Time-dependent block and resurgent tail currents induced by mouse beta4(154-167) peptide in cardiac Na+ channels

Resurgent tail Na(+) currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse beta4 subunit, beta4(154-167), acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons (Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005. Neuron. 45:233-244). To explore these phenotypes in vitro, we characterized beta4(154-167) actions in inactivation-deficient cardiac hNav1.5 Na(+) channels expressed in human embryonic kidney 293t cells. Intracellular beta4(154-167) from 25-250 microM elicited a conspicuous time-dependent block of inactivation-deficient Na(+) currents at 50 mV in a concentration-dependent manner. On and off rates for beta4(154-167) binding were estimated at 10.1 microM(-1)s(-1) and 49.1 s(-1), respectively. Upon repolarization, large tail currents emerged with a slight delay at -140 mV, probably as a result of the rapid unblocking of beta4(154-167). Near the activation threshold (approximately -70 mV), resurgent tail currents were robust and long lasting. Likewise, beta4(154-167) induces resurgent currents in wild-type hNav1.5 Na(+) channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na(+) channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na(+) channels opened persistently but became resistant to beta4(154-167) and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na(+) channels resistant to beta4(154-167). Together, our in vitro studies identify a putative S6-binding site for beta4(154-167) within the inner cavity of hNav1.5 Na(+) channels. Such an S6 receptor readily explains (1) why beta4(154-167) gains access to its receptor as an open-channel blocker, (2), why bound beta4(154-167) briefly prevents the activation gate from closing by a "foot-in-the-door" mechanism during deactivation, (3) why BTX inhibits beta4(154-167) binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates beta4(154-167) binding.     

Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms

We examined the kinetics of voltage-dependent sodium currents in cerebellar Purkinje neurons using whole-cell recording from dissociated neurons. Unlike sodium currents in other cells, recovery from inactivation in Purkinje neurons is accompanied by a sizeable ionic current. Additionally, the extent and speed of recovery depend markedly on the voltage and duration of the prepulse that produces inactivation. Recovery is faster after brief, large depolarizations (e.g., 5 ms at +30 mV) than after long, smaller depolarizations (e.g., 100 ms at -30 mV). On repolarization to -40 mV following brief, large depolarizations, a resurgent sodium current rises and decays in parallel with partial, nonmonotonic recovery from inactivation. These phenomena can be explained by a model that incorporates two mechanisms of inactivation: a conventional mechanism, from which channels recover without conducting current, and a second mechanism, favored by brief, large depolarizations, from which channels recover by passing transiently through the open state. The second mechanism is consistent with voltage-dependent block of channels by a particle that can enter and exit only when channels are open. The sodium current flowing during recovery from this blocked state may depolarize cells immediately after an action potential, promoting the high-frequency firing typical of Purkinje neurons.     

The molecular machinery of resurgent sodium current revealed

Some TTX-sensitive sodium channels open transiently during recovery from inactivation, generating a "resurgent" sodium current that flows immediately following action potentials. In this issue of Neuron, Grieco and colleagues present evidence that resurgent sodium current results from a novel form of inactivation in which the cytoplasmic tail of the beta4 subunit acts as a classic open-channel blocker.     

Interactions among DIV voltage-sensor movement,fast inactivation,and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the Na_Vβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed Na_V1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of Na_Vβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.     

Nav1.6 channels generate resurgent sodium currents in spinal sensory neurons

The Na(v)1.6 voltage-gated sodium channel has been implicated in the generation of resurgent currents in cerebellar Purkinje neurons. Our data show that resurgent sodium currents are produced by some large diameter dorsal root ganglion (DRG) neurons from wild-type mice, but not from Na(v)1.6-null mice; small DRG neurons do not produce resurgent currents. Many, but not all, DRG neurons transfected with Na(v)1.6 produce resurgent currents. These results demonstrate for the first time the intrinsic ability of Na(v)1.6 to produce a resurgent current, and also show that cell background is critical in permitting the generation of these currents.     

