QX-314 restores gating charge immobilization abolished by chloramine-T treatment in squid giant axons.

The gating status of the QX-314 bound Na channels before and after suppressing the fast inactivation by chloramine-T (CT) was investigated by studying the gating charge immobilization using the OFF gating current (Ig,OFF). CT treatment, which abolishes the charge immobilization induced by a prolonged depolarization, altered the kinetics of Ig,OFF: the fast phase became insensitive to the pulse duration and the slow phase became three times faster than the control one. However, internally applied QX-314 (in the presence of external TTX) caused an immediate charge immobilization similar to that observed in the absence of CT treatment. The Ig,OFF exhibited kinetics similar to the inactivated channels, decaying with a very fast time course. We conclude that the charge immobilization is restored by QX-314 in the chloramine-T-treated axon and that the gating state of the QX-314-bound channel is similar to the inactivated one. The role of the gating charge immobilization in the use-dependent block mechanism is discussed.     

Dynamics of 9-aminoacridine block of sodium channels in squid axons

The interactions of 9-aminoacridine with ionic channels were studied in internally perfused squid axons. The kinetics of block of Na channels with 9-aminoacridine varies depending on the voltage-clamp pulses and the state of gating machinery of Na channels. In an axon with intact h gate, the block exhibits frequency- and voltage-dependent characteristics. However, in the pronase-perfused axon, the frequency- dependent block disappears, whereas the voltage-dependent block remains unchanged. A time-dependent decrease in Na currents indicative of direct block of Na channel by drug molecule follows a single exponential function with a time constant of 2.0 +/- 0.18 and 1.0 +/- 0.19 ms (at 10 degrees C and 80 m V) for 30 and 100 microM 9- aminoacridine, respectively. A steady-state block can be achieved during a single 8-ms depolarizing pulse when the h gate has been removed. The block in the h-gate intact axon can be achieved only with multiple conditioning pulses. The voltage-dependent block suggests that 9-aminoacridine binds to a site located halfway across the membrane with a dissociation constant of 62 microM at 0 m V. 9-Aminoacridine also blocks K channels, and the block is time- and voltage-dependent.     

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.     

Interactions of monovalent cations with sodium channels in squid axon. II. Modification of pharmacological inactivation gating

The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.     

Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons.

The time course of recovery from use-dependent block of sodium channels caused by local anesthetics was studied in squid axons. In the presence of lidocaine or its quaternary derivatives, QX-222 and QX-314, or 9-aminoacridine (9-AA), recovery from use-dependent block occurred in two phases: a fast phase and a slow phase. Only the fast phase was observed in the presence of benzocaine. The fast phase had a time constant of several milliseconds and resembled recovery from the fast Na inactivation in the absence of drug. Depending on the drug present, the magnitude of the time constant of the slow phase varied (for example at -80 mV): lidocaine, 270 ms; QX-222, 4.4 s; QX-314, 17 s; and 9-AA, 14 s. The two phases differed in the voltage dependence of recovery time constants. When the membrane was hyperpolarized, the recovery time constant for the fast phase was decreased, whereas that for the slow phase was increased for QX-compounds and 9-AA or unchanged for lidocaine. The fast phase is interpreted as representing the unblocked channels recovering from the fast Na inactivation, and the slow phase as representing the bound and blocked channels recovering from the use-dependent block accumulated by repetitive depolarizing pulse. The voltage dependence of time constants for the slow recovery is consistent with the m-gate trapping hypothesis. According to this hypothesis, the drug molecule is trapped by the activation gate (the m-gate) of the channel. The cationic form of drug molecule leaves the channel through the hydrophilic pathway, when the channel is open. However, lidocaine, after losing its proton, may leave the closed channel rapidly through the hydrophobic pathway.     

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.     

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)     

Frequency domain analysis of asymmetry current in squid axon membrane.

The change in capacity of squid axon membrane during hyper- and depolarizations was investigated in the absence of ionic currents after the membrane was treated with pronase. In the presence of the inactivation process (h parameter), failure to observe the gating current in the frequency domain was attributed to the rapid attenuation of the possible capacity change during depolarizations, which is likely to be due to the sodium activation process. Elimination of the h process would therefore enable us to observe the gating current in the frequency domain as the change in the capacitance component of membrane admittance. However, even after the inactivation process was abolished by pronase, the capacity of the axon membrane remained constant when ionic currents were blocked by external tetrodotoxin and internal Cs+ ion. Actually capacity was observed to decrease slightly with depolarization, contrary to the prediction based on the magnitude of gating currents.     

