Delays in inactivation development and activation kinetics in myxicola giant axons

Na inactivation was studied in Myxicola (two-pulse procedure, 6-ms gap between conditioning and test pulses). Inactivation developed with an initial delay (range 130-817 microseconds) followed by a simple exponential decline (time constant tau c). Delays (deviations from a simple exponential) are seen only for brief conditioning pulses were gNa is slightly activated. Hodgkin-Huxley kinetics with series resistance, Rs, predict deviations from a simple exponential only for conditioning pulses that substantially activate gNa. Reducing INa fivefold (Tris substitution) had no effect on either tau c or delay. Delay in not generated by Rs or by contamination from activation development. The slowest time constant in Na tails is approximately 1 ms (Goldman and Hahin, 1978) and the gap was 6 ms. Shortening the gap to 2 ms had no effect on either tau c or delay. Delay is a true property of the channel. Delay decreased with more positive conditioning potentials, and also decreased approximately proportionally with time to peak gNa during the conditioning pulse, as expected for sequentially coupled activation and inactivation. In a few cases the difference between Na current values for brief conditioning pulses and the tau c exponential could be measured. Difference values decayed exponentially with time constant tau m. The inactivation time course is described by a model that assumes a process with the kinetics of gNa activation as a precursor to inactivation.     

Gating current kinetics in Myxicola giant axons. Order of the back transition rate constants.

Gating current, Ig, was recorded in Myxicola axons with series resistance compensation and higher time resolution than in previous studies. Ig at ON decays as two exponentials with time constants, tau ON-F and tau ON-S, very similar to squid values. No indication of an additional very fast relaxation was detected, but could be still unresolved. Ig at OFF also displays two exponentials, neither reflecting recovery from charge immobilization. Deactivation of the two I(ON) components may proceed with well-separated exponentials at -100 mV. INa tail currents at OFF also display two exponentials plus a third very slow relaxation of 5-9% of the total tail current. The very slow component is probably deactivation of a very small subpopulation of TTX sensitive channels. A -100 mV, means for INa tail component time constants (four axons) are 76 microseconds (range: 53-89 microseconds) and 344 microseconds (range: 312-387 microseconds), and for IOFF (six axons) 62 microseconds (range: 34-87 microseconds) and 291 microseconds (range: 204-456 microseconds) in reasonable agreement. INa ON activation time constant, tau A, is clearly slower than tau ON-F at all potentials. Except for the interval -30 to -15 mV, tau A is clearly faster than tau ON-S, and has a different dependency on potential. tau ON-S is several fold smaller than tau h. Computations with a closed2----closed1----open activation model indicated Na tail currents are consistent with a closed1----open rate constant greater than the closed2----closed1.     

Fast and slow steps in the activation of sodium channels

Kinetic features of sodium conductance (gNa) and associated gating current (Ig) were studied in voltage-clamped, internally perfused squid axons. Following a step depolarization Ig ON has several kinetic components: (a) a rapid, early phase largely preceding gNa turn-on; (b) a delayed intermediate component developing as gNa increases; and (c) a slow component continuing after gNa is fully activated. With small depolarizations the early phase shows a quick rise (less than 40 mus) and smooth decay; the slow component is not detectable. During large pulses all three components are present, and the earliest shows a rising phase or initial plateau lasting approximately 80 mus. Steady-state and kinetic features of Ig are minimally influenced by control pulse currents, provided controls are restricted to a sufficiently negative voltage range. Ig OFF following a strong brief pulse also shows a rising phase. A depolarizing prepulse producing gNa inactivation and Ig immobilization eliminates the rising phase of Ig OFF. gNa, the immobilized portion of Ig ON, and the rising phase reappear with similar time-courses when tested with a second depolarizing pulse after varying periods of repolarization. 30 mM external ZnCl2 delays and slows gNa activation, prolongs the rising phase, and slows the subsequent decay of Ig ON. Zn does not affect the kinetics of gNa tails or Ig OFF as channels close, however. We present a sequential kinetic model of Na channel activation, which adequately describes the observations. The rapid early phase of IgON is generated by a series of several fast steps, while the intermediate component reflects a subsequent step. The slow component is too slow to be clearly associated with gNa activation.     

Charge immobilization caused by modification of internal cysteines in squid Na channels.

