Functional consequences of lidocaine binding to slow-inactivated sodium channels

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use- dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to - 20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow- inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow- inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.     

Voltage-gated sodium channels

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.     

Analysis of the action of lidocaine on insect sodium channels

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

Role of domain 4 in sodium channel slow inactivation

Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.     

Interaction of scorpion alpha-toxins with cardiac sodium channels: binding properties and enhancement of slow inactivation

The effects of the scorpion alpha-toxins Lqh II, Lqh III, and LqhalphaIT on human cardiac sodium channels (hH1), which were expressed in human embryonic kidney (HEK) 293 cells, were investigated. The toxins removed fast inactivation with EC(50) values of <2.5 nM (Lqh III), 12 nM (Lqh II), and 33 nM (LqhalphaIT). Association and dissociation rates of Lqh III were much slower than those of Lqh II and LqhalphaIT, such that Lqh III would not dissociate from the channel during a cardiac activation potential. The voltage dependence of toxin dissociation from hH1 channels was nearly the same for all toxins tested, but it was different from that found for skeletal muscle sodium channels (muI; Chen et al. 2000). These results indicate that the voltage dependence of toxin binding is a property of the channel protein. Toxin dissociation remained voltage dependent even at high voltages where activation and fast inactivation is saturated, indicating that the voltage dependence originates from other sources. Slow inactivation of hH1 and muI channels was significantly enhanced by Lqh II and Lqh III. The half-maximal voltage of steady-state slow inactivation was shifted to negative values, the voltage dependence was increased, and, in particular for hH1, slow inactivation at high voltages became more complete. This effect exceeded an expected augmentation of slow inactivation owing to the loss of fast inactivation and, therefore, shows that slow sodium channel inactivation may be directly modulated by scorpion alpha-toxins.     

Kinetic evidence for two Na channels in frog skeletal muscle

The kinetics of voltage-clamped sodium currents were studied in frog skeletal muscle. Sodium currents in frog skeletal muscle activate and inactivate following an initial delay in response to a depolarizing voltage pulse. Inactivation occurs via a double exponential decay exhibiting fast and slow components for virtually all depolarizing pulses used.The deactivation of Na currents exhibits two exponential components, one decaying rapidly, while the other decays slowly in time; the relative amplitude of the two components changes with the duration of the activating pulse. The two deactivation phases remain after pharmacological elimination of inactivation.In individual fibers, the percent amplitude of the slow inactivation component correlates with the percent amplitude of the slow deactivation component.Tetrodotoxin differentially blocks the slow deactivation component.These observations are interpreted as the activation, inactivation and deactivation of two subtypes (fast and slow) of Na channels.Studies of the slow deactivation phase magnitude vs the duration of the eliciting pulse provide a way to determine the kinetics of the slow Na channel in muscle.Ammonium substitution for Na in the Ringer produces a voltage dependent activation and inactivation of current which exhibits only one decay phase, and eliminates the slow decay phase of current, suggesting that adjustments of the ionic environment of the channels can mask the presence of one of the channel subtypes.     

A negatively charged residue in the outer mouth of rat sodium channel determines the gating kinetics of the channel

Previous studies using combined techniques of site-directed mutagenesis and electrophysiology of voltage-gated Na(+) channels have demonstrated that there are significant overlaps in the regions that are important for the two fundamental properties of the channels, namely gating and permeation. We have previously shown that a pore-lining residue, W402 in S5-S6 region (P loop) in domain I of the micro1 skeletal muscle Na(+) channel, was important in the gating of the channel. Here, we determined the role of an adjacent pore-lining negatively charged residue (E403) in channel gating. Charge neutralization or substitution with positively charged side chain at this position resulted in a marked delay in the rate of recovery from slow inactivation. Indeed, the fast inactivation process appeared intact. Restoration of the negatively charged side chain with a sulfhydryl modifier, MTS-ethylsulfonate, resulted in a reactivation profile from a slow-inactivated state, which was indistinguishable from that of the wild-type channels. We propose an additional functional role for the negatively charged residue. Assuming no major changes in the pore structure induced by the mutations, the negatively charged residue E403 may work in concert with other pore regions during recovery from slow inactivation of the channel. Our data represent the first report indicating the role of negative charge in the slow inactivation of the voltage-gated Na(+) channel.     

