Modified kinetics and selectivity of sodium channels in frog skeletal muscle fibers treated with aconitine

The effect of the plant alkaloid aconitine on sodium channel kinetics, ionic selectivity, and blockage by protons and tetrodotoxin (TTX) has been studied in frog skeletal muscle. Treatment with 0.25 or 0.3 mM aconitine alters sodium channels so that the threshold of activation is shifted 40-50 mV in the hyperpolarized direction. In contrast to previous results in frog nerve, inactivation is complete for depolarizations beyond about -60 mV. After aconitine treatment, the steady state level of inactivation is shifted approximately 20 mV in the hyperpolarizing direction. Concomitant with changes in channel kinetics, the relative permeability of the sodium channel to NH4,K, and Cs is increased. This altered selectivity is not accompanied by altered block by protons or TTX. The results suggest that sites other than those involved in channel block by protons and TTX are important in determining sodium channel selectivity.     

Lysophosphatidic acid-LPA1 receptor-Rho-Rho kinase-induced up-regulation of Nav1.7 sodium channel mRNA and protein in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion

In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.     

Adenosine and Cyclic AMP in Rat Cerebral Cortical Slices: Effects of Adenosine Uptake Inhibitors and Adenosine Deaminase Inhibitors

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.     

藜芦碱和乌头碱在受损背根节神经元诱发不同的放电模式

为了研究钠通道失活门阻断后受损背根节神经元放电模式的变化特征,在大鼠背根节慢性压迫模型上采用单纤维技术记录A类神经元的自发放电。藜芦碱和乌头碱是钠通道失活门的抑制剂,但二者作用于不同的位点,前者结合于D2-S6,后者结合于D3-S6。我们比较了这两种试剂引发的放电模式。结果发现,在同一神经元,藜芦碱(1．5～5．0μmol／L)可以引起放电峰峰间期的慢波振荡,即峰峰间期由大逐渐减小,然后又逐渐增大,形成重复的振荡波形,每个振荡持续约数十秒至数分钟：而乌头碱(10～200μmol／L)则引起强直性放电,即峰峰间期逐渐减小,然后维持在一个稳定的水平。这两种不同的放电模式不因背景放电或试剂浓度的不同而发生明显的改变。实验结果表明,藜芦碱和乌头碱在受损的背根节神经元可以引发不同的放电模式,这可能与它们结合于钠通道上不同位点的抑制作用有关。     

Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

Voltage-dependent K(+) channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel's selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K(+) channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca(2+) or Ba(2+), suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K(+)] (47 mV per 10-fold increase in [K(+)]), suggesting that K(+) binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K(+) ≈ Rb(+) > Cs(+) > Na(+) > Li(+) ≈ NMG(+). Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K(+)] using kinetic schemes in which the open-conductive state is stabilized by K(+) binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K(+) dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K(+)-sensitive inactivation gating, a property that may be common to other K(+) channels.     

Relation between veratridine reaction dynamics and macroscopic Na current in single cardiac cells

Veratridine modification of Na current was examined in single dissociated ventricular myocytes from late-fetal rats. Extracellularly applied veratridine reduced peak Na current and induced a noninactivating current during the depolarizing pulse and an inward tail current that decayed exponentially (tau = 226 ms) after repolarization. The effect was quantitated as tail current amplitude, Itail (measured 10 ms after repolarization), relative to the maximum amplitude induced by a combination of 100 microM veratridine and 1 microM BDF 9145 (which removes inactivation) in the same cell. Saturation curves for Itail were predicted on the assumption of reversible veratridine binding to open Na channels during the pulse with reaction rate constants determined previously in the same type of cell at single Na channels comodified with BDF 9145. Experimental relationships between veratridine concentration and Itail confirmed those predicted by showing (a) half-maximum effect near 60 microM veratridine and no saturation up to 300 microM in cells with normally inactivating Na channels, and (b) half-maximum effect near 3.5 microM and saturation at 30 microM in cells treated with BDF 9145. Due to its known suppressive effect on single channel conductance, veratridine induced a progressive, but partial reduction of noninactivating Na current during the 50-ms depolarizations in the presence of BDF 9145, the kinetics of which were consistent with veratridine association kinetics in showing a decrease in time constant from 57 to 22 and 11 ms, when veratridine concentration was raised from 3 to 10 and 30 microM, respectively. As predicted for a dissociation process, the tail current time constant was insensitive to veratridine concentration in the range from 1 to 300 microM. In conclusion, we have shown that macroscopic Na current of a veratridine-treated cardiomyocyte can be quantitatively predicted on the assumption of a direct relationship between veratridine binding dynamics and Na current and as such can be successfully used to analyze molecular properties of the veratridine receptor site at the cardiac Na channel.     

