Unexpected mexiletine responses of a mutant cardiac Na+ channel implicate the selectivity filter as a structural determinant of antiarrhythmic drug access

Gating properties of Na(+) channels are the critical determinants for the state-dependent block by class I antiarrhythmic drugs; however, recent site-directed mutagenesis studies have shown that the Na(+) channel selectivity filter region controls drug access to and dissociation from the binding site. To validate these observations, we have exploited a naturally occurring cardiac Na(+) channel mutation, S1710L, located next to the putative selectivity filter residue of domain 4, and evaluated the pharmacological properties to mexiletine using whole-cell, patch-clamp recordings. Consistent with the large negative shift of steady-state inactivation and the enhanced slow inactivation, the S1710L channel showed greater mexiletine tonic block than wild-type (WT) channel. In contradiction, S1710L showed attenuated use-dependent block by mexiletine and accelerated recovery from block, suggesting that the drug escape though the external access path is facilitated. Extracellularly applied QX-314, a membrane-impermeant derivative of lidocaine, elicited significantly enhanced tonic block in S1710L similar to mexiletine. However, recovery from internally applied QX-314 was accelerated by 4.4-fold in S1710L compared with WT. These results suggest that the drug access to and dissociation from the binding site through the hydrophilic path are substantially altered. Moreover, K(+) permeability was 1.9-fold increased in S1710L, verifying that the mutated residue is located in the ion-conducting pore. We propose that the Na(+) channel selectivity filter region is a structural determinant for the antiarrhythmic drug sensitivity in addition to gating properties of the indigenous Na(+) channels that govern the state-dependent drug block.     

Preferred mexiletine block of human sodium channels with IVS4 mutations and its pH-dependence

The effects of extracellular pH (6.2, 7.4 and 8.2) and 0.1 mM mexiletine, a channel blocker of the lidocaine type, are studied on two mutations of the fourth voltage sensor of the Nav1.4 sodium channel, R1448H/C. The fast inactivated channel state to which mexiletine preferentially binds is destabilized by the mutations. By contrast to the expected low response of R1448H/C carriers, mexiletine is particularly effective in preventing exercise-induced stiffness and paralysis from which these patients suffer. Our measurements performed in the whole-cell mode on stably transfected HEK cells show for the first time that the mutations strikingly accelerate closed-state inactivation and, as steady-state fast inactivation is shifted to more negative potentials, stabilize the fast inactivated channel state in the potential range around the resting potential. At pH 7.4 and 8.2, the phasic mexiletine block is larger for R1448C (55%) and R1448H (47%) than for wild-type channels (31%) due to slowed recovery from block (tau is approximately 520 ms for R1448C versus 270 ms for wild-type at pH 7.4) although the recovery from inactivation is slightly faster for the mutants (tau is approximately 1.9 ms for R1448C versus 3.8 ms for wild-type at pH 7.4). At pH 6.2, recovery from block is relatively fast (tau is approximately 35 ms for R1448H/C and 14 ms for wild-type) and thus shows no use-dependence. We conclude that enhanced closed-state inactivation expands the concept of a mutation-induced uncoupling of channel inactivation from activation to a new potential range and that the higher mexiletine efficacy in R1448H/C carriers compared to other myotonic patients offers a pharmacogenetic strategy for mutation-specific treatment.     

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.     

Voltage-dependent block of normal and mutant muscle sodium channels by 4-Chloro-m-Cresol

