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1. In voltage clamped myelinated nerve fibres, some properties of the slow K+ current (Dubois, 1981 b) were analysed under different experimental conditions. 2. The instantaneous slow current-voltage relationships are linear in low and high K+ solutions, and the variation of the reversal potential with external K+ concentration agrees with the predicted Nernst changes for the K+ equilibrium potential. 3. Li+, Na+ and Cs+ are virtually impermeant in the slow channels. Rb+ is permeant and the permeability ratio PRb/PK (calculated from the reversal potential and the Goldman-Hodgkin-Katz equation) is a function of the external K+ and Rb+ concentrations. 4. External and internal Cs+ block respectively the slow inward and outward K+ current and the block is voltage dependent. 5. The closing kinetics of the slow channels are independent of the external K+ concentration but depend on the conditioning pulse duration.

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.

Intramembrane charge movement (Q) and sodium current (INa) were monitored in isolated voltage-clamped frog nodes of Ranvier, ON charge movements (QON) for pulses from the holding potential (-100 mV) to potentials V less than or equal to 0 mV followed single exponential time courses, whereas two exponentials were found for pulses to V greater than or equal to 20 mV. The voltage dependence of both QON and its time constant tauON indicated that the two ON components resolved at V greater than or equal to 20 mV were also present, though not resolvable, for pulses to V less than or equal to 0 mV. OFF charge movements (QOFF) monitored at various potentials were well described by single exponentials. When QOFF was monitored at -30 or -40 mV after a 200-microsecond pulse to +20 mV and QON was monitored at the same potential using pulses directly from -100 mV, tauON/tauOFF = 2.5 +/- 0.3. At a set OFF potential (-90 to -70 mV), tauOFF first increased with increasing duration tON of the preceding pulse to a given potential (0 to +30 mV) and then decreased with further increases in tON. The declining phase of tauOFF followed a time course similar to that of the decline in QOFF with tON. For the same pulse protocol, the OFF time constant tauNa for INA also first increased with tON but then remained constant over the tON interval during which tauOFF and QOFF were declining. After 200- or 300-microsecond pulses to +20, +20, or +50 mV, tauOFF/tauNa at -70 to -90 mV was 1.2 +/- 0.1. Similar tauOFF/tauNa ratios were predicted by channel models having three identical charged gating particles that can rapidly and reversibly form an immobile dimer or trimer after independently crossing the membrane from their OFF to their ON locations.

Na current (INa) was monitored in isolated voltage-clamped frog nodes of Ranvier in order to analyse the pharmacological and kinetic properties of fast and slow phases of inactivation. Niflumic acid (0.1-10 mM) and tetrodotoxin (0.3-30 nM) did not alter fast and slow inactivation time courses but preferentially reduced the amplitude of the fast phase of inactivation. The block of both phases of inactivation by niflumic acid and tetrodotoxin was well described if one assumed that more than one molecule of drug reacted with one channel. Fast and slow currents, corresponding respectively to fast and slow phases of inactivation, reversed at different potentials, had different threshold voltages of activation and the slopes of their steady-state inactivation curves were different. The recovery from inactivation of the compound INa could be described by the sum of two exponentials (plus a delay) corresponding respectively to fast and slow currents. When calculated from INa recorded without and with niflumic acid or tetrodotoxin, the slow current activated about three times more slowly than the fast current. Large prehyperpolarizations delayed both the activation and the inactivation of the fast current but only the activation of the slow current. Lowering the temperature decreased the fast current but increased the slow current. We conclude that the inactivatable Na current of the nodal membrane is made up of two components (INa,f and INa,s) corresponding to two different and interconvertible forms of the Na channel.

Capsaicin (10-20 microM) blocks reversibly one component of the K+ current (IKf2) in frog node of Ranvier. The block is enhanced with depolarization but it is not voltage-dependent indicating that the affinity of receptor sites for capsaicin is larger with the channels in the open configuration. The results provide pharmacological evidence of the existence within the nodal membrane of two classes of fast K+ channels, in addition to the slow K+ channels.

