileptic foci. The mechanism of DPH and CBZ action is considered in terms of an allosteric or modulated receptor model of drug binding and action.
Mol Pharmacol 1985 May
PMID:Voltage clamp analysis of the inhibitory actions of diphenylhydantoin and carbamazepine on voltage-sensitive sodium channels in neuroblastoma cells. 258 Nov 24

The determinants of stereospecific binding of type I antiarrhythmic drugs to specific sites associated with the sodium channel were assessed using rat cardiac myocytes. The asymmetric carbon atoms of stereoisomers may be located at two sites within type I drugs. The structure of these drugs can be schematically illustrated as Aromatic-C1-link-C2-Amine, where C1 and C2 represent potentially asymmetric carbon atoms. We used enantiomeric pairs with either C1 or C2 asymmetric carbon atoms to assess the importance of conformation at these sites to drug binding. The affinities of enantiomers of seven sodium channel blockers were measured with a radioligand binding assay using [3H]batrachotoxinin benzoate [( 3H]BTXB) and freshly isolated cardiac myocytes. The enantiomers inhibited [3H]BTXB binding in a dose-dependent manner, with a mean Hill number of 1.0 +/- 0.1. The ratios of affinities [IC50 of (+)-isomer/IC50 of (-)-isomer] were, for the C1 pairs: quinidine, 0.29; cinchonidine, 0.55; disopyramide, 1.11; RAC 109, 5.33; and for C2 pairs: flecainide, 1.03; mexiletine, 2.15; tocainide, 3.01. The stereospecific differences in drug binding suggest that the orientations of both the aromatic and the amine groups to the rest of the drug molecule are important determinants of drug binding to the cardiac sodium channel. This also suggests the presence of at least two stereospecific domains within the binding sites for type I antiarrhythmic drugs.
Mol Pharmacol 1988 Nov
PMID:Determinants of stereospecific binding of type I antiarrhythmic drugs to cardiac sodium channels. 284 86

Local anesthetics inhibited the sodium influx and the inositol phosphate accumulation elicited by the sodium channel activator batrachotoxin in guinea pig cortical synaptoneurosomes. Inhibitory effects of local anesthetics on sodium influx correlated with inhibitory effects on binding of a tritiated batrachotoxin analog to sodium channels in synaptoneurosomes. There was also a correlation between inhibitory effects on sodium influx and on inositol phosphate accumulation; most local anesthetics inhibited sodium influx at concentrations similar to those required for inhibition of inositol phosphate accumulation. Indeed, euprocin, bupivacaine, lidocaine, and certain analogs were nearly equipotent with respect to inhibition of sodium influx and inositol phosphate accumulation. Local anesthetics also inhibited inositol phosphate accumulation that was induced by carbamylcholine through both a tetrodotoxin-sensitive and tetrodotoxin-insensitive pathway. Certain local anesthetics, such as dibucaine, inhibited the tetrodotoxin-sensitive pathway with higher potency than for the tetrodotoxin-insensitive pathway, while others, such as quinacrine, inhibited tetrodotoxin-sensitive and tetrodotoxin-insensitive pathways with equal potency. Diphenhydramine and chlorpromazine appeared to inhibit carbamylcholine-elicited phosphoinositide breakdown through blockade of muscarinic cholinergic receptors rather than because of local anesthetic activity of inhibitory effects on phospholipase C.
Mol Pharmacol 1988 Nov
PMID:Local anesthetics: comparison of effects on batrachotoxin-elicited sodium flux and phosphoinositide breakdown in guinea pig cerebral cortical synaptoneurosomes. 284 89

The alpha subunit of the purified voltage-sensitive sodium channel from rat brain is rapidly phosphorylated to the extent of 3-4 mol phosphate/mol by purified protein kinase C. The alpha subunit of the native sodium channel in synaptosomal membranes is also phosphorylated by added protein kinase C as assessed by specific immunoprecipitation and polyacrylamide gel electrophoresis of labeled membranes. Our results suggest coordinate regulation of sodium channel phosphorylation state by cAMP-dependent and calcium/phospholipid-dependent protein kinases.
Cell Mol Neurobiol 1984 Sep
PMID:Phosphorylation of the alpha subunit of the sodium channel by protein kinase C. 609 71


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