在大鼠受损坐骨神经上由藜芦碱诱发的抛物线簇放电

在大鼠受损坐骨神经上加入 5 μmol L藜芦碱溶液 ,观察到了抛物线簇放电的现象。根据Plant模型 ,发生抛物线簇放电的前提条件必须有两个慢变量所支配的慢振荡过程。结合实验模型 ,从离子通道活动的角度揭示了抛物线簇放电发生的生物物理机制。由藜芦碱诱发的慢变钠内流和钙依赖钾外流被认为是引发实验所观察到的抛物线簇放电的两个慢变量。进而阐明了藜芦碱引起这一放电形式所起的作用 ,即抑制钠通道失活引发慢变钠内流。这种利用非线性动力学理论的分析方法可能会为分析药物的药物动力学提供一种新的途径。     

A study of properties of batrachotoxin modified sodium channels

A further analysis of the effects of the steroidal alkaloid batrachotoxin (BTX) on sodium channels in frog node of Ranvier has been carried out under voltage-clamp conditions. The main properties of modified channels as compared with those of normal ones are as follows: The rate of channel closing is drastically decreased, whereas that of opening is changed slightly if at all; The steady-state voltage dependence of channel activation is shifted towards more negative potentials by 60-70 mV; Currents through modified channels do not show a decay during maintained depolarization as it is typical for normal channels. However modified channels retain the ability to partial inactivation as shown by experiments with depolarizing prepulses; Sodium against potassium selectivity beyond--20 mV suggesting either nonhomogeneity of the modified channels as for their kinetic and selectivity properties or potential-dependence of ionic selectivity for each channel; The selectivity sequence determined from peak current reversal potential measurements is as follows: H: Na :NH4:K = 528:1:0.47: :0.19; The effective pK value of proton block is decreased by about 0.4; 7) The sensitivity of the channels to tetrodotoxin (TTX) block is practically unchanged.     

Veratridine-stimulated central synapses in culture: A quantitative ultrastructural analysis

Synapses in explant cultures of fetal rat neocortex at day 18 in vitro were stimulated by veratridine (10^?4M) for 20 min. The cultures were subsequently processed for electron microscopy and the synapses were analyzed by quantitative techniques, incorporating set mathematical treatment. The mean values of area, perimeter, and form factor of the presynaptic elements significantly increased following veratridine stimulation, compared to the values of control synapses. The length of the postsynaptic thickening also increased, while synaptic curvature did not change significantly in the veratridine group. A fivefold reduction was observed in the mean number of synaptic vesicles per presynaptic element and in the vesicle-terminal area ratio, following veratridine stimulation. The cytoplasm-terminal area ratio and the occurrence of vacuoles/cisternae significantly increased after veratridine application. Planar measurement of membranes (boundary length) of different presynaptic organelles revealed that the total membrane did not change significantly in the veratridine group. The data indicated an increase in volume and swelling of the pre- and postsynaptic elements, considerable depletion of synaptic vesicles, and preservation of the total presynaptic membrane following veratridine stimulation in nerve tissue culture.     

Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.     

Ca2+o-Independent Veratridine-Evoked Acetylcholine Release from Striatal Slices Is Not Inhibited by Vesamicol (AH5183): Mobilization of Distinct Transmitter Pools

The effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol), a compound known to block the uptake of acetylcholine (ACh) into cholinergic synaptic vesicles, on the release of endogenous and [14C]ACh from slices of rat striatum was investigated. ACh release was evoked either by electrical stimulation or by veratridine. The effect of electrical stimulation was entirely dependent on external Ca2+. By contrast, veratridine (40 microM) also enhanced ACh release in the absence of Ca2+. Indeed, with veratridine two components were clearly distinguished: one dependent on external Ca2+ and the other not. Vesamicol inhibited [14C]ACh release evoked by both veratridine and electrical stimulation in the presence of external Ca2+, provided it was added to the tissue prior to loading with [14C]choline. With the same treatment vesamicol only slightly affected the release of endogenous ACh. Under the same conditions the Ca2(+)-independent [14C]ACh release evoked by veratridine was not prevented by vesamicol. The differential responsiveness to vesamicol suggests that ACh pools involved in Ca2+o-dependent ACh release are different from those mobilized during Ca2+o-independent ACh release.     

Contractile inactivation in frog skeletal muscle fibers. The effects of low calcium, tetracaine, dantrolene, D-600, and nifedipine