We studied the effects of modification of native cysteines present in squid giant axon Na channels with methanethiosulfonates. We find that intracellular, but not extracellular, perfusion of axons with positively charged [(2-trimethylammonium)-ethyl]methanethiosulfonate (MTSET), or 3(triethylammonium)propyl]methanethiosulfonate (MTS-PTrEA) irreversibly reduces sodium ionic (INa) and gating (Ig) currents. The rate of modification of Na channels was dependent on the concentration of the modifying agent and the transmembrane voltage. Hyperpolarized membrane potentials (e.g., -110 mV) protected the channels from modification by MTS-PTrEA. In addition to reducing the amplitudes of INa and Ig, MTS-PTrEA also altered their kinetics such that the remaining INa did not appear to inactivate, whereas Ig was made sharper and declined to baseline more quickly. The shape and amplitude of Ig after modification of channels with MTS-PTrEA appeared to be "charge-immobilized," as if the modified channels were inactivated. MTS-PTrEA did not affect INa or Ig when inactivation was removed by internal perfusion of the axon with pronase. In addition, we find that the steady-state inactivation curve of modified Na channels is made much shallower and is markedly shifted to hyperpolarized potentials. The rates of activation, deactivation, or open-state inactivation were not altered in MTS-PTrEA-modified channels. The uncharged sulfhydryl reagent methymethanethiosulfonate (MMTS) did not affect either INa or Ig, but prevented the irreversible effects of MTS-PTrEA or MTSET on Na channels. It is proposed that the positively charged methanethiosulfonates MTS-PTrEA and MTSET modify a native internal cysteine(s) in squid Na channels, and by doing so promote inactivation from closed states, resulting in charge immobilization and reduction of INa.     

Internal cesium and the sodium inactivation gate in Myxicola giant axons.

Internal cesium (CSi), relative to internal potassium (Ki), alters Na current (INa) time course in internally perfused Myxicola giant axons. CSi slows the time to peak INa, slows its decline from peak and increases the steady state to peak current ratio, INainfinity/INapeak. Neither activation nor deactivation kinetics are appreciably affected by CSi. Na current rising phases, times to half maximum and tail current time courses are similar in CSi and Ki. Inactivation time constants determined by both one (tau h) and two (tau c) pulses are also little changed by CSi. The CSi effects are due largely or entirely to an increased INainfinity/INapeak. CSi decreases the steady level of inactivation reached during a step in potential, preventing some fraction of inactivation gates from closing at all, the rest apparently closing normally. Inactivation block in CSi decreases with increasing inward current magnitude and in Ki inactivation block is appreciable only for outward Na channel current, suggesting the site of action is located somewhere in the current pathway. If this site mediates the normal operation of the inactivation gate, then a possible mechanism for gate closure could involve a positively charged structure moving to associate with a negative site near or into the inner channel mouth.     

Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells.

Study of the excitatory sodium current (INa) intact heart muscle has been hampered by the limitations of voltage clamp methods in multicellular preparations that result from the presence of large series resistance and from extracellular ion accumulation and depletion. To minimize these problems we voltage clamped and internally perfused freshly isolated canine cardiac Purkinje cells using a large bore (25-microns diam) double-barreled flow-through glass suction pipette. Control of [Na+]i was demonstrated by the agreement of measured INa reversal potentials with the predictions of the Nernst relation. Series resistance measured by an independent microelectrode was comparable to values obtained in voltage clamp studies of squid axons (less than 3.0 omega-cm2). The rapid capacity transient decays (tau c less than 15 microseconds) and small deviations of membrane potential (less than 4 mV at peak INa) achieved in these experiments represent good conditions for the study of INa. We studied INa in 26 cells (temperature range 13 degrees-24 degrees C) with 120 or 45 mM [Na+]o and 15 mM [Na+]i. Time to peak INa at 18 degrees C ranged from 1.0 ms (-40 mV) to less than 250 microseconds (+ 40 mV), and INa decayed with a time course best described by two time constants in the voltage range -60 to -10 mV. Normalized peak INa in eight cells at 18 degrees C was 2.0 +/- 0.2 mA/cm2 with [Na+]o 45 mM and 4.1 +/- 0.6 mA/cm2 with [Na+]o 120 mM. These large peak current measurements require a high density of Na+ channels. It is estimated that 67 +/- 6 channels/micron 2 are open at peak INa, and from integrated INa as many as 260 Na+ channels/micron2 are available for opening in canine cardiac Purkinje cells.     

Geographical distribution and inactivation kinetics in internally perfused Myxicola giant axons.