Properties of slow, cumulative sodium channel inactivation in rat hippocampal CA1 pyramidal neurons.

Sodium channels in the somata and dendrites of hippocampal CA1 pyramidal neurons undergo a form of long-lasting, cumulative inactivation that is involved in regulating back-propagating action potential amplitude and can influence dendritic excitation. Using cell-attached patch-pipette recordings in the somata and apical dendrites of CA1 pyramidal neurons, we determined the properties of slow inactivation on response to trains of brief depolarizations. We find that the amount of slow inactivation gradually increases as a function of distance from the soma. Slow inactivation is also frequency and voltage dependent. Higher frequency depolarizations increase both the amount of slow inactivation and its rate of recovery. Hyperpolarized resting potentials and larger command potentials accelerate recovery from slow inactivation. We compare this form of slow inactivation to that reported in other cell types, using longer depolarizations, and construct a simplified biophysical model to examine the possible gating mechanisms underlying slow inactivation. Our results suggest that sodium channels can enter slow inactivation rapidly from the open state during brief depolarizations or slowly from a fast inactivation state during longer depolarizations. Because of these properties of slow inactivation, sodium channels will modulate neuronal excitability in a way that depends in a complicated manner on the resting potential and previous history of action potential firing.     

Involvement of Methionine Residues in the Fast Inactivation Mechanism of the Sodium Current from Toad Skeletal Muscle Fibers

The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal muscle fibers was studied with the mild oxidant chloramine-T (CT). Isolated segments of fibers were voltage clamped in a triple Vaseline? gap chamber. Sodium current was isolated by replacing potassium ions by tetramethylammonium ions in the external and internal solutions. Externally applied chloramine-CT was found to render noninactivating a large fraction of sodium channels and to slow down the fast inactivation mechanism of the remainder fraction of inactivatable channels. The action of CT appeared to proceed first by slowing and then removing the fast inactivation mechanism. The voltage dependence of the steady-state inactivation of the inactivatable CT-treated currents was shifted +10 mV. CT also had a blocking effect on the sodium current, but was without effect on the activation mechanism. The effects of CT were time and concentration dependent and irreversible. The use of high CT concentrations and/or long exposure times was found to be deleterious to the fiber. This side effect precluded the complete removal of fast inactivation. The effects of CT on the fast inactivation of the sodium current can be explained assuming that at least two methionine residues are critically involved in the mechanism underlying this process. Received: 10 November 1998/Revised: 4 January 1999     

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.     

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.     

Differential Effects of TipE and a TipE-Homologous Protein on Modulation of Gating Properties of Sodium Channels from Drosophila melanogaster

β subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of β subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. In this study, we compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNa_v9-1, DmNa_v22, and DmNa_v26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNa_v26. Further analysis of the gating kinetics of DmNa_v9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo.     

Slow inactivation in human cardiac sodium channels.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.     

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.     

Effect of depolarization on binding kinetics of scorpion alpha-toxin highlights conformational changes of rat brain sodium channels.

Binding of scorpion alpha-toxins to receptor site 3 on voltage-gated sodium channels inhibits sodium current inactivation and is voltage-dependent. To reveal the direct effect of depolarization, we analyzed binding kinetics of the alpha-toxin Lqh-II (from Leiurus quinquestriatus hebraeus) to rat brain synaptosomes and effects on rat brain II (rBII) channels expressed in mammalian cells. Our results indicated that the 33-fold decrease in toxin affinity for depolarized (0 mV, 90 mM [K(+)](out), K(d) = 5.85 +/- 0.5 nM) versus polarized (-55 mV, 5 mM [K(+)](out), K(d) = 0.18 +/- 0.04 nM) synaptosomes at steady state results from a 48-fold reduction in the association rate (k(on) at 5 mM [K(+)] = (12.0 +/- 4) x 10(6) M(-1) s(-1) and (0.25 +/- 0.03) x 10(6) M(-1) s(-1) at 90 mM [K(+)](out)) with nearly no change in the dissociation rate. Electrophysiological analyses of rBII channels expressed in mammalian cells revealed that approximately 75% and 40% of rBII occupied fast- and slow-inactivated states, respectively, at resting membrane potential of synaptosomes (-55 mV), and Lqh-II markedly increased the steady-state fast and slow inactivation. To mimic electrophysiological conditions we induced fast depolarization of toxin-bound synaptosomes, which generated a biphasic unbinding of Lqh-II from toxin-receptor complexes. The first fast off rate closely resembled values determined electrophysiologically for rBII in mammalian cells. The second off rate was similar to the voltage-independent steady-state value, attributed to binding to the slow-inactivated channel states. Thus, the Lqh-II voltage-dependent affinity highlights two independent mechanisms representing conformational changes of sodium channels associated with transitions among electrically visible and invisible inactivated states.     