In batrachotoxin (BTX)-treated frog node of Ranvier, in spite of a marked reduction in Na inactivation, the Na current still presents a time- and voltage-dependent inactivation that could induce a 50-60% decrease in the current. The inactivation was found to be modified by changing the amplitude of a conditioning pulse, adding tetrodotoxin in the external solution, or replacing NaCl with KCl in the external solution. Conditioning pulses were able to alter the reversal potential of the BTX-modified Na current (Vrev). Vrev was shifted toward negative values for inward conditioning currents and was shifted toward positive values for outward conditioning currents. The change in Vrev was proportional to the conditioning current amplitude. Large inward currents induced 15-25 mV shifts of Vrev. During a 10-20-ms depolarizing pulse, the inactivation and change in Vrev were proportional to the time integral of the current. For longer depolarizations, Vrev reached a steady state level proportional to the current amplitude. The conductance, as calculated from the current and the actual Vrev, showed an inactivation proportional to exp(Vrev F/RT). These observations suggest that the BTX-modified Na current induces a decrease in local Na concentrations, which results in an alteration of the driving force and the conductance. During a pulse that induced a large inward current, the Na space concentration [( Na]s) changed from 114 to 50-60 mM. In normal fibers, the reversal potential of Na current was also shifted toward negative values by a prepulse that induced a large inward current. The change in Vrev reached 5-15 mV, which corresponded to a decrease in [Na]s of 20-50 mM. This change in Vrev slightly altered the time course of Na current. On the basis of a three-compartment model (axoplasm-perinodal space-bulk solution), a Na permeability of the barrier between the space and the bulk solution (PNa,s) and a mean thickness of the space (theta) were calculated. The mean value of PNa,s was 0.0051 cm X s-1 in both normal and BTX-treated fibers, whereas the value of theta was 0.29 micron in BTX-treated fibers and 0.05 micron in normal fibers. When compared with the values calculated during K accumulation, PNa,s was 10 times smaller than PK,s and theta Na-BTX was equal to theta K.(ABSTRACT TRUNCATED AT 400 WORDS)

Sodium current and intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). Sodium current activation followed a single-exponential time course, provided a delay was interposed between the onset of the step ON depolarization and that of the current change. The delay decreased with increased ON depolarization and, for a constant ON depolarization, increased with prehyperpolarization. ON charge movement followed a single-exponential time course with time constants tau Q,ON slightly larger than tau Na, ON. For pulses between -70 and -50 mV, tau Q,ON/tau Na,ON = 1.14 +/- 0.08. The OFF charge movement and OFF sodium current tails after a depolarizing pulse followed single-exponential time courses, with tau Q, OFF larger than tau Na, OFF. tau Q,OFF/tau Na,OFF increased with OFF voltage from 1 near -100 mV to 2 near -160 mV. At a set OFF potential (-120 mV), both tau Q,OFF and tau Na,OFF increased with ON pulse duration. The delay in INa activation and the effect of ON pulse duration on tau Q,OFF and tau Na,OFF are inconsistent with a simple two-state, single-transition model for the gating of batrachotoxin-modified sodium channels.

The steady state effects and rates of action of tetrodotoxin (TTX) on sodium current were studied in the voltage clamped frog node of Ranvier. Inactivation of the sodium current was separated into fast and slow phases. Both phases were assumed to correspond to two different currents (fast and slow) flowing through fast and slow channels (Benoit et al. 1985). The dose-response curve of the steady state effect of tetrodotoxin on the fast current was sigmoid. An analysis of this effect in double logarithmic coordinates gave a Hill coefficient of 1.74. The rates of tetrodotoxin action on the fast current were determined by the reversible reduction of the peak current recorded at a potential (+20 mV) at which the slow current was relatively small. After an initial delay, the onset of TTX effect followed an exponential function of time whose constant decreased with increasing tetrodotoxin concentrations. Expressed as the time corresponding to a reduction of 2% of the current, the delay (delta t2%) increased from about 100 ms with 300 nM-TTX to about 30 s with 1 nM-TTX. When tetrodotoxin was removed, the offset developed quasi-instantaneously and followed an exponential function of time whose constant was independent of the toxin concentration. Both steady state and rates of tetrodotoxin effects could be fitted well if one assumed that the block of one fast channel occurred after binding of two TTX molecules to two cooperative sites.

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.

Voltage clamp experiments carried out on nodes of Ranvier of myelinated fibres of Rana esculenta showed that a small fractoon of sodium channels fail to inactivate. Thus during long lasting depolarizing pulses there is a small Na-current superimposed on the leakage and potassium currents. This late Na-current appears more marked in sensory fibres than in motor ones.

Oenanthotoxin (OETX) is known to induce convulsions and epileptic spikes in rat. It is shown that OETX reversibly blocks the action potential and the sodium, potassium and gating currents in frog myelinated nerve fibres. These observations suggest that the block of ionic currents by OETX is due to an immobilization of the channel gates with the channel in a closed configuration. Before complete block, it is shown that OETX differentially affects the sodium current and the charge movement and blocks to different extents the different ionic currents. The results are discussed by comparison with the effects of agents known to immobilize the gating particles and in relation to the pharmacological actions of the toxin on the central nervous system.

Voltage clamp experiments done on nodes of Ranvier show that external application of Cesium blocks the inward but not the outward potassium currents. Internal application of Cs ions reduces the outward K-current and the inward K-current is not affected. These results support the hypotheses that K ions cross th- K-channel after dehydration at superficial sites where competition may occur with Cs-ions.

