State- and use-dependent block of muscle Nav1.4 and neuronal Nav1.7 voltage-gated Na+ channel isoforms by ranolazine

Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na(+) channels. However, ranolazine block of muscle and neuronal Na(+) channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na(+) currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na(+) channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC(50)) >/= 60 microM. Use-dependent block of Na(+) channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 microM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na(+) currents by ranolazine with estimated IC(50) values of 2.4, 6.2, and 1.7 microM, respectively. On- and off-rates of ranolazine were 8.2 microM(-1) s(-1) and 22 s(-1), respectively, for Nav1.4 open channels and 7.1 microM(-1) s(-1) and 14 s(-1), respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na(+) channels reduced the potency of ranolazine approximately 17-fold. We conclude that: 1) both muscle and neuronal Na(+) channels are as sensitive to ranolazine block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na(+) channels; and 3) ranolazine block of open Na(+) channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.     

Tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ channels differ in their sensitivity to Cd2+ and Zn2+

The sensitivity of Na+ channels to inhibition by Cd2+ and Zn2+ was studied in 22Na+ uptake experiments after stabilization of an open conformation of the Na+ channels with different neurotoxins and in voltage clamp experiments. Six different cell types of neuronal, cardiac or skeletal muscle origin were surveyed. Three cell types possess Na+ channels that are highly sensitive to tetrodotoxin (TTX) (Kd = 1-5 nM) and three possess Na+ channels that are resistant to TTX (Kd = 0.3-1 microM). The 22Na+ uptake experiments using veratridine or batrachotoxin to activate Na+ channels indicated that TTX-resistant Na+ channels are more sensitive to the inhibitory action of Cd2+ (IC50(Cd2+) = 0.2 mM) and of Zn2+ (IC50(Zn2+) = 50 microM) than TTX-sensitive Na+ channels (IC50(Cd2+) = 5 mM, IC50(Zn2+) = 2 mM). Electrophysiological experiments showed that high concentrations of Cd2+ (IC50 = 2 mM) are necessary to inhibit both TTX-sensitive and TTX-insensitive Na+ channels when the channels are activated by voltage steps. The results suggest that Cd2+ acts competitively with veratridine or batrachotoxin and that the difference in the effects of Cd2+ and Zn2+ on 22Na+ fluxes in TTX-sensitive and TTX-resistant cells is related to differences at the site of action of alkaloid neurotoxins.     

Ketamine impairs excitability in superficial dorsal horn neurones by blocking sodium and voltage-gated potassium currents

Ketamine shows, besides its general anaesthetic effect, a potent analgesic effect after spinal administration. We investigated the local anaesthetic-like action of ketamine and its enantiomers in Na+ and K+ channels and their functional consequences in dorsal horn neurones of laminae I-III, which are important neuronal structures for pain transmission receiving most of their primary sensory input from Adelta and C fibres. Combining the patch-clamp recordings in slice preparation with the 'entire soma isolation' method, we studied action of ketamine on Na+ and voltage-activated K+ currents. The changes in repetitive firing behaviour of tonically firing neurones were investigated in current-clamp mode after application of ketamine. Concentration-effect curves for the Na+ peak current revealed for tonic block half-maximal inhibiting concentrations (IC50) of 128 microM and 269 microM for S(+) and R(-)-ketamine, respectively, showing a weak stereoselectivity. The block of Na+ current was use-dependent. The voltage-dependent K+ current (K(DR)) was also sensitive to ketamine with IC50 values of 266 microM and 196 microM for S(+) and R(-)-ketamine, respectively. Rapidly inactivating K+ currents (K(A)) were less sensitive to ketamine. The block of K(DR) channels led to an increase in action potential duration and, as a consequence, to lowering of the discharge frequency in the neurones. We conclude that ketamine blocks Na+ and K(DR) channels in superficial dorsal horn neurones of the lumbar spinal cord at clinically relevant concentrations for local, intrathecal application. Ketamine reduces the excitability of the neurones, which may play an important role in the complex mechanism of its action during spinal anaesthesia.     

