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

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

Differential modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by the local anesthetic lidocaine

1 Voltage-gated Na+ channels are transmembrane proteins that are essential for the propagation of action potentials in excitable cells. Nav1.7 and Nav1.8 dorsal root ganglion Na+ channels exhibit different kinetics and sensitivities to tetrodotoxin (TTX). We investigated the properties of both channels in the presence of lidocaine, a local anesthetic (LA) and class I anti-arrhythmic drug. 2 Nav1.7 and Nav1.8 Na+ channels were coexpressed with the beta1-subunit in Xenopus oocytes. Na+ currents were recorded using the two-microelectrode voltage-clamp technique. 3 Dose-response curves for both channels had different EC50 (dose producing 50% maximum current inhibition) (450 microm for Nav1.7 and 104 microm for Nav1.8). Lidocaine enhanced current decrease in a frequency-dependent manner. Steady-state inactivation of both channels was also affected by lidocaine, Nav1.7 being the most sensitive. Only the steady-state activation of Nav1.8 was affected while the entry of both channels into slow inactivation was affected by lidocaine, Nav1.8 being affected to a larger degree. 4 Although the channels share homology at DIV S6, the LA binding site, they differ in their sensitivity to lidocaine. Recent studies suggest that other residues on DI and DII known to influence lidocaine binding may explain the differences in affinities between Nav1.7 and Nav1.8 Na+ channels. 5 Understanding the properties of these channels and their pharmacology is of critical importance to developing drugs and finding effective therapies to treat chronic pain.     

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.     

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.     

Mechanism of inhibition of the sodium current by bepridil in guinea-pig isolated ventricular cells.

1. Effects of bepridil, a sodium-, calcium-, and potassium-antagonistic agent, on the Na+ current were studied by the whole cell voltage clamp technique (tip resistance = 0.5 MOhm, [Na]i and [Na]o 10 mmol l-1 at 20 degrees C). 2. Bepridil produced tonic block (Kdrest = 295.44 mumol l-1, Kdi = 1.41 mumol l-1; n = 4). 3. Bepridil (100 mumol l-1) shifted the inactivation curve in the hyperpolarization direction by 13.4 +/- 2.7 mV (n = 4) without change in the slope factor. 4. In the presence of 50 mumol l-1 bepridil, bepridil showed use-dependent block at 2 Hz, whereas changes in pulse duration did not significantly effect this use-dependent block (81% +/- 2% at 10 ms, 84% +/- 3% at 30 ms, 86% +/- 3% at 100 ms; n = 4). 5. After removal of fast inactivation of the Na+ current by 3 mmol l-1 tosylchloramide sodium, bepridil (50 mumol l-1) still showed use-dependent block which was independent of the holding potential. 6. The recovery time constant from the bepridil-induced use-dependent block was 0.48 s at holding potential of -100 mV and 0.51 s at holding potential of -140 mV. 7. These results indicate that bepridil could bind to the receptor in the sodium channel through the hydrophobic and the hydrophilic pathway and leave the receptor through the hydrophobic pathway in the lipid bilayer. The binding and dissociation kinetics of this drug were shown to be fast, and the accumulation of the drug in the sodium channel appeared to be small.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of three local anaesthetics: lidocaine, bupivacaine and ropivacaine on guinea pig papillary muscle sodium channels (Vmax)