1 The effects of 4-Chloro-m-Cresol (4-CmC) were examined on heterologously expressed wild type (WT), Paramyotonia Congenita (R1448H) and Hyperkalemic Periodic Paralysis (M1360V) mutant alpha-subunits of human muscle sodium channels. 2 Block of rested sodium channels caused by 4-CmC was concentration-dependent with an ECR50 of 0.40 mM in WT, 0.45 mM in R1448H and 0.49 mM in M1360V. 3 Inactivation significantly promoted 4-CmC-induced sodium channel block in all clones indicated by 4-CmC-induced shifts of steady-state availability curves, reflecting a higher proportion of channel block at depolarized membrane potentials. Channel block was almost complete (>90%) at concentrations close to the ECR50 (0.5 mM) on application of an inactivating prepulse before the test pulse. 4 4-CmC accelerated the current decay following depolarization and prolonged recovery from inactivation in all clones. Of these, R1448H, the mutant which displayed severely impaired inactivation in the controls, responded to 4-CmC with the most pronounced acceleration of inactivation. Control experiments revealed enhanced recovery from inactivation in the mutants, which was restored to normal in 0.1 mM 4-CmC. 5 4-CmC induced no additional frequency-dependent block. 6 Our results clearly demonstrate that 4-CmC is as effective as lidocaine (Fan et al., 1996) in blocking muscle sodium channels. Low concentrations of the compound (相似文献   

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.     

Voltage-dependent inhibition of brain Na(+) channels by American ginseng

American ginseng (Panax quinquefolius) is a major species of ginseng that has many pharmacological effects. Studies have demonstrated that constituents of ginseng have neuroprotective effects during ischemia. Neuronal damage during ischemic episodes has been associated with abnormal Na(+) fluxes. Drugs that block voltage-dependent Na(+) channels provide cytoprotection during cerebral ischemia. We thus hypothesized that American ginseng may block Na(+) channels. In this study, effects of an American ginseng aqueous extract was evaluated in tsA201 cells transfected with cDNA expressing alpha subunits of the Brain(2a) Na(+) channel using the whole-cell patch clamp technique. We found that American ginseng extract tonically and reversibly blocked the channel in a concentration- and voltage-dependent manner. It shifted the voltage-dependence of inactivation by 14 mV (3 mg/ml) in the hyperpolarizing direction and delayed recovery from inactivation, whereas activation of the channel was unaffected. Ginsenoside Rb(1), a major constituent of the American ginseng extract, produced similar effects. The data were compared with the actions of lidocaine, a Na(+) channel blocker. Our results suggest that Na(+) channel block by American ginseng extract and Rb(1) was primarily due to interaction with the inactive state of the channel. Inhibition of the Na(+) channel activity by American ginseng extract may contribute to its neuroprotective effect during ischemia.     

Increased hindrance on the chiral carbon atom of mexiletine enhances the block of rat skeletal muscle Na+ channels in a model of myotonia induced by ATX

1 The antiarrhythmic drug mexiletine (Mex) is also used against myotonia. Searching for a more efficient drug, a new compound (Me5) was synthesized substituting the methyl group on the chiral carbon atom of Mex by an isopropyl group. Effects of Me5 on Na+ channels were compared to those of Mex in rat skeletal muscle fibres using the cell-attached patch clamp method. 2 Me5 (10 microM) reduced the maximal sodium current (INa) by 29.7+/-4.4 % (n=6) at a frequency of stimulation of 0.3 Hz and 65.7+/-4.4 % (n=6) at 1 Hz. At same concentration (10 microM), Mex was incapable of producing any effect (n=3). Me5 also shifted the steady-state inactivation curves by -7. 9+/-0.9 mV (n=6) at 0.3 Hz and -12.2+/-1.0 mV (n=6) at 1 Hz. 3 In the presence of sea anemone toxin II (ATX; 5 microM), INa decayed more slowly and no longer to zero, providing a model of sodium channel myotonia. The effects of Me5 on peak INa were similar whatever ATX was present or not. Interestingly, Me5 did not modify the INa decay time constant nor the steady-state INa to peak INa ratio. 4 Analysis of ATX-induced late Na+ channel activity shows that Me5 did not affect mean open times and single-channel conductance, thus excluding open channel block property. 5 These results indicate that increasing hindrance on the chiral atom of Mex increases drug potency on wild-type and ATX-induced noninactivating INa and that Me5 might improve the prophylaxis of myotonia.     