Stimulation of the hypothalamo-pituitary-adrenal axis in the rat by the type 4 phosphodiesterase (PDE-4) inhibitor, denbufylline.

1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.     

Ca2+ entry via P/Q-type Ca2+ channels and the Na+/Ca2+ exchanger in rat and human neocortical synaptosomes

Rat or human neocortical synaptosomes were used to study the role of voltage-gated Ca(2+) channels and the Na(+)/Ca(2+) exchanger in (45)Ca(2+) influx into nerve terminals. K(+) depolarization-induced (45)Ca(2+) influx through voltage-gated Ca(2+) channels into rat or human synaptosomes was completely blocked by mibefradil 30 microM or Cd(2+) 100 microM but was not affected by tetrodotoxin 1 microM. It was reduced by omega-agatoxin IVA 0.2 microM by 68% in synaptosomes of either species, whereas omega-conotoxin GVIA 0.1 microM and nifedipine 1 microM had no effect. Veratridine-induced (45)Ca(2+) entry into rat neocortical synaptosomes was completely blocked by mibefradil 30 microM, reduced by 80% by Cd(2+) 100 microM, by 90% by tetrodotoxin 1 microM and by 53% by omega-agatoxin IVA 0.2 microM but not by omega-conotoxin GVIA 0.1 microM or nifedipine 1 microM. Na(+)/Ca(2+) exchanger-mediated (45)Ca(2+) uptake into rat neocortical synaptosomes evoked by replacement of Na(+) by choline(+) in the incubation buffer was reduced by KB-R7943 (3-50 microM), an inhibitor of the Na(+)/Ca(2+) exchanger, in a concentration-dependent manner (maximal inhibition by 46% at 50 microM; IC(23%)=7.1 microM). Mibefradil also inhibited the Na(+)/Ca(2+) exchanger-mediated Ca(2+) uptake, although at 3.7 times lower potency (IC(23%)=26 microM). It is concluded that in rat and human neocortical nerve terminals Ca(2+) entry is mediated under physiological conditions by P/Q-type, but not by N- or L-type Ca(2+) channels or the Na(+)/Ca(2+) exchanger. If the cytosolic Na(+) concentration is increased, Ca(2+) is also taken up via the Na(+)/Ca(2+) exchanger. In addition to the ability of mibefradil to block all voltage-operated Ca(2+) channels, this drug is a low potency inhibitor of the Na(+)/Ca(2+) exchanger.     

Stereoselective modulatory actions of oleamide on GABA(A) receptors and voltage-gated Na(+) channels in vitro: a putative endogenous ligand for depressant drug sites in CNS

1. cis-9,10-octadecenoamide ('oleamide') accumulates in CSF on sleep deprivation. It induces sleep in animals (the trans form is inactive) but its cellular actions are poorly characterized. We have used electrophysiology in cultures from embryonic rat cortex and biochemical studies in mouse nerve preparations to address these issues. 2. Twenty microM cis-oleamide (but not trans) reversibly enhanced GABA(A) currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks. 3. cis-oleamide stereoselectively blocked veratridine-induced (but not K(+)-induced) depolarisation of mouse synaptoneurosomes (IC(50), 13. 9 microM). 4. The cis isomer stereoselectively blocked veratridine-induced (but not K(+)-induced) [(3)H]-GABA release from mouse synaptosomes (IC(50), 4.6 microM). 5. At 20 microM cis-oleamide, but not trans, produced a marked inhibition of Na(+) channel-dependent rises in intrasynaptosomal Ca(2+). 6. The physiological significance of these observations was examined by isolating Na(+) spikes in cultured pyramidal neurones. Sixty-four microM cis-oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz). 7. cis-Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC(50) of 4.1 microM suggesting a frequency- or state-dependent block of voltage-gated Na(+) channels. 8. Oleamide is a stereoselective modulator of both postsynaptic GABA(A) receptors and presynaptic or somatic voltage-gated Na(+) channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics. 9. Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal.     