Short muscle fibers (approximately 1.5 mm) of Rana pipiens were voltage-clamped with a two-microelectrode technique at a holding potential of -100 mV. Using conditioning depolarizing ramps, with slopes greater than 0.2 mV/s, partially inactivated responses are obtained at threshold values between -55 and -35 mV. With slopes equal to or slower than 0.1 mV/s, one inactivates contraction without ever activating it. When the membrane potential is brought slowly to values more positive than about -40 mV, test pulses, applied on top of the ramps, bringing the membrane potential to values up to +100 mV, are ineffective in eliciting contractile responses, which indicates complete inactivation. After inactivation, contractile threshold is shifted by perhaps 10 mV, to about -40 mV. The sensitivity of fibers to depolarizing ramps is increased by D-600 (50 microM), dantrolene (50 microM), tetracaine (100 microM), and low calcium (10(-8) M). In the presence of these agents, complete inactivation was obtained using ramp slopes of 1, 0.8, 0.4, and 0.2 mV/s, respectively. Nifedipine was less effective. With D-600, once inactivation had been induced, no repriming occurred after repolarization to -100 mV, and partial recovery occurred after washing out the drug. With low calcium, tetracaine, and nifedipine, the tension-voltage relationship was not affected, whereas the steady state inactivation curve (obtained in repriming experiments) was shifted by 10-25 mV toward more negative potentials. With D-600, the activation curve was not modified, whereas the inactivation curve could not be obtained, because of repriming failure. With dantrolene, the inactivation curve was not affected, whereas the activation curve was shifted toward less negative potentials and peak tension diminished, depending on the pulse duration. The results indicate that it is possible to induce complete inactivation without activation, and to differentiate activation and inactivation parameters pharmacologically, which suggests that the two are separate processes.     

Mutation D384N Alters Recovery of the Immobilized Gating Charge in Rat Brain IIA Sodium Channels

Rat brain (rBIIA) sodium channel fast inactivation kinetics and the time course of recovery of the immobilized gating charge were compared for wild type (WT) and the pore mutant D384N heterologously expressed in Xenopus oocytes with or without the accessory beta1-subunit. In the absence of the beta1-subunit, WT and D384N showed characteristic bimodal inactivation kinetics, but with the fast gating mode significantly more pronounced in D384N. Both, for WT and D384N, coexpression of the beta1-subunit further shifted the time course of inactivation to the fast gating mode. However, the recovery of the immobilized gating charge (Qg) of D384N was clearly faster than in WT, irrespective of the presence of the beta1-subunit. This was also reflected by the kinetics of the slow Ig OFF tail. On the other hand, the voltage dependence of the Qg-recovery was not changed by the mutation. These data suggest a direct interaction between the selectivity filter and the immobilized voltage sensor S4D4 of rBIIA sodium channels.     

Aluminum enhances the voltage activated sodium currents in the neurons of the pond snail Lymnaea stagnalis L

1. The effects of aluminum on voltage activated sodium currents (VASCs) were investigated by using the conventional two-electrode voltage clamp technique in Lymnaea stagnalis L. neurons. The peak amplitude, kinetics, and voltage-dependence of activation and inactivation of the sodium currents were studied in the presence of 5-500 microM AlCl3, at pH = 7.7. 2. There was a significant concentration-dependent increase in the peak amplitude of sodium currents after Al treatment, ED50 = 67 microM. The threshold concentration of the enhancement was 50 microM. The maximal peak increase of 143% was caused by a 500 microM aluminum. The action of aluminum on VASCs developed slowly, and it is not recovered by washing within 20 min. 3. There was little alteration of the voltage-dependence of the current. It was not a significant effect on the activation- and inactivation time constants of INa, but the steady-state inactivation curve shifted to negative direction on the voltage axis in the presence of Al. 4. The leak currents were not influenced by aluminum up to the highest concentration applied.     

Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).     

Competition for Binding Between Veratridine and KIFMK: An Open Channel Blocking Peptide of the RIIA Sodium Channel

Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer & Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel. Received: 19 November 1996/Revised: 31 July 1997     

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.     

Properties of aconitine-modified sodium channels in single cells of mouse ventricular myocardium

Effects of the plant alkaloid Aconitine on the kinetics of sodium channels were studied in enzymatically isolated single cells of the mouse ventricular myocardium. Aconitine (1 mumol/l) induced a prolongation of the 90% repolarization of action potentials from 52.4 +/- 3.7 ms to 217.0 +/- 12.5 ms. Delayed terminal repolarization and oscillatory afterpotentials preceded spontaneous activity with high frequencies. Peak sodium currents were diminished from 28.0 +/- 9.0 to 14.0 +/- 6.0 nA. The reversal potential of the sodium current was shifted from 16.0 +/- 11.0 to -8.0 +/- 6.0 mV (52.5 mmol/l extracellular sodium concentration) suggesting a decreased selectivity of the Aconitine-modified Na channels. The m-affinity-curves were shifted 31 mV towards more negative potentials at a constant slope. The h affinity-curves were shifted in the same direction by 13 mV. The slope parameter of the h affinity-voltage relationship was enlarged from 9.1 +/- 2.2 mV to 15.6 +/- 4.4 mV. Shifts in m affinity and h affinity resulted in an increased "window". The alkaloid modified channels inactivated extremely slowly at potentials negative to -40 mV, but showed a fast and complete inactivation at potentials positive to -40 mV.     

Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel

KcsA is a proton-activated K⁺ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K⁺, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K⁺ selectivity, and permitted Na⁺ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.     

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.