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.     

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.     

The role of the putative inactivation lid in sodium channel gating current immobilization

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge-voltage relationships were not different between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the development of charge immobilization assayed at -180 mV after depolarization to 0 mV was similar to the time constant of inactivation of I(Na) at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a(MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.     

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.     

Fast Inactivation in Shaker K+ Channels : Properties of Ionic and Gating Currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH₂-terminus region.     

Sodium channel gating currents in frog skeletal muscle

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.     

Gating current and potassium channels in the giant axon of the squid.

Gating current (Ig) underlying Na-channel activation is large enough to enable resolution of components both preceding and paralleling Na conductance (gNa) turn-on. For large depolarizations (beyond +20 mV), an additional "slow phase" of Ig is observed during a time when Na activation is already complete, but when K-channel opening is just becoming detectable. If Na- and K-channel gating are similar, the slow kinetics and long delay for K activation predict that K channel Ig must be relatively small and slow. Externally applied dibucaine almost totally blocks gNa and greatly reduces the fast (Na channel) Ig without altering gK or the Ig slow phase. The slow phase of Ig depends in part of the presence of functional K channels. Selective diminution in amplitude of the slow phase is consistently observed after a 30-min perfusion with both external and internal K-free media, a procedure which destroys nearly all K channels. This decrease of Ig amounts to approximately 10% of the total charge movements at +40 to +80 mV, with gating charge and K channels disappearing in a ratio of less than 1 e- per picosiemens of gK. These findings are consistent with the idea that part of the Ig slow phase represents gating current generated by the early steps in K-channel activation.     

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.     

Inactivation of the sodium channel. II. Gating current experiments

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.     

Differential effects of paramyotonia congenita mutations F1473S and F1705I on sodium channel gating

We investigated effects of paramyotonia congenita mutations F1473S and F1705I on gating of skeletal muscle Na+ channels. We used on-cell recordings from Xenopus oocytes to compare fast inactivation and deactivation in wild type and mutant channels. Then, we used gating current recordings to determine how these actions of PC mutants might be reflected in their effects on charge movement and its immobilization. F1473S, but not F1705I, accelerated deactivation from the inactivated state and enhanced the remobilization of gating charge. F1473S and F1705I decreased the completion of closed-state fast inactivation, and each mutant decreased charge movement over the voltage range at which channels did not activate. An unexpected result was that F1705I increased the extent of charge immobilization in response to strong depolarization. Our results suggest that the DIV S4-S5 linker mutation F1473S promotes the hyperpolarized position of DIVS4 to accelerate recovery. Inhibition of charge movement by F1473S and F1705I in the absence of channel opening is discussed with respect to their effects on closed-state fast inactivation.     

Differential effects of paramyotonia congenita mutations F1473S and F1705I on sodium channel gating

We investigated effects of paramyotonia congenita mutations F1473S and F1705I on gating of skeletal muscle Na+ channels. We used on-cell recordings from Xenopus oocytes to compare fast inactivation and deactivation in wild-type and mutant channels. Then, we used gating current recordings to determine how these actions of PC mutants might be reflected in their effects on charge movement and its immobilization. F1473S, but not F1705I, accelerated deactivation from the inactivated state and enhanced the remobilization of gating charge. F1473S and F1705I decreased the completion of closed-state fast inactivation, and decreased charge movement over the voltage range at which channels did not activate. An unexpected result was that F1705I increased the extent of charge immobilization in response to strong depolarization. Our results suggest that the DIV S4-S5 linker mutation F1473S promotes the hyperpolarized position of DIVS4 to accelerate recovery. Inhibition of charge movement by F1473S and F1705I in the absence of channel opening is discussed with respect to their effects on closed-state fast inactivation.     

Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine,N-bromoacetamide,and N-bromosuccinimide

Summary We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding.     

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.     

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.     

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.     

Two components of voltage-dependent inactivation in Ca(v)1.2 channels revealed by its gating currents

Voltage-dependent inactivation (VDI) was studied through its effects on the voltage sensor in Ca(v)1.2 channels expressed in tsA 201 cells. Two kinetically distinct phases of VDI in onset and recovery suggest the presence of dual VDI processes. Upon increasing duration of conditioning depolarizations, the half-distribution potential (V(1/2)) of intramembranous mobile charge was negatively shifted as a sum of two exponential terms, with time constants 0.5 s and 4 s, and relative amplitudes near 50% each. This kinetics behavior was consistent with that of increment of maximal charge related to inactivation (Qn). Recovery from inactivation was also accompanied by a reduction of Qn that varied with recovery time as a sum of two exponentials. The amplitudes of corresponding exponential terms were strongly correlated in onset and recovery, indicating that channels recover rapidly from fast VDI and slowly from slow VDI. Similar to charge "immobilization," the charge moved in the repolarization (OFF) transient became slower during onset of fast VDI. Slow VDI had, instead, hallmarks of interconversion of charge. Confirming the mechanistic duality, fast VDI virtually disappeared when Li(+) carried the current. A nine-state model with parallel fast and slow inactivation pathways from the open state reproduces most of the observations.     

Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.     

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.     

Voltage dependence of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier

Sodium current and sodium channel intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). BTX caused an approximately threefold increase in steepness of the Q vs. voltage relationship and a 50-mV negative shift in its midpoint. The maximum amount of intramembrane charge was virtually identical before and after BTX treatment. BTX treatment eliminated the charge immobilization observed in untreated nodes after relatively long depolarizing pulses and slowed the rate of OFF charge movement after a pulse. After BTX treatment, the voltage dependence of charge movement was the same as the steady-state voltage dependence of sodium conductance activation. The observations are consistent with the hypothesis that BTX induces an aggregation of the charged gating particles associated with each channel and causes them to move as a unit having approximately three times the average valence of the individual particles. Movement of this single aggregated unit would open the BTX-modified sodium channel.