Effects of dextrorotatory morphinans on brain Na+ channels expressed in Xenopus oocytes

We previously demonstrated that dextromethorphan (DM; 3-methoxy-17-methylmorphinan) analogs have neuroprotective effects. Here, we investigated the effects of DM, three of its analogs (DF, 3-methyl-17-methylmorphinan; AM, 3-allyloxy-17-methoxymorphian; and CM, 3-cyclopropyl-17-methoxymorphinan) and one of its metabolites (HM; 3-methoxymorphinan), on Na(+) channel activity. We used the two-microelectrode voltage-clamp technique to test the effects of DM, DF, AM, CM and HM on Na(+) currents (I(Na)) in Xenopus oocytes expressing cRNAs encoding rat brain Nav1.2 alpha and beta1 or beta2 subunits. In oocytes expressing Na(+) channels, DM, DF, AM and CM, but not HM, induced tonic and use-dependent inhibitions of peak I(Na) following low- and high-frequency stimulations. The order of potency for the inhibition of peak I(Na) was AM-CM > DM=DF. The DM, DF, AM and CM-induced tonic inhibitions of peak I(Na) were voltage-dependent, dose-dependent and reversible. The IC(50) values for DM, DF, AM and CM were 116.7+/-14.9, 175.8+/-16.9, 38.6+/-15.5, and 42.5+/-8.5 microM, respectively. DM and its analogs did not affect the steady-state activation and inactivation voltages. AM and CM, but not DM and DF, inhibited the plateau I(Na) more effectively than the peak I(Na) in oocytes expressing inactivation-deficient I1485Q-F1486Q-M1487Q (IFMQ3) mutant channels; the IC(50) values for AM and CM in this system were 8.4+/-1.3 and 8.7+/-1.3 microM, respectively, for the plateau I(Na) and 43.7+/-5.9 and 32.6+/-7.8 microM, respectively, for the peak I(Na). These results collectively indicate that DM and its analogs could be novel Na(+) channel blockers acting on the resting and open states of brain Na(+) channels.     

Stimulation of phosphoinositide breakdown in brain synaptoneurosomes by agents that activate sodium influx: antagonism by tetrodotoxin, saxitoxin, and cadmium

Agents that increase intracellular concentrations of Na+ stimulate phosphoinositide breakdown in guinea pig cerebral cortical synaptoneurosomes. When combined, these agents did not have additive effects on phosphoinositide breakdown but did have additive or greater than additive effects with carbamylcholine. Scorpion venom (Leiurus quinquestriatus) and pumiliotoxin B, which induce small increases in influx of 22Na+ in synaptoneurosomes, stimulate phosphoinositide breakdown by about 6- and 3-fold, respectively; both effects are inhibited by tetrodotoxin (TTX). Batrachotoxin (BTX) and veratridine, which cause a large increase in influx of 22Na+ through activation of voltage-dependent sodium channels, induce a 5- to 6-fold dose-dependent increase in phosphoinositide breakdown, which appears competitively inhibited by 5 microM TTX. BTX- and veratridine-elicited influx of 22Na+ into synaptoneurosomes is virtually completely blocked by 5 microM TTX. Agents that block voltage-dependent calcium channels, such as D-600, nifedipine, and Co2+, do not inhibit either influx of 22Na+ or stimulation of phosphoinositide breakdown elicited by scorpion venom, pumiliotoxin B, or BTX. Cadmium ions (200 microM), which are known to block TTX-resistant sodium channels, block phosphoinositide breakdown induced by agents that activate sodium influx through sodium channels. Cadmium blocks BTX-induced phosphoinositide breakdown with an IC50 value of 48 microM, while blocking BTX-induced 22Na+ influx in synaptoneurosomes with a 13-fold lower potency (IC50, 610 microM). In the presence of 0.5 microM TTX, the IC50 for Cd2+ inhibition of BTX-induced 22Na+ influx is now 430 microM. Neither TTX nor Cd2+ antagonize neurotransmitter- or monensin-induced phosphoinositide breakdown. It appears that BTX-induced phosphoinositide breakdown in guinea pig synaptoneurosomes is dependent primarily on activation of TTX-resistant, Cd2+-sensitive sodium channels that account for only a small fraction of the total sodium influx induced by BTX in synaptoneurosomes. However, cadmium also may in some way inhibit phosphoinositide breakdown elicited by sodium channel agents at a point subsequent to sodium influx.     