Comparative actions of mexiletine on sodium channels in nerve, skeletal and cardiac muscle

Mexiletine's actions on voltage-clamped sodium channels of frog myelinated nerve and skeletal muscle are described. Mexiletine blocks half the sodium channels (infrequent depolarizations) of single myelinated nerves at a 83 micro M concentration while only 26 micro M is required to do the same in skeletal muscle preparations where similar vaseline-gap techniques are utilized. Mexiletine's potency for block of sodium current in nerve is clearly related to its lipid distribution characteristics given proper consideration of the drug class to which it belongs. Hyperpolarizing prepulses, which are typically used to remove normal sodium inactivation, appear to reduce drug blocking potency suggesting that noninactive channels have a considerably lower affinity for the drug than do inactive channels. Direct evidence supporting selective drug block of inactive channels is also given. In addition the effects of this drug on sodium channels of guinea pig papillary muscle have been studied using measurements of maximum upstroke velocity of intracellularly recorded action potentials. In these myocardial studies 5 to 20 micro M mexiletine depressed upstroke velocity of papillary muscle action potentials in a frequency-dependent fashion. No basal (nonfrequency-dependent) block was observed in heart at these therapeutic concentrations of mexiletine. comparisons are made between skeletal and cardiac muscle effects of mexiletine, especially relating to the important role played by sodium channel inactivation.     

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.     

Inhibition of cardiac Na+ current by primaquine

The electrophysiological effects of the anti-malarial drug primaquine on cardiac Na(+) channels were examined in isolated rat ventricular muscle and myocytes. In isolated ventricular muscle, primaquine produced a dose-dependent and reversible depression of dV/dt during the upstroke of the action potential. In ventricular myocytes, primaquine blocked I(Na)(+) in a dose-dependent manner, with a K(d) of 8.2 microM. Primaquine (i) increased the time to peak current, (ii) depressed the slow time constant of I(Na)(+) inactivation, and (iii) slowed the fast component for recovery of I(Na)(+) from inactivation. Primaquine had no effect on: (i) the shape of the I - V curve, (ii) the reversal potential for Na(+), (iii) the steady-state inactivation and g(Na)(+) curves, (iv) the fast time constant of inactivation of I(Na)(+), and (v) the slow component of recovery from inactivation. Block of I(Na)(+) by primaquine was use-dependent. Data obtained using a post-rest stimulation protocol suggested that there was no closed channel block of Na(+) channels by primaquine. These results suggest that primaquine blocks cardiac Na(+) channels by binding to open channels and unbinding either when channels move between inactivated states or from an inactivated state to a closed state. Cardiotoxicity observed in patients undergoing malaria therapy with aminoquinolines may therefore be due to block of Na(+) channels, with subsequent disturbances of impulse conductance and contractility.     

The cardiac persistent sodium current: an appealing therapeutic target?

The sodium current in the heart is not a single current with a mono-exponential decay but rather a mixture of currents with different kinetics. It is not clear whether these arise from distinct populations of channels, or from modulation of a single population. A very slowly inactivating component, [(INa(P))] I(Na(P)) is usually about 1% of the size of the peak transient current [I(Na(T))], but is enhanced by hypoxia. It contributes to Na(+) loading and cellular damage in ischaemia and re-perfusion, and perhaps to ischaemic arrhythmias. Class I antiarrhythmic agents such as flecainide, lidocaine and mexiletine generally block I(NA(P)) more potently than block of I(Na(T)) and have been used clinically to treat LQT3 syndrome, which arises because mutations in SCN5A produce defective inactivation of the cardiac sodium channel. The same approach may be useful in some pathological situations, such as ischaemic arrhythmias or diastolic dysfunction, and newer agents are being developed with this goal. For example, ranolazine blocks I(Na(P)) about 10 times more potently than I(Na(T)) and has shown promise in the treatment of angina. Alternatively, the combination of I(Na(P)) block with K(+) channel block may provide protection from the induction of Torsades de Pointe when these agents are used to treat atrial arrhythmias (eg Vernakalant). In all of these scenarios, an understanding of the role of I(Na(P)) in cardiac pathophysiology, the mechanisms by which it may affect cardiac electrophysiology and the potential side effects of blocking I(Na(P)) in the heart and elsewhere will become increasingly important.     

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.     