In some preparations the time constant of Na current inactivation determined with two pulses (tau c) is larger over some range of potentials than that determined from the current decay during a single pulse (tau h), while in others tau c(V) and tau h(V) are the same. Myxicola giant axons obtained from specimens collected from coastal waters of northeastern North America display a tau c - tau h difference under all conditions we have tested. In these axons tau c(V) and tau h(V) are unchanged by reduction of Na current density, addition of K-channel blockers, or internal perfusion. Specimens of the same species, Myxicola infundibulum, collected from a different geographical location, the south coast of England, have been studied under internal perfusion with K as the major cation internally, with reduced external Na concentration and in the presence of K-channel blockers. In these axons tau c(V) and tau h(V) approximately superpose, raising the possibility that dramatic differences in Na current kinetics may not necessarily reflect basic differences in the organization of the Na channel gating machinery.     

Kinetic analysis of sodium channel block by internal methylene blue in pronased crayfish giant axons

The cationic dye methylene blue (MB+) blocks INa in a voltage and time-dependent manner and exhibits no frequency dependent block at 1 Hz when internally perfused in normal or pronase-treated crayfish axons. Peak INa decreases with increasing MB+ concentrations in the range 50 microM to 5 mM, but the blocking time constant approaches an asymptote at concentrations above 500 microM. IgON is not noticeably affected by internal MB+ at concentrations of 500 microM or below, in the absence of external tetrodotoxin (TTX). However, 5 mM MB+ produces a visible suppression of IgON that is reversible following washout. A pseudo-first-order analysis of MB+ blocking kinetics suggests a drug binding site deep in the transmembrane voltage field (dz = 0.85, KD = 11 microM at 0 mV). The voltage sensitivity of the individual rate constants is highly asymmetric, suggesting that the major energy barrier for MB+ is very close to the axoplasmic margin of the voltage field. Reversing the Na+ gradient and direction of INa has little effect on the kinetics of MB+ block. The kinetic properties of state-dependent vs. state-independent blocking schemes are investigated and compared with our observations of MB+ block. Analysis of hooked sodium tail currents following depolarization to various test potentials demonstrates quantitatively that MB+ binds in a state-dependent manner to open sodium channels. The appropriateness of first-order kinetic analysis of drug block is then considered in light of these observations.     

Anticonvulsants modify inactivation but not activation processes of sodium channels in Myxicola axons

The effects of pronase and the anticonvulsant drugs diphenylhydantoin, bepridil, and sodium valproate on fast and slow Na+ inactivation were examined in cut-open Myxicola giant axons with loose patch-clamp electrodes applied to the internal surface. Pronase completely eliminated fast Na+ inactivation without affecting the kinetics of Na+ activation or the maximum Na+ conductance. The time and voltage dependences of slow inactivation following pronase treatment were identical to those measured before enzyme application in the same axons. All three anticonvulsants slowed the time course of recovery from fast Na+ inactivation in untreated axons, and shifted the steady-state fast inactivation curve in the hyperpolarizing direction along the voltage axis. Anticonvulsants enhanced steady-state slow inactivation and retarded recovery from slow inactivation in both untreated and pronase-treated axons. Although some quantitative differences were seen, the order of potency of the anticonvulsants on slow Na+ inactivation was the same as that for recovery from fast inactivation.     

Slowing of sodium channel opening kinetics in squid axon by extracellular zinc

The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e., the amount of additional depolarization required to overcome the Zn2+ effect. In these terms the mean shifts caused by 30 mM Zn2+ were +29.5 mV for Na channel opening (on) kinetics (t1/2 on), +2 mV for closing (off) kinetics (tau off), and +8.4 mV for the gNa-V curve. Zn2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of or approximately 0.67. Outward current is unaffected. Effects of Zn2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNa. On gating current kinetics are shifted by +27.5 mV, off kinetics by +6 mV, and the Q-V distribution by +6.5 mV. Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants. More than one of the several steps in activation must be affected. The results are not compatible with the usual simple theory of uniform fixed surface charge. They suggest instead that Zn2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation.     

Modification of cardiac Na+ channels by batrachotoxin: effects on gating, kinetics, and local anesthetic binding.

The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.     

Kinetics of veratridine action on Na channels of skeletal muscle

Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations.     

Proton and zinc effects on HERG currents.