Molecular determinants of mexiletine structure for potent and use-dependent block of skeletal muscle sodium channels

On the basis of the information about drug receptor on voltage-gated sodium channels, mexiletine (Mex) analogs with substitutions at either the asymmetric carbon atom or the aromatic ring were synthesized as pure enantiomers. The compounds were tested in vitro for their ability to produce voltage- and use-dependent block of sodium currents (I(Na)) of frog muscle fibers by the vaseline-gap voltage-clamp method. In all experimental conditions, the drug potency was highly correlated with the lipophilicity of the group on the asymmetric center, the derivative with a benzyl moiety (Me6) having IC(50) values more than 10 times lower than those of Mex, followed by the phenyl (Me4) and the isopropyl (Me5) derivative. All of the compounds showed a further reduction of IC(50) values at depolarized membrane potentials and at high frequency of stimulation (10 Hz). Mex and Me5, but not Me4, produced a stereoselective tonic block of I(Na), the R-(-) isomers being 2-fold more potent than the S-(+) ones. The removal of both methyl groups from the aromatic ring of Mex (Me3) caused a 7-fold reduction of the potency, whereas similar substitutions on the phenyl derivative Me4 (Me7 and Me8) produced opposite effects. In fact, the IC(50) of R-(-) Me7 for use-dependent block of I(Na) was 30 times lower than that of R-(-) Mex. Me8 and Me7 were stereoselective during both tonic and use-dependent blockade. All of the compounds left-shifted the steady-state inactivation curves in relation to their potency and to the duration of the inactivating prepulse. Finally, the presence of apolar groups on the asymmetric center of mexiletine is pivotal to reinforce hydrophobic interactions with the proposed aromatic residues at the receptor, and lead to potent and therapeutically interesting inactivated channel blockers.     

Reversal of neuropathic pain by alpha-hydroxyphenylamide: a novel sodium channel antagonist

Sodium (Na) channel blockers are known to possess antihyperalgesic properties. We have designed and synthesized a novel Na channel antagonist, alpha-hydroxyphenylamide, and determined its ability to inhibit both TTX-sensitive (TTX-s) and TTX-resistant (TTX-r) Na currents from small dorsal root ganglion (DRG) neurons. alpha-Hydroxyphenylamide tonically inhibited both TTX-s and TTX-r Na currents yielding an IC(50) of 8.2+/-2.2 microM (n=7) and 28.9+/-1.8 microM (n=8), respectively. In comparison, phenytoin was less potent inhibiting TTX-s and TTX-r currents by 26.2+/-4.0% (n=8) and 25.5+/-2.0%, respectively, at 100 microM. alpha-Hydroxyphenylamide (10 microM) also shifted equilibrium gating parameters of TTX-s Na channels to greater hyperpolarized potentials, slowed recovery from inactivation, accelerated the development of inactivation and exhibited use-dependent block. In the chronic constriction injury (CCI) rat model of neuropathic pain, intraperitoneal administration of alpha-hydroxyphenylamide attenuated the hyperalgesia by 53% at 100mg/kg, without affecting motor coordination in the Rotorod test. By contrast, the reduction in pain behavior produced by phenytoin (73%; 100mg/kg) was associated with significant motor impairment. In summary, we report that alpha-hydroxyphenylamide, a sodium channel antagonist, exhibits antihyperalgesic properties in a rat model of neuropathic pain, with favorable sedative and ataxic side effects compared with phenytoin.     