The new local anaesthetic ropivacaine (LEA 103) like lidocaine and bupivacaine used as references, blocked cardiac sodium channels in a use-dependent fashion. At equimolar concentrations lidocaine had the lowest efficacy and bupivacaine the highest. The action potential was shortened and the plateau was depressed at high concentrations of each drug. Pacing a papillary muscle at 3.3 Hz in the presence of all three drugs resulted in a marked use-dependent accumulation of block (P less than 0.01). The accumulated block slowly dissipated after rest. At -90 mV holding (= resting) potential, and at a concentration of 10 microM, the time constant for recovery from block was 186 msec. in lidocaine (n = 4), 1.4 sec. in ropivacaine (n = 7), and 2.1 sec. in bupivacaine (n = 7). Lidocaine decreased Vmax progressively at high rates of stimulation, but not significantly at rates below 2 Hz. Ropivacaine progressively decreased Vmax significantly at rates above 1 Hz, but to a lesser degree than bupivacaine. The use-dependent action of the drugs was increased at more depolarized (less negative) holding potentials, whereas at more hyperpolarized potentials the block was diminished. Lidocaine and ropivacaine could be readily dissociated from the receptors at more hyperpolarized membrane potentials (-100 to -120 mV), whereas bupivacaine bound much harder. All three drugs blocked sodium channels more effectively after a long single conditioning pulse. Bupivacaine had the most prominent effect, and lidocaine was least effective. Bupivacaine and ropivacaine seem to interact with the inactivated state of the sodium channels, whereas lidocaine acted on both the open and on the inactivated state of the channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency and voltage-dependent inhibition of type IIA Na+ channels, expressed in a mammalian cell line, by local anesthetic, antiarrhythmic, and anticonvulsant drugs.

This study examined the actions of phenytoin, carbamazepine, lidocaine, and verapamil on rat brain type IIA Na+ channels functionally expressed in mammalian cells, using the whole-cell voltage-clamp recording technique. The drugs blocked Na+ currents in both a tonic and use-dependent manner. Tonic block was more pronounced at depolarized holding potentials and reduced at hyperpolarized membrane potentials, reflecting an overall negative shift in the relationship between membrane potential and steady state inactivation. Dose-response relationships with phenytoin supported the hypothesis that the voltage dependence of tonic block resulted from the higher affinity of the drugs for inactivated than for resting channels. At -62 mV, approximately 50% of the Na+ channels were blocked by phenytoin at 13 microM, compared with therapeutic brain levels of 4-8 microM. The use-dependent component of block developed progressively during a 2-Hz train of 40-msec-long stimulus pulses from -85 mV to 0 mV. At 2 Hz, verapamil was the most potent use-dependent blocker, lidocaine and phenytoin had intermediate potencies, and carbamazepine was least effective. The use-dependent block resulted from drug binding to open and inactivated channels during the depolarizing pulses and the slow repriming of drug-bound channels during the interpulse intervals. Verapamil, lidocaine, and phenytoin all bound preferentially to open channels, but this open channel block was most striking for verapamil. Use-dependent block was less pronounced at hyperpolarized membrane potentials, due to more rapid repriming of drug-bound channels. The results indicate that type IIA Na+ channels expressed in a mammalian cell line retain the complex pharmacological properties characteristic of native Na+ channels. These channels are likely to be an important site of the anticonvulsant action of phenytoin and carbamazepine. Lidocaine and verapamil, drugs with well characterized effects on peripheral Na+ and Ca2+ channels, are also effective blockers of these brain Na+ channels.     

Role of the local anesthetic receptor in the state-dependent inhibition of voltage-gated sodium channels by the insecticide metaflumizone

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Na(v)) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Na(v)1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Na(v)1.4/F1579A and Na(v)1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Na(v)1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Na(v)1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class.     

Actions of Three Local Anaesthetics: Lidocaine,Bupivacaine and Ropivacaine on Guinea Pig Papillary Muscle Sodium Channels (V̇max)