In nerve membrane, the non-linear capacity current (displacement current) is assumed to reflect the movement of intrinsic membrane charges which control the opening of specific pathways for sodium ions (Na channels). However, various discrepancies have been reported between the effects of pharmacological agents on sodium and displacment currents (for a review see ref. 1). It is generally supposed that the opening and closing of Na channels constitutes one step of multi-step system in which each configuration change may or not give rise to a measurable charge movement. New drugs affecting either sodium or displacement currents may elucidate the relationship between charge movement and the control of sodium conductance. We therefore now report a comparison of the effects of a vegetable toxin (oenanthotoxin or OETX) on both sodium current (INa) and intra-membrane charge movement (Q) in Ranvier nodes. We show that OETX reversibly blocks both sodium and displacement current. Studies of INa and Q during partial supression by the toxin reveal differences in the effects of OETX on the remaining INa and Q. The findings are discussed in relation to recent models for the Na-channel gating process and for Na-channel block by local anaesthetics.

1. In voltage clamped myelinated nerve fibres, the K+ conductance has been calculated from current recordings obtained in low and high K+ media, taking into account the changes in EK resulting from accumulation of depletion of K+ ions near to nodal membrane. 2. At the end of a depolarization, the instantaneous K+ current reverses at a potential (instantaneous reversal potential) differing from the Nernst potential calculated using the external and internal bulk concentrations (theoretical Nernst potential). During a depolarization, EK, as estimated from the instantaneous reversal potential, changes continuously. This change depends on the size, the duration and the direction of the time dependent K+ current. The variation of EK is attributed to continuous changes in K+ concentration near the membrane during voltage pulses which turn on the K+ conductance. 3. The chord conductance [GK = IK/(E-EK), as calculated using the instantaneous reversal potential values for EK, has been analysed as a function of time and membrane potential. As previously reported it increases with the initial K+ concentration in the external medium. 4. The time course of the K+ current depends on both the kinetics of the conductance increase and the rate of change in the driving force for K. The kinetics of the conductance increase can satisfactorily be described by a single exponential function following a delay after the onset of the depolarizing voltage clamp pulse. 5. This delay increases when the holding potential is made more negative. It decreases with membrane depolarization and it is independent of the external K+ concentration. At a given membrane potential, the turning on of the K+ conductance is found to be faster at high than at low external K+ concentrations. 6. At repolarization the turning off of the conductance cannot be described by a single exponential function. It is faster at low than at high external K+ concentrations. 7. The results suggest that the change in K+ conductance proceeds in a multi-step transition or (and) that the K+ conductance is determined by several types of K+ channels.

Palytoxin is a highly toxic compound isolated form several zoanthid Palythoa species. The effects of palytoxin on the nodal membrane of frog myelinated fiber have been studied under current clamp and under voltage clamp conditions. Under current clamp conditions, palytoxin (0.1 microng/ml, 3 x 10(-8)M) induces a depolarization which is not reversed by washing. The resting potential reaches a value of -35 mV after 10 minutes. During the same period, the evoked action potential shows a gradual decline and finally disapears after about 30 minutes. The membrane depolarization is suppressed by removal of Na ions from the external medium, but only slightly diminished when tetrodotoxin (10(-6)M) is subsequently added to the external medium. When the potential of the nodal membrane is maintained at -70 mV, palytoxin (0.1 microng/ml) causes the appearance of an inward current that increases in magnitude during 30 minutes before attaining a steady-state value. The kinetics of development of that current is modified in the presence of tetrodotoxin or saxitoxin. Voltage clamp analysis shows that palytoxin causes an increase of the resting sodium permeability that is accompanied by a shift of the voltage dependence of the transient sodium permeability in the direction of membrane hyperpolarization. The shift in the voltage dependence of the transient permeability is accompanied by a decrease of the peak transient permeability. A similar shift in the potential dependence of the sodium inactivation is observed. During and after the application of palytoxin, the internal sodium concentration increases. The steady-state (potassium) conductance is also decreased at the same time as the leak current is increasing.

Toxin I from the venom of the black mamba snake Dendroaspis polylepis polylepis specifically blocks one component of the K-current (IKf1) in the frog node of Ranvier, with a high affinity (Kd = 4 X 10(-10) M) without significantly affecting either the others components of the K current (IKf2 and IKs) or the Na-current. Moreover, for toxin concentrations corresponding to 10- or 100-fold the Kd value, the block was almost irreversible.