The proton and Zn2+ effects on the human ether-a-go-go related gene (HERG) channels were studied after expression in Xenopus oocytes and stable transfection in the mammalian L929 cell line. Experiments were carried out using the two-electrode voltage clamp at room temperature (oocytes) or the whole-cell patch clamp technique at 35 degrees C (L929 cells). In oocytes, during moderate extracellular acidification (pHo = 6.4), current activation was not shifted on the voltage axis, the time course of current activation was unchanged, but tail current deactivation was dramatically accelerated. At pHo < 6.4, in addition to accelerating deactivation, the time course of activation was slower and the midpoint voltage of current activation was shifted to more positive values. Protons and Zn2+ accelerated the kinetics of deactivation with apparent Kd values about one order of magnitude lower than for tail current inhibition. For protons, the Kd values for the effect on tail current amplitude versus kinetics were, respectively, 1.8 microM (pKa = 5.8) and 0.1 microM (pKa = 7.0). In the presence of Zn2+, the corresponding Kd values were, respectively, 1.2 mM and 169 microM. In L929 cells, acidification to pHo = 6.4 did not shift the midpoint voltage of current activation and had no effect on the time course of current activation. Furthermore, the onset and recovery of inactivation were not affected. However, the acidification significantly accelerated tail current deactivation. We conclude that protons and Zn2+ directly interact with HERG channels and that the interaction results, preferentially, in the regulation of channel deactivation mechanism.     

Interactions of monovalent cations with sodium channels in squid axon. I. Modification of physiological inactivation gating

Inactivation of Na channels has been studied in voltage-clamped, internally perfused squid giant axons during changes in the ionic composition of the intracellular solution. Peak Na currents are reduced when tetramethylammonium ions (TMA+) are substituted for Cs ions internally. The reduction reflects a rapid, voltage-dependent block of a site in the channel by TMA+. The estimated fractional electrical distance for the site is 10% of the channel length from the internal surface. Na tail currents are slowed by TMA+ and exhibit kinetics similar to those seen during certain drug treatments. Steady state INa is simultaneously increased by TMA+, resulting in a "cross-over" of current traces with those in Cs+ and in greatly diminished inactivation at positive membrane potentials. Despite the effect on steady state inactivation, the time constants for entry into and exit from the inactivated state are not significantly different in TMA+ and Cs+. Increasing intracellular Na also reduces steady state inactivation in a dose-dependent manner. Ratios of steady state INa to peak INa vary from approximately 0.14 in Cs+- or K+-perfused axons to approximately 0.4 in TMA+- or Na+-perfused axons. These results are consistent with a scheme in which TMA+ or Na+ can interact with a binding site near the inner channel surface that may also be a binding or coordinating site for a natural inactivation particle. A simple competition between the ions and an inactivation particle is, however, not sufficient to account for the increase in steady state INa, and changes in the inactivation process itself must accompany the interaction of TMA+ and Na+ with the channel.     

Modification of inactivation in cardiac sodium channels: ionic current studies with Anthopleurin-A toxin

The site 3 toxin, Anthopleurin-A (Ap-A), was used to modify inactivation of sodium channels in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Although Ap-A toxin markedly prolonged decay of sodium current (INa) in response to step depolarizations, there was only a minor hyperpolarizing shift by 2.5 +/- 1.7 mV (n = 13) of the half-point of the peak conductance- voltage relationship with a slight steepening of the relationship from - 8.2 +/- 0.8 mV to -7.2 +/- 0.8 mV (n = 13). Increases in Gmax were dependent on the choice of cation used as a Na substitute intracellularly and ranged between 26 +/- 15% (Cs, n = 5) to 77 +/- 19% (TMA, n = 8). Associated with Ap-A toxin modification time to peak INa occurred later, but analysis of the time course INa at multiple potentials showed that the largest effects were on inactivation with only a small effect on activation. Consistent with little change in Na channel activation by Ap-A toxin, INa tail current relaxations at very negative potentials, where the dominant process of current relaxation is deactivation, were similar in control and after toxin modification. The time course of the development of inactivation after Ap-A toxin modification was dramatically prolonged at positive potentials where Na channels open. However, it was not prolonged after Ap-A toxin at negative potentials, where channels predominately inactivate directly from closed states. Steady state voltage-dependent availability (h infinity or steady state inactivation), which predominately reflects the voltage dependence of closed-closed transitions equilibrating with closed-inactivated transitions was shifted in the depolarizing direction by only 1.9 +/- 0.8 mV (n = 8) after toxin modification. The slope factor changed from 7.2 +/- 0.8 to 9.9 +/- 0.9 mV (n = 8), consistent with a prolongation of inactivation from the open state of Ap-A toxin modified channels at more depolarized potentials. We conclude that Ap-A selectively modifies Na channel inactivation from the open state with little effect on channel activation or on inactivation from closed state(s).     