Optically active mexiletine analogues as stereoselective blockers of voltage-gated Na(+) channels

Optically active mexiletine analogues were synthesized and evaluated in vitro as use-dependent blockers of skeletal muscle sodium channels. The mexiletine analogues were obtained by replacing either the methyl group on the stereogenic center of mexiletine [1-(2,6-dimethylphenoxy)propan-2-amine] with a phenyl group or modifying the phenoxy moiety (by removal of one or both of the methyl groups, or introducing a chlorine atom), or both. The voltage clamp recordings showed that, regardless of the substitution pattern of the aryloxy moiety, all the compounds bearing a phenyl group on the stereogenic center (3a-f) were more active than mexiletine both in tonic and phasic block. This observation was in contrast with what was observed for mexiletine, where the removal of both methyls from the aryloxy moiety caused a dramatic reduction of potency. The most potent congener, (R)-2-(2-methylphenoxy)-1-phenylethanamine [(R)-3b], was 27-fold more potent than (R)-mexiletine in producing a tonic block, i.e., the reduction of peak sodium current in resting conditions after application of the compound. (R)-3b maintained a use-dependent behavior, being 23-fold more potent in condition of high frequency of stimulation (phasic block). Despite what was observed with mexiletine, the stereoselectivity held in phasic block conditions. Stereoselectivity indexes were generally low, ranging from 1 to 4, but except for that of the 2,6-xylyloxy congener 3c, they were higher for the congeners bearing a phenyl ring on the stereogenic center than for mexiletine and its strictly related analogue 1-methyl-2-phenoxyethanamine (1). This finding was in agreement with Pfeiffer's rule. The introduction of a chlorine atom in the 4-position of the aryloxy moiety caused a reduction of potency and a reversal of stereoselectivity as well. On the basis of the model to date accepted for the sodium channel local anesthetic-like molecule receptor, some possible explanations of our observations will be proposed.     

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.     

Inhibition of tetrodotoxin (TTX)-resistant and TTX-sensitive neuronal Na(+) channels by the secretolytic ambroxol

Ambroxol has a long history for the treatment of airway diseases because of its beneficial effects on surfactant synthesis and mucus-modifying properties. Some findings, however, point to an additional effect on neuronal signal transduction: ambroxol can suppress reflexes such as the cough or the corneal reflex. The airways and the cornea are innervated by C-fibers, which express voltage-gated Na(+) channels with and without sensitivity to tetrodotoxin (TTX). In this study, we performed voltage-clamp experiments to investigate whether ambroxol affects these channel types. In rat dorsal root ganglia, TTX-resistant Na(+) currents were suppressed in a concentration-dependent manner with IC(50) values of 35.2 and 22.5 microM for tonic and phasic block, respectively. Depolarizing prepulses increased the potency of ambroxol, and steady-state inhibition curves were shifted to more negative values. The inhibition was not frequency-dependent. TTX-sensitive currents were inhibited with lower potency (approximately 50% inhibition with 100 microM). Recombinant rat brain IIA channels in Chinese hamster ovary cells were blocked with IC(50) values of 111.5 and 57.6 microM for tonic and phasic block, respectively; in contrast to TTX-resistant channels the block was frequency-dependent. Thus, ambroxol indeed blocks neuronal voltage-gated Na(+) channels, and TTX-resistant channels in sensory neurons were more sensitive than TTX-sensitive. Compared with known local anesthetics (e.g., lidocaine or benzocaine), the potency for Na(+) channel block was relatively high. A recent clinical trial has further confirmed that ambroxol relieved pain and was beneficial in patients who suffered from sore throat.     