Abstract: The new local anaesthetic ropivacaine (LEA 103) like lidocaine and bupivacaine used as references, blocked cardiac sodium channels in a use-dependent fashion. At equimolar concentrations lidocaine had the lowest efficacy and bupivacaine the highest. The action potential was shortened and the plateau was depressed at high concentrations of each drug. Pacing a papillary muscle at 3.3 Hz in the presence of all three drugs resulted in a marked use-dependent accumulation of block (P < 0.01). The accumulated block slowly dissipated after rest. At ?90 mV holding (= resting) potential, and at a concentration of 10 μM, the time constant for recovery from block was 186 msec. in lidocaine (n = 4), 1.4 sec. in ropivacaine (n = 7), and 2.1 sec. in bupivacaine (n = 7). Lidocaine decreased V?_max progressively at high rates of stimulation, but not significantly at rates below 2 Hz. Ropivacaine progressively decreased V?_max significantly at rates above 1 Hz, but to a lesser degree than bupivacaine. The use-dependent action of the drugs was increased at more depolarized (less negative) holding potentials, whereas at more hyperpolarized potentials the block was diminished. Lidocaine and ropivacaine could be readily dissociated from the receptors at more hyperpolarized membrane potentials (?100 to ?120 mV), whereas bupivacaine bound much harder. All three drugs blocked sodium channels more effectively after a long single conditioning pulse. Bupivacaine had the most prominent effect, and lidocaine was least effective. Bupivacaine and ropivacaine seem to interact with the inactivated state of the sodium channels, whereas lidocaine acted on both the open and on the inactivated state of the channels. The peak force of contraction was only moderately diminished after treatment with ropivacaine, whereas bupivacaine, even at 1 Hz, had a pronounced depressant action. Lidocaine at 10 μM had almost no effect on depolarization induced automaticity (DIA), whereas ropivacaine at 10 μM had a small depressant effect. Bupivacaine dampened the automaticity as well as peak force at 5 μM. At 10 μM no DIA could be initiated in the presence of bupivacaine.     

Altered gating and local anesthetic block mediated by residues in the I-S6 and II-S6 transmembrane segments of voltage-dependent Na+ channels

The cytoplasmic side of the voltage-dependent Na+ channel pore is putatively formed by the S6 segments of domains I to IV. The role of amino acid residues of I-S6 and II-S6 in channel gating and local anesthetic (LA) block was investigated using the cysteine scanning mutagenesis of the rat skeletal muscle Na+ channel (Nav1.4). G428C uniquely reduced sensitivity to rested state or first-pulse block by lidocaine without alterations in the voltage dependence or kinetics of gating that would otherwise account for the increase in the IC50 for block. Mutations in I-S6 (N434C and I436C) and in II-S6 (L785C and V787C) increased sensitivity to first-pulse block by lidocaine. Enhanced inactivation accounted for the increased sensitivity of N434C to lidocaine and hastening of inactivation of I436C in the absence of drug could account for higher affinity first-pulse block. Mutations in I-S6 (I424C, I425C, and F430C) and in II-S6 (I782C and V786C) reduced the use-dependent lidocaine block. The reduction in use-dependent block of F430C was consistent with alterations in inactivation gating; the other mutants did not exhibit gating changes that could explain the reduced sensitivity to lidocaine. Therefore, several amino acids (I424, I425, G428, I782, and V786), in addition to those previously identified (Yarov-Yarovoy et al., 2002), alter the sensitivity of Nav1.4 to lidocaine, independent of mutation-induced changes in gating. The magnitude of the change in the IC50 values, the isoform, and LA dependence of the changes in affinity suggest that the determinants of binding in I-S6 and II-S6 are subsidiary to those in IV-S6.     

Effect of dextromethorphan on human K(v)1.3 channel activity: involvement of C-type inactivation