1. The effects of toxin II from scorpion Androctonus australis Hector (AaH II) on the Na current of frog myelinated nerve fibres were analysed under voltage-clamp conditions. 2. Like other alpha-scorpion toxins and Anemonia toxin II, AaH II both increased the inactivation time constants of peak Na current and induced a non-inactivatable Na current (maintained current). 3. In the presence of AaH II, the slope of the maintained conductance-voltage curve was less steep than that corresponding to the peak conductance and the maintained current reversed at a voltage about 20 mV more negative than the peak current. 4. When the peak current was inactivated by pre-depolarizations, 'on' and 'off' relaxation kinetics of the maintained current were an exponential function whose time constant changed with voltage in a bell-shaped manner. At 0 mV, the time constant was about 10 ms. 5. The effects of AaH II could be decomposed into fast effects (increase in inactivation time constants of the peak current) which developed within about 5 s and slow effects (increase in maintained current and changes in initial amplitudes of fast and slow phases of peak current inactivation) which developed within about 30 s. 6. These two types of AaH II effects could be completely removed by conditioning depolarizations giving rise to outward currents. 7. A model is proposed in which the binding of the toxin with its receptor is modulated by membrane potential and internal cations, the appearance of the maintained current is modulated by the environment of channels and the change in inactivation time constants is modulated by membrane potential. The maintained current would correspond to the transformation of a fraction of channels into a non-inactivable (late) form.

1. In voltage clamped myelinated fibres, the K+ current was recorded in high-K+ media to allow analysis without complications due to K+ accumulation. 2. After a depolarization, the tail of K+ current following repolarization decreases in two phases: a fast phase lasting about 20 msec and a slow exponential phase lasting several hundred milliseconds. When the duration of the depolarization is increased, the amplitude at time zero of the fast phase increases (activation of the conductance) and then decreases slowly (inactivation of the conductance). Simultaneously, the amplitude of the slow phase, extrapolated to time zero of repolarization, increases slowly and reaches a steady-state level (about 20% of the maximum instantaneous current) after about 600 msec of depolarization. 3. The fast phase of the tail current is blocked by external application of 4-aminopyridine (4-AP) (KD = 10(-5)M). The slow phase is unaltered by 4-AP (10(-7)-10(-2)M). 4. In the presence of 4-AP (10-3M), the remaining slow K+ current, activated by depolarizations, does not inactivate. 5. During depolarizations and repolarizations, the conductance of the slow current (GKs) varies exponentially. The steady-state value of the slow conductance and its time constant of activation vary with voltage. The variation of the slow conductance with time and voltage can be described by a closed-open mode, assuming that each channel is gated by one particle. The activation kinetics of the slow current is unaltered by long lasting (500 msec) prepolarizations. 6. The fast K+ conductance, calculated from the fast tail current, is fully inactivated at the end of a 3 min depolarization to 0 mV. 7. The fast K+ conductance can be decomposed into two components: one component (GKf1) activating between -80 and -30 mV and inactivating very slowly (tau = 45 sec at E = 0 mV); one component (GKf2) activating between -40 mV and +30 mV and inactivating slowly (tau = 2 sec at E = 0 mV). t = 12 degrees C. 8. The maximum slow and fast conductances increase with [K]0. While the maximum fast conductance tends to saturate at high external K+ concentrations, the maximum slow conductance shows no sign of saturation. 9. A comparison between motor and sensory fibres shows that, while the amplitude of maximum slow and fast conductances are identical for both types of fibres, the amplitude of fast-1 conductance is larger and consequently the amplitude of fast-2 is smaller in motor than in sensory fibres. The different spike frequency adaptations observed on both types of fibres are discussed in relation to these different relative fast conductances amplitudes. 10. It is concluded that the K+ conductance of the nodal membrane is composed of three components (GKS, GKf1 and GKf2) corresponding to three different and distinct types of K+ channels.

The effects of two toxins extracted from the tentacles of Anemonia sulcata on ionic currents have been tested on the nodal membrane of myelinated nerve fibres from Rana esculenta. While external application of Toxin I at 100 muM leaves both specific ionic currents unmodified, Toxin II at 10 muM reacts with a receptor site associated with the sodium conductance inactivation gating. Since internal application by diffusion of Toxin II at a concentration of 700 muM leaves the ionic currents unchanged, the receptor site is most likely located on the external side of the nodal membrane. An equilibrium dissociation constant for the effects of Toxin II was estimated as 20 muM. The on-reaction is fast (rate constant for the on-reaction roughly equal to 3.103 M-1) suggesting a readily accesible receptor site for the toxin. The kinetics characteristics of the sodium currents recorded in the presence of Toxin II suggest that there are at least two steps in the reaction leading to Na+ -channels with the inactivation gate completely immobilized. The relatively fast reversibility of the intermediate stage of the reaction and the rather slow but, in the end, complete reversal of the toxin effects suggest that the toxin acts by modifying the energy profile for the transition "inactivation gate in the open configuration to inactivation gate in the closed configuration." Toxin II at higher concentrations (greater than 100 muM) also inhibits the potassium currents but these effects were not studied in any detail.