Internal cations, membrane current, and sodium inactivation gate closure in Myxicola giant axons.

Steady state to peak Na current ratio (INa,/INapeak) in Myxicola is greater, under some conditions, in internal Cs than in K, indicating less steady state inactivation in Csi. Csi effects are selective for steady state inactivation, with negligible effects on single-pulse inactivation time constants (Th). Mean Th ratios (Csi to Ki) were 1.04 and 1.02 at 0 and 10 mV. Two pulse inactivation time constants were also little affected. Inactivation is blocked in an all or none manner. Ki has little effect on steady state inactivation in the presence of inward INa, with INa/INapeak often declining to zero at positive potentials and independent of external Na concentration from 1/4 to 2/3 artificial sea water (ASW). Cs also has little effect at more negative potentials, but more with either more positive potentials or Na reduction, both reducing inward INa. K effects are evident when Na channel current is outward. A site in the current pathway when occupied selectively blocks inactivation gate closure. As occupancy does not depend significantly on potential, the site must not be very deep into the membrane field. Inactivation gates may associate with these sites on closure. The inactivated state may consist of a positively-charged structure occluding the inner channel mouth.     

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

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

Simulation of Na channel inactivation by thiazine dyes

Some dyes of the methylene blue family serve as artificial inactivators of the sodium channels when present inside squid axons at a concentration of approximately 0.1 mM. The dyes restore a semblance of inactivation after normal inactivation has been destroyed by pronase. In fibers that inactivate normally, the dyes hasten the decay of sodium current. Many dye-blocked channels conduct transiently on exit of the dye molecule after repolarization to the holding potential. In contrast, normally inactivated channels do not conduct during recovery from inactivation. Kinetic evidence shows that inactivation of a dye-blocked channel is unlikely or impossible, which suggests that dye molecules compete with inactivation "particles" for the same site. In the absence of tetrodotoxin, the dyes do not affect the ON gating current unless the interpulse interval is very short. If sufficient equilibration time is allowed during a pulse, the initial amplitude of the OFF gating current is reduced to near zero. This suggests that a dye molecule is a Na channel completely blocks that channel's gating current, even the fraction that is resistant to normal inactivation. Dyes block INa and Ig with the same time course. This provides the strongest evidence to date that virtually all of recorded "gating current" is associated with Na channels. Tetrodotoxin greatly slows dissociation of dye molecules from Na channels and reduced gating current during both opening and closing of the channels.     

Blockade of sodium channels by Bistramide A in voltage-clamped frog skeletal muscle fibres.

The effect of Bistramide A, a toxin isolated from Bistratum lissoclinum Sluiter (Urochordata), on the peak sodium current (INa) of frog skeletal muscle fibres was studied with the double sucrose gap voltage clamp technique. External or internal application of Bistramide A inhibited INa without alteration of the kinetic parameters of the current nor of the apparent reversal potential for Na. The steady-state activation curve of INa was unchanged while the steady-state inactivation curve of INa was shifted towards more negative membrane potentials. Dose-response curves indicated an apparent dissociation constant for Bistramide A of 3.3 microM and a Hill coefficient of 1.2 which suggested a one to one relation between the toxin and Na channel. The inhibition of INa occurred at rest, and was more important at more positive holding potentials. Bistramide A exhibited only a weak frequency-dependent effect. The toxin did not interact with the use-dependent effect of lidocaine. It mainly blocked Na channels at more depolarized holding potentials. The toxin blocked Na channels when it was internally applyed and when the inactivation gating system has been previously destroyed by internal diffusion of iodate. The data suggest that Bistramide A inhibited the Na channel both at rest and in the inactivated state and occupied a site which was not located on the inactivation gate.     

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

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

Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.     

Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

Delays in inactivation development and activation kinetics in myxicola giant axons

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

Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.     

The Na+ channel inactivation gate is a molecular complex: a novel role of the COOH-terminal domain

Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated Na+ channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the alpha subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH terminus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the Na+ channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process.     

Phosphorylation of S1505 in the cardiac Na+ channel inactivation gate is required for modulation by protein kinase C

Inactivation of both brain and cardiac Na+ channels is modulated by activation of protein kinase C (PKC) but in different ways. Previous experiments had shown that phosphorylation of serine 1506 in the highly conserved loop connecting homologous domains III and IV (LIII/IV) of the brain Na+ channel alpha subunit is necessary for all effects of PKC. Here we examine the importance of the analogous serine for the different modulation of the rH1 cardiac Na+ channel. Serine 1505 of rH1 was mutated to alanine to prevent its phosphorylation, and the resulting mutant channel was expressed in 1610 cells. Electrophysiological properties of these mutant channels were indistinguishable from those of wild-type (WT) rH1 channels. Activation of PKC with 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced WT Na+ current by 49.3 +/- 4.2% (P < 0.01) but S1505A mutant current was reduced by only 8.5 +/- 5.4% (P = 0.29) when the holding potential was -94 mV. PKC activation also caused a -17-mV shift in the voltage dependence of steady-state inactivation of the WT channel which was abolished in the mutant. Thus, phosphorylation of serine 1505 is required for both the negative shift in the inactivation curve and the reduction in Na+ current by PKC. Phosphorylation of S1505/1506 has common and divergent effects in brain and cardiac Na+ channels. In both brain and cardiac Na+ channels, phosphorylation of this site by PKC is required for reduction of peak Na+ current. However, phosphorylation of S1506 in brain Na+ channels slows and destabilizes inactivation of the open channel. Phosphorylation of S1505 in cardiac, but not S1506 in brain, Na+ channels causes a negative shift in the inactivation curve, indicating that it stabilizes inactivation from closed states. Since LIII/IV containing S1505/S1506 is completely conserved, interaction of the phosphorylated serine with other regions of the channel must differ in the two channel types.     