Resurgent current and voltage sensor trapping enhanced activation by a beta-scorpion toxin solely in Nav1.6 channel. Significance in mice Purkinje neurons

Resurgent currents are functionally crucial in sustaining the high frequency firing of cerebellar Purkinje neurons expressing Na(v)1.6 channels. Beta-scorpion toxins, such as CssIV, induce a left shift in the voltage-dependent activation of Na(v)1.2 channels by "trapping" the IIS4 voltage sensor segment. We found that the dangerous Cn2 beta-scorpion peptide induces both the left shift voltage-dependent activation and a transient resurgent current only in human Na(v)1.6 channels (among 1.1-1.7), whereas CssIV did not induce the resurgent current. Cn2 also produced both actions in mouse Purkinje cells. These findings suggest that only distinct beta-toxins produce resurgent currents. We suggest that the novel and unique selectivity of Cn2 could make it a model drug to replace deep brain stimulation of the subthalamic nucleus in patients with Parkinson disease.     

Amine blockers of the cytoplasmic mouth of sodium channels: a small structural change can abolish voltage dependence.

Many drugs block sodium channels from the cytoplasmic end (Moczydlowski, E., A. Uehara, X, Guo, and J. Heiny. 1986. Isochannels and blocking modes of voltage-dependent sodium channels. Ann. N.Y. Acad. Sci. 479:269-292.). Lidocaine, applied to either side of the membrane, induces two blocking modes, a rapid, voltage-dependent open-channel block, and a block of the inactivated channel that occurs on a 1000-fold slower timescale. Here we describe the actions of several lidocaine-related amines on batrachotoxin(BTX)-activated bovine cardiac sodium channels incorporated into planar lipid bilayers. We applied blocking amines from the intracellular side and examined the structural determinants of fast, open-channel block. Neither hydroxyl nor carbonyl groups, present in the aryl-amine link of lidocaine, were necessary, indicating that hydrogen bonding between structures in the aryl-amine link and the channel is not required. Block, however, was significantly enhanced by addition of an aromatic ring, or by the lengthening of aliphatic side chains, suggesting that a hydrophobic domain strengthens binding while the amine group blocks the pore. For most blockers, depolarizing potentials enhanced block, with the charged amine group apparently traversing 45-60% of the transmembrane voltage. By contrast, block by phenylhydrazine was essentially voltage-independent. The relatively rigid planar structure of phenylhydrazine may prevent the charged amino end from entering the electric field when the aromatic ring is bound. The relation between structural features of different blockers and their sensitivity to voltage suggests that the transmembrane voltage drops completely over less than 5 A. We raise the possibility that the proposed hydrophobic binding domain overlaps the endogenous receptor for the inactivation gate. If so, our data place limits on the distance between this receptor and the intrapore site at which charged amines bind.     

Dissecting lidocaine action: diethylamide and phenol mimic separate modes of lidocaine block of sodium channels from heart and skeletal muscle.

We have investigated block of sodium channels by diethylamide and phenol, which resemble the hydrophilic tertiary amine head and the hydrophobic aromatic tail of the lidocaine molecule, respectively. Diethylamide and phenol separately mimicked the fast and slow modes of block caused by lidocaine. Experiments were performed using single batrachotoxin-activated bovine cardiac and rat skeletal muscle sodium channels incorporated into neutral planar lipid bilayers. Diethylamide, only from the intracellular side, caused a voltage-dependent reduction in apparent single channel amplitude ('fast' block). Block was similar for cardiac and skeletal muscle channels, and increased in potency when extracellular sodium was replaced by N-methylglucamine, consistent with an intrapore blocking site. Thus, although occurring at 15-fold higher concentrations, block by diethylamide closely resembles the fast mode of block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80-90). For cardiac sodium channels, phenol bound to a closed state causing the appearance of long blocked events whose duration increased with phenol concentration. This slow block depended neither on voltage nor on the side of application, and disappeared upon treatment of the channel with trypsin. For skeletal muscle channels, slow phenol block occurred with only very low probability. Thus, phenol block resembles the slow mode of block observed for lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:91-100). Our data suggest that there are separate sites for fast lidocaine block of the open channel and slow block of the "inactivated" channel. Fast block by diethylamide inhibited the long, spontaneous, trypsin-sensitive (inactivation-like) closures of cardiac channels, and hence secondarily antagonized slow block by phenol or lidocaine. This antagonism would potentiate shifts in the balance between the two modes of action of a tertiary amine drug caused by changes in the relative concentrations of the charged (fast blocking) and neutral (slow blocking) forms of the drug.     

Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Subunit dependence of Na channel slow inactivation and open channel block in cerebellar neurons

Purkinje and cerebellar nuclear neurons both have Na currents with resurgent kinetics. Previous observations, however, suggest that their Na channels differ in their susceptibility to entering long-lived inactivated states. To compare fast inactivation, slow inactivation, and open-channel block, we recorded voltage-clamped, tetrodotoxin-sensitive Na currents in Purkinje and nuclear neurons acutely isolated from mouse cerebellum. In nuclear neurons, recovery from all inactivated states was slower, and open-channel unblock was less voltage-dependent than in Purkinje cells. To test whether specific subunits contributed to this differential stability of inactivation, experiments were repeated in Na(V)1.6-null (med) mice. In med Purkinje cells, recovery times were prolonged and the voltage dependence of open-channel block was reduced relative to control cells, suggesting that availability of Na(V)1.6 is quickly restored at negative potentials. In med nuclear cells, however, currents were unchanged, suggesting that Na(V)1.6 contributes little to wild-type nuclear cells. Extracellular Na(+) prevented slow inactivation more effectively in Purkinje than in nuclear neurons, consistent with a resilience of Na(V)1.6 to slow inactivation. The tendency of nuclear Na channels to inactivate produced a low availability during trains of spike-like depolarization. Hyperpolarizations that approximated synaptic inhibition effectively recovered channels, suggesting that increases in Na channel availability promote rebound firing after inhibition.     

Block of sodium conductance and gating current in squid giant axons poisoned with quaternary strychnine.

Quaternary strychnine blocks sodium channels from the axoplasmic side, probably by insertion into the inner channel mouth. Block is strongly voltage dependent, being more pronounced in depolarized than in resting axons. Using potential steps as a means to modulate the level of block, we investigate strychnine effects on sodium and gating currents at +50 and -50 mV. We analyze our data in terms of the simplest possible model, wherein only an open channel may receive and retain a strychnine molecule. Our main findings are (a) block by strychnine and inactivation resemble each other and (b) block of sodium and gating currents by strychnine happen with closely similar time-courses. Our data support the hypothesis of Armstrong and Bezanilla (1977) wherein an endogenous blocking particle causes inactivation by inserting itself into the inner mouth of the sodium channel. Quaternary strychnine may act as an artificial substitute for the hypothetical endogenous blocking particle. Further, we suggest that at least 90% of the rapid asymmetrical displacement current in squid axons is sodium channel gating current, inasmuch as quaternary strychnine can block 90% of the displacement current simultaneously with sodium current.     

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers.

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.     

The second hydrophobic domain contributes to the kinetic properties of epithelial sodium channels

The epithelial sodium channel (ENaC) is the prototype of a new class of ion channels known as the ENaC/Deg family. The hallmarks of ENaC are a high selectivity for Na(+), block by amiloride, small conductance, and slow kinetics that are voltage-independent. We have investigated the contribution of the second hydrophobic domain of each of the homologous subunits alpha, beta, and gamma to the kinetic properties of ENaC. Chimeric subunits were constructed between alpha and beta subunits (alpha-beta) and between gamma and beta subunits (gamma-beta). Chimeric and wild-type subunits were expressed in various combinations in Xenopus oocytes. Analysis of whole-cell and unitary currents made it possible to correlate functional properties with specific sequences in the subunits. Functional channels were generated without the second transmembrane domain from alpha subunits, indicating that it is not essential to form functional pores. The open probability and kinetics varied with the different channels and were influenced by the second hydrophobic domains. Amiloride affinity, Li(+)/Na(+) selectivity, and single channel conductance were also affected by this segment.     