Voltage-dependent blockade of normal and mutant muscle sodium channels by benzylalcohol

1. We studied the effects of benzylalcohol on heterologously expressed wild type (WT), paramyotonia congenita (R1448H) and hyperkalaemic periodic paralysis (M1360V) mutant alpha-subunits of human skeletal muscle sodium channels. 2. Benzylalcohol blocked rested channels at -150 mV membrane potential, with an ECR(50) of 5.3 mM in wild type, 5.1 mM in R1448H, and 6.2 mM in M1360V. When blockade was assessed at -100 mV, the ECR(50) was reduced in R1448H (2 mM) compared with both wild type (4.3 mM; P<0.01) and M1360V (4.3 mM). 3. Membrane depolarization before the test depolarization significantly promoted benzylalcohol-induced sodium channel blockade. The values of K(D) for the fast-inactivated state derived from benzylalcohol-induced shifts in steady-state availability curves were 0.66 mM in wild type and 0.58 mM in R1448H. In the presence of slow inactivation induced by 2.5 s depolarizing prepulses, the ECI(50) for benzylalcohol-induced current inhibition was 0.59 mM in wild type and 0.53 mM in R1448H. 4. Recovery from fast inactivation was prolonged in the presence of drug in all clones. 5. Benzylalcohol induced significant frequency-dependent block at stimulating frequencies of 10, 50, and 100 Hz in all clones. 6. Our results clearly show that benzylalcohol is an effective blocker of muscle sodium channels in conditions that are associated with membrane depolarization. Mutants that enter voltage-dependent inactivation at more hyperpolarized membrane potentials compared with wild type are more sensitive to inhibitory effects at the normal resting potential.     

Characteristics of ginsenoside Rg3-mediated brain Na+ current inhibition

We demonstrated previously that ginsenoside Rg(3) (Rg(3)), an active ingredient of Panax ginseng, inhibits brain-type Na(+) channel activity. In this study, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced Na(+) channel inhibition. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on Na(+) currents (I(Na)) in Xenopus laevis oocytes expressing wild-type rat brain Na(V)1.2 alpha and beta1 subunits, or mutants in the channel entrance, the pore region, the lidocaine/tetrodotoxin (TTX) binding sites, the S4 voltage sensor segments of domains I to IV, and the Ile-Phe-Met inactivation cluster. In oocytes expressing wild-type Na(+) channels, Rg(3) induced tonic and use-dependent inhibitions of peak I(Na). The Rg(3)-induced tonic inhibition of I(Na) was voltage-dependent, dose-dependent, and reversible, with an IC(50) value of 32 +/- 6 microM. Rg(3) treatment produced a 11.2 +/- 3.5 mV depolarizing shift in the activation voltage but did not alter the steady-state inactivation voltage. Mutations in the channel entrance, pore region, lidocaine/TTX binding sites, or voltage sensor segments did not affect Rg(3)-induced tonic blockade of peak I(Na). However, Rg(3) treatment inhibited the peak and plateau I(Na) in the IFMQ3 mutant, indicating that Rg(3) inhibits both the resting and open states of Na(+) channel. Neutralization of the positive charge at position 859 of voltage sensor segment domain II abolished the Rg(3)-induced activation voltage shift and use-dependent inhibition. These results reveal that Rg(3) is a novel Na(+) channel inhibitor capable of acting on the resting and open states of Na(+) channel via interactions with the S4 voltage-sensor segment of domain II.     

Disparate role of Na(+) channel D2-S6 residues in batrachotoxin and local anesthetic action

Batrachotoxin (BTX) stabilizes the voltage-gated Na(+) channels in their open conformation, whereas local anesthetics (LAs) block Na(+) conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha-subunit Na(+) channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na(+) channel ((mu)1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na(+) currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na(+) currents during repetitive pulses. Two of these four mutants, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na(+) channels (K(I)) of approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.     

Inhibition of frog skeletal muscle sodium channels by newly synthesized chiral derivatives of mexiletine and tocainide