NA+- and K+-channels as molecular targets of the alkaloid ajmaline in skeletal muscle fibres

BACKGROUND AND PURPOSE: Ajmaline is a widely used antiarrhythmic drug. Its action on voltage-gated ion channels in skeletal muscle is not well documented and we have here elucidated its effects on Na(+) and K(+) channels. EXPERIMENTAL APPROACH: Sodium (I(Na)) and potassium (I(K)) currents in amphibian skeletal muscle fibres were recorded using 'loose-patch' and two-microelectrode voltage clamp techniques (2-MVC). Action potentials were generated using current clamp. KEY RESULTS: Under 'loose patch' clamp conditions, the IC(50) for I(Na) was 23.2 microM with Hill-coefficient h=1.21. For I(K), IC(50) was 9.2 microM, h=0.87. Clinically relevant ajmaline concentrations (1-3 microM) reduced peak I(Na) by approximately 5% but outward I(K) values were reduced by approximately 20%. Na(+) channel steady-state activation and fast inactivation were concentration-dependently shifted towards hyperpolarized potentials ( approximately 10 mV at 25 microM). Inactivation curves were markedly flattened by ajmaline. Peak-I(K) under maintained depolarisation was reduced to approximately 30% of control values by 100 microM ajmaline. I(K) activation time constants were increased at least two-fold. Lower concentrations (10 or 25 microM) reduced steady-state-I(K) slightly but peak-I(K) significantly. Action potential generation threshold was increased by 10 microM ajmaline and repolarisation prolonged. CONCLUSIONS AND IMPLICATIONS: Ajmaline acts differentially on Na(+) and K(+) channels in skeletal muscle. This suggests at least multiple sites of action including the S4 subunit. Our data may provide a first insight into specific mechanisms of ajmaline-ion channel interaction in tissues other than cardiac muscle and could suggest possible side-effects that need to be further evaluated.     

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.     

Effect of trimipramine on depolarization-induced and Na(+)-Ca2+ exchange-induced 45calcium uptake in synaptosomes from the cortex of the rat brain.

The present study examined the inhibition of synaptosomal uptake of 45calcium by racemic trimipramine and nortrimipramine and by enantiomers of trimipramine. Trimipramine, nortrimipramine, (+)-trimipramine and (-)-trimipramine inhibited the net K(+)-induced uptake of 45calcium with IC50 values of 31, 39, 17 and 95 microM, respectively. No significant difference could be detected between the parent compound trimipramine and the metabolite nortrimipramine; however, the levorotatory isomer had an IC50 value significantly larger than the dextrorotatory isomer. At normal therapeutic doses, a 25-40% inhibition of net K(+)-induced uptake of 45calcium, could be expected with trimipramine or 30-50% inhibition for trimipramine and nortrimipramine combined; these data, therefore, do not exclude the possibility that inhibition of voltage-dependent calcium channels could contribute to the therapeutic effect of trimipramine. The order of potency of stereoisomers of trimipramine, for inhibition of calcium channels, was the same as their reported order of potency in the clinic; this parallelism adds support to the possible involvement of blockade of calcium channels in the antidepressant effect. With respect to uptake of 45calcium induced by the Na(+)-Ca2+ exchange process, all drugs inhibited this mechanism with a similar potency (IC50 74-91 microM); the drugs are not expected to have a significant effect on this exchange process, at therapeutic antidepressant doses.     

Mibefradil potently blocks ATP-activated K(+) channels in adrenal cells

Mibefradil is a novel Ca(2+) channel antagonist that preferentially blocks T-type Ca(2+) channels in many cells. Using whole-cell and single-channel patch-clamp recording, we found that mibefradil also potently blocked an ATP-activated K(+) channel (I(AC)) expressed by adrenal zona fasciculata cells. I(AC) channels were inhibited by mibefradil with an IC(50) value of 0.50 microM, a concentration 2-fold lower than that required to inhibit T-type Ca(2+) channels under similar conditions in the same cells. The inhibition of I(AC) by mibefradil was independent of the membrane potential. Mibefradil also reversibly blocked, with similar potency, unitary I(AC) currents recorded in outside-out membrane patches. An analysis of dwell time histograms indicated the presence of two closed and one open state. Mibefradil (1 microM) increased the duration of the two closed time constants (tau(c1) and tau(c2)) from 2.30 +/- 0.18 and 27.9 +/- 4.7 ms to 4.32 +/- 0.61 and 62.5 +/- 13.8 ms, respectively, but did not alter the open time constant (tau(o)). Mibefradil also failed to reduce the size of the unitary I(AC) current. A voltage-gated A-type K(+) current was also inhibited by mibefradil at concentrations approximately 10-fold higher than those required to block I(AC) (IC(50) = 4.65 microM). These results identify mibefradil as a potent inhibitor of ATP-activated K(+) channels in adrenal zona fasciculata cells. It appears to function by stabilizing closed states of these channels. In contrast to its selective block of T-type Ca(2+) channels, mibefradil may be a potent but less-selective K(+) channel blocker. In this regard, the block of K(+) channels may produce some of the toxicity associated with mibefradil in cardiovascular pharmacology.     