Dextromethorphan exhibits neuroprotective effects against inflammation-mediated neurodegeneration. However, relatively little is known regarding the molecular mechanism for this inflammation-mediated neuroprotection. Human K(v)1.3 channels, one of the voltage-gated potassium channels, are widely expressed in the immune and nervous systems. Activation of human K(v)1.3 channels causes neuroglia-mediated neurodegeneration. Agents that inhibit human K(v)1.3 channel activity have been developed as novel drugs for immunosuppression. In the present study, we investigated the effects of dextromethorphan on human K(v)1.3 or K(v)1.2 channel activity heterologously expressed in Xenopus laevis oocytes. The channel currents were measured with the two-electrode voltage clamp technique. Activation of both channels induced outward peak and steady-state currents. Dextromethorphan treatment induced a slight inhibition of peak currents in human K(v)1.2 and K(v)1.3 channels, whereas dextromethorphan profoundly inhibited the steady-state currents of human K(v)1.3 channels compared to K(v)1.2 channel currents. Dextromethorphan's action on steady-state currents of human K(v)1.3 channels was in a concentration-dependent manner. The half-maximal inhibitory concentration (IC(50)) on steady-state currents of human K(v)1.3 channels was 12.8±1.6μM. Dextromethorphan also accelerated the C-type inactivation rate, increased the current decay rate, and inhibited currents in a use-dependent manner. These results indicate that dextromethorphan accelerates C-type inactivation of human K(v)1.3 channels and acts as an open-channel blocker. These results further suggest the possibility that dextromethorphan-mediated acceleration of C-type inactivation of human K(v)1.3 channels might be one of the cellular bases of dextromethorphan-mediated protection against inflammation-mediated neurodegeneration.     

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.     

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.     

Phenol derivatives accelerate inactivation kinetics in one inactivation-deficient mutant human skeletal muscle Na(+) channel

Altered inactivation kinetics in skeletal muscle Na(+) channels due to mutations in the encoding gene are causal for the alterations in muscle excitability in nondystrophic myotonia. Na(+) channel blockers like lidocaine and mexiletine, suggested for therapy of myotonia, do not reconstitute inactivation in channels with defective inactivation in vitro. We examined the effects of four methylated and/or halogenated phenol derivatives on one heterologously expressed inactivation-deficient Paramyotonia congenita-mutant (R1448H) muscle Na(+) channel in vitro. All these compounds accelerated delayed inactivation of R1448H-whole-cell currents during a depolarization and delayed accelerated recovery from inactivation. The potency of these effects paralleled the potency of the drugs to block the peak current amplitude. We conclude that the investigated phenol derivatives affect inactivation-deficient Na(+) channels more specifically than lidocaine and mexiletine. However, for all compounds, the effect on inactivation was accompanied by a substantial block of the peak current amplitude.     

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.     

Local anesthetic inhibits hyperpolarization-activated cationic currents

Systemic administration of local anesthetics has beneficial perioperative properties and an anesthetic-sparing and antiarrhythmic effect, although the detailed mechanisms of these actions remain unclear. In the present study, we investigated the effects of a local anesthetic, lidocaine, on hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels that contribute to the pacemaker currents in rhythmically oscillating cells of the heart and brain. Voltage-clamp recordings were used to examine the properties of cloned HCN subunit currents expressed in Xenopus laevis oocytes and human embryonic kidney (HEK) 293 cells under control condition and lidocaine administration. Lidocaine inhibited HCN1, HCN2, HCN1-HCN2, and HCN4 channel currents at 100 μM in both oocytes and/or HEK 293 cells; it caused a decrease in both tonic and maximal current (～30-50% inhibition) and slowed current activation kinetics for all subunits. In addition, lidocaine evoked a hyperpolarizing shift in half-activation voltage (ΔV(1/2) of ～-10 to -14 mV), but only for HCN1 and HCN1-HCN2 channels. By fitting concentration-response data to logistic functions, we estimated half-maximal (EC(50)) concentrations of lidocaine of ～30 to 40 μM for the shift in V(1/2) observed with HCN1 and HCN1-HCN2; for inhibition of current amplitude, calculated EC(50) values were ～50 to 70 μM for HCN1, HCN2, and HCN1-HCN2 channels. A lidocaine metabolite, monoethylglycinexylidide (100 μM), had similar inhibitory actions on HCN channels. These results indicate that lidocaine potently inhibits HCN channel subunits in dose-dependent manner over a concentration range relevant for systemic application. The ability of local anesthetics to modulate I(h) in central neurons may contribute to central nervous system depression, whereas effects on I(f) in cardiac pacemaker cells may contribute to the antiarrhythmic and/or cardiovascular toxic action.     