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.     

Activation, inactivation and recovery in the sodium channels of the squid giant axon dialysed with different solutions.

Comparisons were made between families of ion currents recorded in voltage-clamped squid axons dialysed with 20 mM NaF and 330 mM CsF or TMAF, and bathed in a solution in which four fifths of the Na was replaced by Tris. The permeability coefficient PNa,fast for the fast-inactivating current in the initial open state was calculated as a function of test potential from the size of the initial peak of INa. The permeability coefficient PNa,non for the non-inactivating open state was calculated from the steady-state INa that persisted until the end of the test pulse. Dialysis with TMA had no direct effect on the QV curve for gating charge. The reversal potential for INa,non was always lower than that for INa,fast, the mean difference being about -9 mV when dialysing with Cs, but only about -1 mV with TMA. Except close to threshold, PNa,fast was roughly halved by dialysis with TMA as compared with Cs, but PNa,non was substantially increased. The time constant tau h inactivation of the sodium system was slightly increased during dialysis with TMA in place of Cs, and there were small shifts in the steady-state inactivation curve, but the rate of recovery from inactivation was not measurably altered. The flattening off of the tau h curve at increasingly positive test potentials corresponded to a steady reduction of the apparent inactivation charge until a value of about 0.2e was reached for pulses to 100 mV. The instantaneous I-V relationship in the steady state was also investigated. The results have a useful bearing on the effects of dialysis with TMA, on the differences between the initial and steady open states of the sodium channel, and on the relative voltage-dependences of the transitions in each direction between the resting and inactivated states.     

Local anesthetics. Effect of pH on use-dependent block of sodium channels in frog muscle.

Sodium currents were studied under voltage clamp in the presence of neutral, amine, and quaternary local anesthetic compounds. Use-dependent block was observed as a cumulative depression of INa seen with repetitive depolarizing test pulses applied at frequencies of 2-10s-1. With quaternary QX-314, the time constant of use dependence was long, and with neutral benzocaine, very short. With lidocaine and procaine, increasing external pH (pHo) changed the time constant from long to short, but alterations of internal pH have no effect. Inactivation in Na channels was measured by the influence of prepulses on peak INa during test pulses. Single-stimulus inactivation curves were shifted more with lidocaine at high pHo than at low pHo, but inactivation curves measured during pulse trains with any of the drugs and at any pHo were strongly shifted. All measurements show that the drug-receptor reaction was slow for amine drugs at low pHo, as for quaternary drugs at any pHo, and fast for amine drugs at high pHo, as for neutral drugs at any pHo. The major effect of low pHo on amine drugs was to reduce the concentration of drugs in the fiber and to protonate drug molecules on the receptor, thus trapping them in the blocking position for a longer time. Direct effects of pH on the receptor seemed minimal.     

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

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

Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent

We have determined the time course of Na channel inactivation in clonal pituitary (GH3) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole-cell patch clamp, with papain included in the internal medium. Inactivation was slowly removed over the course of 10 min, making it possible to obtain control records before the enzyme acted. Papain caused a large (4-100x) increase in current magnitude for small depolarizations (near -40 mV), and a much smaller increase for large ones (approximately 1.5x at +40 mV). For technical reasons it was sometimes convenient to study outward INa recorded with no Na+ outside. The instantaneous I-V (IIV) curve in this condition was nonlinear before papain, and more nearly linear afterwards. The gNa-V curve after papain, obtained by dividing the INa-V curve by the IIV curve, was left-shifted by at least 20 mV and steepened. A spontaneous 5-10 mV left shift occurred in the absence of papain. The rate of the inactivation step was found to vary only slightly from -100 mV to +60 mV, based on the following evidence. (a) Before papain, inactivation rate saturated with voltage and was constant from +20 to +60 mV. (b) We activated the channels with a brief pulse, and studied the time course of the current on changing the voltage to a second, usually more negative level (Na+ present internally and externally). The time course of inactivation at each voltage was obtained by comparing control traces with those after inactivation was removed. When the 5-10-mV spontaneous shift was taken into account, inactivation rate changed by less than 10% from -100 to +60 mV. The data are considered in terms of existing models of the Na channel.     

Aminopyridine block of transient potassium current

The blocking action of 4-aminopyridine (4-AP) and 3, 4-diaminopyridine (Di-AP) on transient potassium current (IA) in molluscan central neurons was studied in internal perfusion voltage-clamp experiments. Identical blocking effects were seen when the drugs were applied either externally or internally. It was found that aminopyridines have two kinds of effects on IA channels. The first involves block of open channels during depolarizing pulses and results in a shortening of the time to peak current and an increase in the initial rate of decay of current. This effect of the drug is similar to the block of delayed potassium current by tetraethylammonium (TEA). The other effect is a steady block that increases in strength during hyperpolarization, is removed by depolarization, and is dependent on the frequency of stimulation. The voltage dependence of steady state block approximates the voltage dependence of inactivation gating a changes e-fold in approximately 10 mV. These data suggest that the strength of block may depend on the state of IA gating such that the resting state of the channel with open inactivation gate is more susceptible to block than are the open or inactivated states. A multistate sequential model for IA gating and voltage-dependent AP block is developed.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

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.