Structural requirements for voltage-dependent block of muscle sodium channels by phenol derivatives

We have studied the effects of four different phenol derivatives, with methyl and halogen substituents, on heterologously expressed human skeletal muscle sodium channels, in order to find structural determinants of blocking potency. All compounds blocked skeletal muscle sodium channels in a concentration-dependent manner. The methylated phenol 3-methylphenol and the halogenated phenol 4-chlorophenol blocked sodium currents on depolarization from -100 mV to 0 mV with IC(50) values of 2161 and 666 microM respectively. Methylation of the halogenated compound further increased potency, reducing the IC(50) to 268 microM in 2-methyl-4-chlorophenol and to 150 microM in 3,5-dimethyl-4-chlorophenol. Membrane depolarization before the test depolarization increased sodium channel blockade. When depolarizations were started from -70 mV or when a 2.5 s prepulse was introduced before the test pulse inducing slow inactivation, the IC(50) was reduced more than 3 fold in all compounds. The values of K(D) for the fast-inactivated state derived from drug-induced shifts in steady-state availability curves were 14 microM for 3,5-dimethyl-4-chlorophenol, 19 microM for 2-methyl-4-chlorophenol, 26 microM for 4-chlorophenol and 115 microM for 3-methylphenol. All compounds accelerated the current decay during depolarization and slowed recovery from fast inactivation. No relevant frequency-dependent block after depolarizing pulses applied at 10, 50 and 100 Hz was detected for any of the compounds. All the phenol derivatives that we examined are effective blockers of skeletal muscle sodium channels, especially in conditions that are associated with membrane depolarization. Blocking potency is increased by halogenation and by methylation with increasing numbers of methyl groups.     

Inhibitory effects of artemisinin on voltage-gated ion channels in intact nodose ganglion neurones of adult rats

Recent data show that artemisinin has anti-arrhythmic and local anaesthetic effects. To better understand the mechanisms, the effects of artemisinin on action potential discharge and voltage-gated ion channels properties were studied on nodose ganglion neurones of adult rats with known sensory afferent fibre type using whole cell patch and vagus nodose slice preparation. The present data show that both depolarization and repolarization of action potentials were markedly inhibited by artemisinin in a concentration- and time-dependent manner in either A-type or C-type nodose ganglion neurones without change in conduction velocity. Both tetrodotoxin-sensitive (TTX-S) Na+ and tetrodotoxin-resistant (TTX-R) Na+ currents were significantly reduced by micro-perfusion of artemisinin; the steady-state half-activation and half-inactivation for both TTX-S and TTX-R Na+ currents were shifted towards the right without changing slope factors. Median inhibition concentration (IC50) are 68.1 microM and 236.2 microM for TTX-S and TTX-R Na+ currents, respectively. Total outward K+ currents from C-type nodose ganglion neurones were blocked by artemisinin 30-300 microM concentration-dependently, IC50 being 104.7 microM. This effect was mimicked by tetraethylammonium 15 mM. Peak currents of N-type Ca2+ channels were also reduced significantly (IC50=344.6 microM) in the presence of artemisinin, which was less effective than that induced by 1 microM omega-conotoxin (CTX) GIVA. Our data demonstrate that depolarization and repolarization of action potentials recorded from either A- or C-type nodose ganglion neurones were inhibited by artemisinin in a concentration- and time-dependent manner, and that this inhibitory effect of artemisinin is probably due to the non-selective inhibition of all major ion channels functionally expressed in nodose ganglion neurones.     