Analysis of the action of lidocaine on insect sodium channels

A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are not important for the action of SCBIs on insect sodium channels, suggesting unique interactions of SCBIs with insect sodium channels. However, the mechanism of action of LAs on insect sodium channels has not been investigated. In this study, we examined the effects of lidocaine on a cockroach sodium channel variant, BgNa(v)1-1a, and determined whether F1817 and Y1824 are also critical for the action of LAs on insect sodium channels. Lidocaine blocked BgNa(v)1-1a channels in the resting state with potency similar to that observed in mammalian sodium channels. Lidocaine also stabilized both fast-inactivated and slow-inactivated states of BgNa(v)1-1a channels, and caused a limited degree of use- and frequency-dependent block, major characteristics of LA action on mammalian sodium channels. Alanine substitutions of F1817 and Y1824 reduced the sensitivity of the BgNa(v)1-1a channel to the use-dependent block by lidocaine, but not to tonic blocking and inactivation stabilizing effects of lidocaine. Thus, similar to those on mammalian sodium channels, F1817 and Y1824 are important for the action of lidocaine on cockroach sodium channels. Our results suggest that the receptor sites for lidocaine and SCBIs are different on insect sodium channels.     

Interaction of n-alkylguanidines with the sodium channels of squid axon membrane

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.     

Voltage-dependent block by tetrodotoxin of the sodium channel in rabbit cardiac Purkinje fibers.

The two-microelectrode, voltage-clamp technique was applied to rabbit cardiac Purkinje fibers to study the interaction of tetrodotoxin (TTX) with the slowly inactivating Na current. Binding of TTX to rested, inactivated, and activated channels was estimated by measuring the relative decrease of current at the beginning (rested and inactivated channels) and the end (activated channels) of a 1 s depolarizing clamp to -45 mV. The accelerated decline of the Na current in the presence of a submaximal dose of TTX was interpreted as an increase in blocking efficiency upon depolarization. The experiments show that activated as well as inactivated channels are more sensitive to TTX than are rested channels. The dissociation equilibrium constants for the three states are 3.5 X 10(-6) M for the rested, 0.94 X 10(-6) M for the activated, and 0.75 X 10(-6) M for the inactivated channels. The time course of activation block was dependent on TTX concentration. Rate constants for association and dissociation of the activated state are 1.3 X 10(6) M-1 X s-1 and 1.5 s-1, respectively.     

Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states.     

Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states.     

Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin.

A voltage clamp technique was used to study sodium currents and gating currents in squid axons internally perfused with the membrane impermeant sodium channel blocker, QX-314. Block by QX-314 is strongly and reversibly enhanced if a train of depolarizing pulses precedes the measurement. The depolarization-induced block is antagonized by external sodium. This antagonism provides evidence that the blocking site for the drug lies inside the channel. Depolarization-induced block of sodium current by QX-314 is accompanied by nearly twofold reduction in gating charge movement. This reduction does not add to a depolarization-induced immobilization of gating charge normally present and believed to be associated with inactivation of sodium channels. Failure to act additively suggests that both, inactivation and QX-314, affect the same component of gating charge movement. Judged from gating current measurement, a drug-blocked channel is an inactivated channel. In the presence of external tetrodotoxin and internal QX-314, gating charge movement is always half its normal size regardless of conditioning, as it QX-314 is then permanently present in the channel.     

Stimulus-dependent blockade of sodium channels of the Ranvier node membrane by the phenothiazine derivative ethmozine

The action of the antiarrhythmic drug ethmozine on sodium channels of the membrane was studied in experiments on single from Ranvier nodes by the voltage clamp method. Application of ethmozine to both the outer and the inner side of the membrane reduced the amplitude of the sodium current INa; the kinetics of this current and steady-state inactivation of the sodium channels were unchanged. Tonic and phasic (transient, stimulus-dependent) components can be distinguished in the ethmozine block of the sodium current. Tonic blockage of the sodium current develops slowly and can be potentiated by high-frequency stimulation of the membrane. The possible nature of the tonic block is discussed. The stimulus-dependent blockade of the sodium current deepens with an increase in the frequency and amplitude of depolarizing stimuli. Prolonged membrane depolarization does not evoke any additional blocking of the sodium current. It is concluded that the stimulus-dependent blockade is due to interaction between ethomizine and open sodium channels. Modification of the channels by batrachotoxin (preventing inactivation of the sodium channels) makes them insensitive to ethmozine. Increasing the potassium ion concentration on the outer side of the membrane was found to reduce the tonic effect of ethmozine and to potentiate the stimulus-dependent blockade. The action of ethmozine was compared with the effects of tertiary and quaternary local anesthetics.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 380–389, July–August, 1981.