A multifunctional aromatic residue in the external pore vestibule of Na+ channels contributes to the local anesthetic receptor

Voltage-gated Na(+) (Na(v)) channels are responsible for initiating action potentials in excitable cells and are the targets of local anesthetics (LA). The LA receptor is localized to the cytoplasmic pore mouth formed by the S6 segments from all four domains (DI-DIV) but several outer pore-lining residues have also been shown to influence LA block (albeit somewhat modestly). Many of the reported amino acid substitutions, however, also disrupt the inactivated conformations that favor LA binding, complicating the interpretation of their specific effects on drug block. In this article, we report that an externally accessible aromatic residue in the Na(v) channel pore, DIV-Trp1531, when substituted with cysteine, completely abolished LA block (e.g., 300 microM mexiletine induced a use-dependent block with 65.0 +/- 2.9% remaining current and -11.0 +/- 0.6 mV of steady-state inactivation shift of wild-type (WT) channels versus 97.4 +/- 0.7% and -2.4 +/- 2.1 mV of W1531C, respectively; p < 0.05) without destabilizing fast inactivation (complete inactivation at 20 ms at -20 mV; V(1/2) = -70.0 +/- 1.6 mV versus -48.6 +/- 0.5 mV of WT). W1531C also abolished internal QX-222 block (200 microM; 98.4 +/- 3.4% versus 54.0 +/- 3.2% of WT) without altering drug access. It is interesting that W1531Y restored WT blocking behavior, whereas W1531A channels exhibited an intermediate phenotype. Together, our results provide novel insights into the mechanism of drug action, and the structural relationship between the LA receptor and the outer pore vestibule.     

Electrophysiologic properties of lidocaine, cocaine, and n-3 fatty-acids block of cardiac Na+ channels

Lidocaine and cocaine, two local anesthetics, and n-3 polyunsaturated fatty acids in fish oils, inhibit the voltage-gated Na(+) channels of cardiomyocytes. This inhibition by lidocaine and n-3 fish oil is associated with antiarrhythmic effects, whereas with cocaine lethal arrhythmias may occur. These electrophysiologic studies show that at the concentrations tested, the n-3 fish oil fatty acids and lidocaine share three actions on I(Na): a potent inhibition of I(Na); a strong voltage-dependence of this inhibition; and a large shift of the steady-state inactivation to hyperpolarized potentials. By contrast cocaine shares only the potent inhibition of I(Na). The voltage-dependence of the inhibition is much decreased with cocaine, which produces only a very small leftward shift of the voltage-dependence of inactivation. The large leftward shift of the steady-state inactivation seems very important in the prevention of fatal arrhythmias by the n-3 fatty acids. Thus, we suggest that it is lack of this effect by cocaine, which is one factor, that eliminates its ability to prevent fatal cardiac arrhythmias. Further we report that in cultured neonatal rat cardiomyocytes n-3 fish oil fatty acids terminate the tachycardia induced by the alpha(1) adrenergic agonist, phenylephrine, whereas cocaine accelerates the tachycardia and causes bouts of tachyarrhythmias.     

Electrophysiological interactions between quinidine-lidocaine and quinidine-phenytoin in guinea-pig papillary muscle

The interactions between quinidine and lidocaine or phenytoin at the sodium channel level have been studied in the present work. The maximum upstroke velocity (Vmax) of the guinea-pig papillary muscle action potential has been used as a measure of the sodium current. Lidocaine interfered with the use-dependent blocking effects on Vmax of quinidine, by decreasing the fraction of sodium channels blocked by quinidine during the conditioning action potential, in an apparently competitive way. These results strongly suggest that quinidine and lidocaine bind to a common receptor site. Alternatively, it has been suggested that lidocaine and quinidine bind to different but related receptor sites, since lidocaine may induce allosteric changes in quinidine's receptor. Phenytoin increased the use-dependent blocking effects on Vmax of quinidine by slowing the time course of the slow component of reactivation of Vmax induced by quinidine. Phenytoin did not change the fraction of sodium channels blocked by quinidine during the conditioning action potential. These results suggest that phenytoin binds to a different receptor site than quinidine.