Characteristics of sodium tail currents in Myxicola axons. Comparison with membrane asymmetry currents.

Sodium currents after repolarization to more negative potentials after initial activation were digitally recorded in voltage-clamped Myxicola axons compensated for series resistance. The results are inconsistent with a Hodgkin-Huxley-type kinetic scheme. At potentials more negative than -50 mV, the Na+ tails show two distinct time constants, while at more positive potentials only a single exponential process can be resolved. The time-course of the tail currents was totally unaffected when tetrodotoxin (TTX) was added to reduce gNa to low values, demonstrating the absence of any artifact dependent on membrane current. Tail currents were altered by [Ca++] in a manner consistent with a simple alteration in surface potential. Asymmetry current "off" responses are well described by a single exponential. The time constant for this response averaged 2.3 times larger than that for the rapid component of the Na+ repolarization current and was not sensitive to pulse amplitude or duration, although it did vary with holding potential. Other asymmetry current observations confirm previous reports on Myxicola.     

Sodium channel activation in the squid giant axon. Steady state properties

Treatment of giant axons from the squid, Loligo pealei, with pronase removes Na channel inactivation. It was found that the peak Na current is increased, but the activation kinetics are not significantly altered, by pronase. Measurements of the fraction of open channels as a function of voltage (F-V) showed an e-folding at 7 mV and a center point near -15 mV. The rate of e-folding implies that a minimum of 4 e-/channel must cross the membrane field to open the channel. The charge vs. voltage (Q-V) curve measured in a pronase-treated axon is not significantly different from that measured when inactivation is intact: approximately 1,850 e-/micron2 were measured over the voltage range -150 to 50 mV, and the center point was near -30 mV. Normalizing these two curves (F-V and Q-V) and plotting them together reveals that they cross when inactivation is intact but saturate together when inactivation is removed. This illustrates the error one makes when measuring peak conductance with intact inactivation and interpreting that to be the fraction of open channels. A model is described that was used to interpret these results. In the model, we propose that inactivation must be slightly voltage dependent and that an interaction occurs between the inactivating particle and the gating charge. A linear sequence of seven states (a single open state with six closed states) is sufficient to describe the data presented here for Na channel activation in pronase-treated axons.     

Molecular and structural basis of resting and use-dependent block of sodium current defined using disopyramide analogues.

The effects of disopyramide (Norpace) and 14 closely related structural analogues on the Na current of voltage clamped squid axons were examined to determine which physico-chemical properties and which changes in the structure of the Norpace molecule can alter the nature of its sodium channel blocking actions. Conventional voltage clamp technique for internally perfused giant axons was used. Axons were exposed to 100 microM concentrations via the internal perfusion solution, and the actions of the 15 analogues to produce resting and use-dependent block of Na current were assessed. The roles of Na ions and the activation and inactivation processes in the development of and recovery from use-dependent block of Na current induced by the Norpace analogues were also examined. The results indicate that for both mono-tertiary and bis-tertiary amines the potency to produce use-dependent block was proportional to molecular weight, whereas the correlation between potency to produce resting block and molecular weight was significant only for bis-tertiary amines. The mono- were more potent than the bis-compounds. However, comparisons between compounds having similar molecular weights and/or pKa values indicate that other factors also can influence blocking potency. For compounds within each homologous mono- or bis-tertiary amine series, hydrophobicity as estimated from log P values (P = octanol/water partition coefficient) was found to influence the potency to produce use dependent block of Na current. Use-dependent block was extant in axons internally exposed to pronase to remove the inactivation process, which indicates that inactivation is not an obligate condition for development of use-dependent block of Na current. An important role for the activation process in the development of use-dependent block of Na current is suggested by the finding that, in general, the voltage dependence of Na current activation paralleled that of use-dependent block. However, the potential dependence of use-dependent block produced by less hydrophobic but not by more hydrophobic compounds was shifted in the hyperpolarizing direction by removing Na+ from the external solution. Compounds with intermediate hydrophobicities altered the time course of Na current during its activating and inactivating phases. This finding can be explained by the kinetics of association and dissociation of drug molecules with channel receptor sites during the development and relaxation of use-dependent block rather than by postulating any major effect of drug to alter channel gating kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of sodium currents by local anesthetics in chloramine-T- treated squid axons. The role of channel activation

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.