BTX modification of Na channels in squid axons. I. State dependence of BTX action

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.     

TRPV4 links inflammatory signaling and neuroglial swelling

The activity of voltage-gated sodium channels contributes to onset and duration of the cardiac action potential through an intricate balance with the activity of other ion channels. Activation of sodium channels leads to membrane depolarization and Phase 0 of the cardiac action potential. Sodium channel fast inactivation contributes to Phase 1, the initial repolarization. Slow inactivation and closed state fast inactivation determine channel availability and, thus, overall membrane excitability. Defects in any of these biophysical states or transitions between them, imparted by (over 170 reported thus far, including both Long QT3 and Brugada syndromes) mutations in the (over 2000) amino acids that compose the sodium channel protein, can lead to channel dysfunction that manifests as an abnormal cardiac action potential and electrocardiogram. A causal relationship between several such abnormalities and the panoply of sodium channel mutations have led to a greater understanding of the molecular underpinnings of cardiac arrhythmias as well as a deeper appreciation for the intricacies of sodium channel function. Here, we review the literature regarding these causal relationships from a perspective of the biophysical properties of sodium channels.     

Biophysical defects in voltage.gated sodium channels associated with long QT and Brugada syndromes

The activity of voltage-gated sodium channels contributes to onset and duration of the cardiac action potential through an intricate balance with the activity of other ion channels. Activation of sodium channels leads to membrane depolarization and Phase 0 of the cardiac action potential. Sodium channel fast inactivation contributes to Phase 1, the initial repolarization. Slow inactivation and closed state fast inactivation determine channel availability and, thus, overall membrane excitability. Defects in any of these biophysical states or transitions between them, imparted by (over 170 reported thus far, including both Long QT3 and Brugada syndromes) mutations in the (over 2000) amino acids that compose the sodium channel protein, can lead to channel dysfunction that manifests as an abnormal cardiac action potential and electrocardiogram. A causal relationship between several such abnormalities and the panoply of sodium channel mutations have led to a greater understanding of the molecular underpinnings of cardiac arrhythmias as well as a deeper appreciation for the intricacies of sodium channel function. Here, we review the literature regarding these causal relationships from a perspective of the biophysical properties of sodium channels.     

Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin.

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.     

Rate of opening of a population of Na channels in frog skeletal muscle fibers

Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.Predictions of channel opening rate during voltage clamp pulses show that sodium channel inactivation arises coincident with a decline in channel opening rate.Sodium currents pharmacologically modified with Chloramine-T treatment so that they do not inactivate, show a predicted sustained channel opening rate.Large depolarizing voltage clamp pulses produce channel opening rate functions that resemble gating currents.The predicted channel opening rate functions are best described by kinetic models for Na channels which confer most of the charge movement to transitions between closed states.Comparisons of channel opening rate functions with gating currents suggests that there may be subtypes of Na channel with some contributing more charge movement per channel opening than others.Na channels open on average, only once during the transient period of Na activation and inactivation.After transiently opening during the activation period and then closing by entering the inactivated state, Na channels reopen if the voltage pulse is long enough and contribute to steady-state currents.The convolution model overestimates the opening rate of channels contributing to the steady-state currents that remain after the transient early Na current has subsided.