Charged local anesthetics block ionic conduction in the sheep cardiac sarcoplasmic reticulum calcium release channel.

We have examined the effect of the charged local anesthetics QX314, QX222, and Procaine on monovalent cation conduction in the Ca2+ release channel of the sheep cardiac sarcoplasmic reticulum. All three blockers only affect cation conductance when present at the cytoplasmic face of the channel. QX222 and Procaine act as voltage-dependent blockers. With 500 Hz filtering, this is manifest as a relatively smooth reduction in single-channel current amplitude most prominent at positive holding potentials. Quantitative analysis gives an effective valence of approximately 0.9 for both ions and Kb(0)s of 9.2 and 15.8 mM for QX222 and Procaine, respectively. Analysis of the concentration dependence of block suggests that QX222 is binding to a single site with a Km of 491 microM at a holding potential of 60 mV. The use of amplitude distribution analysis, with the data filtered at 1 to 2 kHz, reveals that the voltage and concentration dependence of QX222 block occurs largely because of changes in the blocker on rate. The addition of QX314 has a different effect, leading to the production of a substate with an amplitude of approximately one-third that of the control. The substate's occurrence is dependent on holding potential and QX314 concentration. Quantitative analysis reveals that the effect is highly voltage dependent, with a valence of approximately 1.5 caused by approximately equal changes in the on and off rates. Kinetic analysis of the concentration dependence of the substate occurrence reveals positive cooperativity with at least two QX314s binding to the conduction pathway, and this is largely accounted for by changes in the on rate. A paradoxical increase in the off rate at high positive holding potentials and with increasing QX314 concentration at 80 mV suggests the existence of a further QX314-dependent reaction that is both voltage and concentration dependent. The substate block is interpreted physically as a form of partial occlusion in the vestibule of the conduction pathway giving a reduction in single-channel current by electrostatic means.     

Sodium inactivation mechanism modulates QX-314 block of sodium channels in squid axons.

Blocking action of Na channels by QX-314, a quaternary derivative of lidocaine, was studied in internally perfused and voltage-clamped axons of squid. In axons with intact Na inactivation, QX-314 exhibited both a frequency- and a voltage-dependent block of Na channels. Repetitive pulsing to more positive potentials enhanced the degree of block. Both frequency- and voltage-dependent blocks disappeared in axons in which Na inactivation had been destroyed by either pronase or N-bromoacetamide treatment. These results support the notion that Na inactivation not only modulates the frequency-dependent block but also involves the voltage-dependent binding reaction between QX-314 and Na channels.     

Batrachotoxin uncouples gating charge immobilization from fast Na inactivation in squid giant axons.

The fast inactivation of sodium currents and the immobolization of sodium gating charge are thought to be closely coupled to each other. This notion was tested in the squid axon in which kinetics and steady-state properties of the gating charge movement were compared before and after removal of the Na inactivation by batrachotoxin (BTX), pronase, or chloramine-T. The immobilization of gating charge was determined by measuring the total charge movement (QON) obtained by integrating the ON gating current (Ig,ON) using a double pulse protocol. After removal of the fast inactivation with pronase or chloramine-T, the gating charge movement was no longer immobilized. In contrast, after BTX modification, the channels still exhibited an immobilization of the gating charge (QON) with an onset time course and voltage dependence similar to that for the activation process. These results show that BTX can uncouple the charge immobilization from the fast Na inactivation mechanism, suggesting that the Na gating charge movement can be immobilized independently of the inactivation of the channel.     

Current-dependent block of nerve membrane sodium channels by paragracine.

Paragracine, isolated from the coelenterate species Parazoanthus gracilis, selectively blocks sodium channels of squid axon membranes in a frequency-dependent manner. The blocking action depends on the direction and magnitude of the sodium current rather than on the absolute value of the membrane potential. Paragracine blocks the channels only from the axoplasmic side and does so only when the current is in the outward direction. This block may be reversed by generating inward sodium currents. In axons in which sodium inactivation has been removed by pronase, the frequency-dependent block persists, and a slow time-dependent block is observed. A slow interaction with its binding site in the channel may account for the frequency-dependent block.     