To search for potent use-dependent blockers of skeletal muscle sodium channels as potential antimyotonic agents, the actions of newly synthesized chiral analogs of mexiletine and tocainide were tested in vitro on sodium currents of single fibers of frog semitendinosus muscle by vaseline-gap voltage clamp method. The effect of each drug on the maximal peak Na⁺ transient (I_{Na max}) was evaluated as both tonic and use-dependent block by using infrequent depolarizing stimulation and trains of pulses at 2–10 Hz frequency, respectively. The mexiletine analog 3-(2,6-dimethylphenoxy)-2-methylpropanamine (Me2), having an increased distance between the phenyl and the amino groups, was less potent than mexiletine in producing a tonic block but produced a remarkable use-dependent block. In fact, the half-maximal concentration (IC₅₀) for tonic block of S(–)-Me2 was 108 μM vs. 54.5 μM of R(–)-mexiletine, but the IC₅₀ was 6.2 times lowered by the 10 Hz stimulation with respect to the 2.4fold decrease observed with mexiletine. The R(–)-mexiletine and the S(–)-Me2 were about twofold more potent than the corresponding enantiomers in producing a tonic block, but the stereoselectivity attenuated during use-dependent blockade. The more lipophilic 2-(4-chloro-2-methylphenoxy)-1-phenylethylamine (Me1), presently available as raceme, produced a potent and irreversible tonic block of the sodium currents with an IC₅₀ of 29 μM, but had a less pronounced use-dependent inhibition, with a 1.9fold decrease of the IC₅₀ at 10 Hz. The R(–) isomer of 2′,6′-valinoxylidide (To1), a tocainide derivative with an increased hindrance on the chiral carbon atom, was twofold (IC₅₀ = 209 μM) and tenfold (IC₅₀ = 27.4 μM) more potent than R(–)-tocainide in tonic and use-dependent block, respectively. Tocainide was almost devoid of stereoselectivity, whereas the eudismic ratio of To1 [(IC₅₀ S(+)-To1/IC₅₀ R(–)-To1] was 1.7. As for mexiletine and Me2, the stereoselectivity of To1 was the weaker the higher the frequency of stimulation. The cyclic pyrrolo-imidazolonic tocainide analog To2 produced a small tonic block at 500 μM, and 1 min stimulation at 10 Hz was needed to show up a 50% block of I_{Na max}. All the compounds produced a left-shift of the steady-state inactivation curve correlated positively with the extent of use-dependent inhibition, with the exception of the cyclic To2 that acted as an open-channel blocker. The highly use-dependent blockers Me2 and To1 might be promising drugs to solve high frequency discharges of action potentials typical of myotonic muscles. Concomitantly the high potency of Me1 and the open-channel block exerted by To2 can represent important features to get selective blockers for skeletal muscle sodium channels. Received: 12 March 1997 / Accepted: 7 August 1997     

Halothane attenuates the cerebroprotective action of several Na+ and Ca2+ channel blockers via reversal of their ion channel blockade

We have previously shown the involvement of Na(+) channel as well as N-type and P/Q-type Ca(2+) channels in the oxygen and glucose deprivation-induced injury in rat cerebrocortical slices. In the present study, we investigated the influence of halothane on the cerebroprotective effects of a variety of Na(+) and Ca(2+) channel blockers in rat cerebrocortical slices. The hypoxic injury was attenuated by Na(+) channel blockers including tetrodotoxin, lidocaine and dibucaine, and Ca(2+) channel blockers, such as verapamil, omega-agatoxin IVA and omega-conotoxin GVIA. Halothane abolished the protective effects of lidocaine, dibucaine and verapamil, all of which block the respective cation channels in a voltage-dependent manner, without affecting the actions of tetrodotoxin, omega-agatoxin IVA and omega-conotoxin GVIA, which reveal voltage-independent blockade. On the other hand, the nitric oxide synthesis estimated from the extracellular cyclic GMP formation was elevated during exposure to hypoxia. All channel blockers tested here attenuated hypoxia-evoked nitric oxide synthesis. Halothane blocked almost completely these actions of lidocaine and verapamil. Moreover, the Na(+) and Ca(2+) channel blockade by these compounds, as determined by veratridine- and KCl-stimulated nitric oxide synthesis, respectively, was also reversed by halothane. These findings suggest that an anesthetic agent halothane reversed the Na(+) and Ca(2+) channel blockade of several voltage-dependent ion channel blockers, leading to the attenuation of their cerebroprotective actions. Therefore, the influence of halothane anesthesia should be taken into consideration for the evaluation of neuroprotective action of Na(+) and Ca(2+) channel blockers.