Inhibitory effects of diprafenone stereoenantiomers on cardiac Na+ channels

The potency of (-)- and (+)-diprafenone to depress the Vmax of Na+-dependent action potentials and to block single cardiac Na+ channels was analyzed in microelectrode experiments with guinea pig papillary muscles and in patch clamp experiments with DPI-modified Na+ channels using neonatal cardiocytes. Within 20-30 min, both optical enantiomers caused a Vmax depression which occurred predominantly as a phasic blockade at a low dosage (10 mumol/l). (-)- and (+)-diprafenone were equally effective in evoking a tonic and phasic depression of Vmax. Exposing the cytoplasmic side of inside-out patches to 10 mumol/l of (-)- or (+)-diprafenone evoked a flicker block of DPI-modified Na+ channels within 1-2 s. Kinetic analysis of the latter revealed a KD value for the blocking action of 6.3 X 10(-5) mol for the (-) enantiomer and 7.1 X 10(-5) mol for the (+) enantiomer. Nevertheless, larger association and dissociation rate constants were obtained with (+)-diprafenone than with (-)-diprafenone. This indicates that there are stereoselective reaction kinetics in blocking open modified Na+ channels.     

In vitro potency and mode of action of ANQ9040: a novel fast acting muscle relaxant.

1. The in vitro potency and mode of action of the novel, rapid-onset steroidal relaxant ANQ9040 were characterized in the rat isolated phrenic nerve hemidiaphragm. 2. At 32 degrees C, ANQ9040 antagonized neurally evoked contractures with EC50s of 21.5 microM for unitary twitches; 14.4 microM for 2 Hz 'trains of four'; and 7.5 microM for 50 Hz (2 s) tetanic stimulus trains. 3. (+)-Tubocurarine was 22-24 times more potent than ANQ9040 in comparative organ bath experiments. 4. Intracellular recording from endplates revealed that ANQ9040 (0.53-10.0 microM) dose-dependently and reversibly decreased the amplitude of miniature-endplate potentials (IC50 of circa 0.95 microM) without changing transmembrane potential. 5. Surmountable antagonism of subthreshold responses to exogenous (ionophoretic) acetylcholine provided evidence for a non-depolarizing and competitive blockade of post-junctional nicotinic receptors. 6. Sucrose gap recordings of phrenic nerve action potentials revealed that, at concentrations up to 32 microM, ANQ9040 produced no tonic or frequency-dependent antagonism of axonic Na+ channels. 7. We conclude that ANQ9040 is a relatively low-affinity, non-depolarizing, nicotinic antagonist. The in vitro results are discussed in relation to factors impinging on relaxant kinetics and current models for frequency-dependent fade.     

Two novel sodium channel inhibitors from Heriaeus melloteei spider venom differentially interacting with mammalian channel's isoforms

Two new polypeptide toxins named Hm-1 and Hm-2 were isolated from the venom of the crab spider Heriaeus melloteei. These toxins consist of 37 and 40 amino acid residues, respectively, contain three intramolecular disulfide bonds, and presumably adopt the inhibitor cystine knot motif. Hm-1 is C-terminally amidated and shows a low degree of homology to spider toxins agelenin and mu-agatoxin-II, whereas Hm-2 has no relevantly related peptide sequences. Hm-1 and Hm-2 were found to act on mammalian voltage-gated Na(+) channels. Both toxins caused a strong decrease of Na(+) current peak amplitude, with IC(50) values of 336.4 and 154.8nM, respectively, on Na(V)1.4. Hm-1 and Hm-2 did not shift the voltage-dependence of activation, nor did they change the kinetics of fast inactivation of the Na(+) currents. Interestingly, both toxins negatively shifted the steady-state inactivation process, which might have important functional consequences in vivo. However, this hyperpolarizing shift cannot by itself explain the observed inhibition of the Na(+) current, indicating that the two presented toxins could provide important structural information about the interaction of polypeptide inhibitors with voltage-gated Na(+) channels.