A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V_1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.     

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.     

A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes.

We have utilized molecular biological techniques to demonstrate that rat IIA sodium channels expressed in Xenopus oocytes were blocked by tetrodotoxin (TTX) in a use-dependent manner. This use dependence was the result of an increased affinity of the channels for TTX upon depolarization, most likely due to a conformational change in the channel. Using a mutant with a slower macroscopic rate of inactivation, we have demonstrated that this conformational change is not the transition into the fast-inactivated state. The transition is probably one occurring during activation of the channel, as suggested by the fact that one sodium channel mutant demonstrated comparable depolarizing shifts in the voltage dependence of both activation and use-dependent block by TTX. The transition occurred at potentials more negative than those resulting in channel conductance, suggesting that the conformational change that causes use-dependent block by TTX is a closed-state voltage-dependent gating transition.     

Comparison of Gating Properties and Use-Dependent Block of Nav1.5 and Nav1.7 Channels by Anti-Arrhythmics Mexiletine and Lidocaine

Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Na_v1.5 and Na_v1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Na_v1.5 or Na_v1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Na_v1.5 and Na_v1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Na_v1.5 or Na_v1.7. The recovery from inactivation of Na_v1.5 or Na_v1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Na_v1.5 than lidocaine, but not Na_v1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Na_v1.5, as compared to Na_v1.7; and mexiletine exhibits stronger use-dependent block of Na_v1.5. The differential gating properties of Na_v1.5 and Na_v1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain.     

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     

Mechanisms of blockade of glutamate receptor ionic channels: Paradox of 9-aminoacridine

9-Aminoacridine and tacrine differ from other channel blockers of NMDA receptors in that their binding prevents the closing of blocked channels and subsequent dissociation of the agonist. Structural determinants of aminoacridine derivatives underlying the blocking mechanism are still unknown. The aim of this study was to elucidate the effects of a dicationic 9-aminoacridine derivative and some other tricyclic compounds on NMDA receptors of rat hippocampal pyramidal neurons. All the compounds under study are voltage-dependent blockers of NMDA channels; their IC₅₀ values recorded at −80 mV vary from 1 to 50 μM. The dicationic derivatives demonstrate the same voltage dependence of the block as the monocationic derivatives. The monoand dicationic tricyclic compounds under study are weak blockers of AMPA receptor channels and differ from adamantane, phenylcyclohexyl and other dicationic derivatives that exhibit greater voltage dependence of the NMDA channel block and are able to induce effective suppression of AMPA channels. We conclude that the mechanisms of action of the tricyclic and dicationic 9-aminoacridine derivatives are different from that of 9-aminoacridine, since these compounds do not prevent closing of the blocked channels. This suggests that the binding site for 9-aminoacridine has specific properties and high selectivity with respect to ligand structure. Original Russian Text ? K.H. Kim, V.E. Gmiro, D.B. Tikhonov, L.G. Magazanik, 2007, published in Biologicheskie Membrany, 2007, Vol. 24, No. 1, pp. 96–104.     

Block of sodium channels by internal mono- and divalent guanidinium analogues. Modulation by sodium ion concentration.

We have investigated the block of squid axon sodium channels by mono- and divalent guanidinium analogues. The action of these compounds on steady state sodium currents was independent of the presence or absence of the normal inactivation process. Block by both mono- and divalent analogues was voltage-dependent, but was a steeper function of potential for divalent molecules. The voltage-dependence could not, in general, be reproduced by a simple model based on Boltzmann's equation. Inhibition of steady state currents by guanidinium ions with 50 mM internal sodium was reasonably well described by a 1:1 drug/channel binding function. Increasing the internal sodium ion concentration increased both the degree and voltage-dependence of current inhibition. This is in sharp contrast to the decrease in inactivation caused by internal sodium. Changes in the external sodium concentration had very little effect on drug block. These results are consistent with a model of the sodium channel as a multi-ion pore. Only a small increase in block can be produced by increased internal sodium in a three-barrier two-site model, but a four-barrier three-site model can reproduce these experimental findings. The implications of these results for physical models of inactivation are discussed.     

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.