Some Relations between Action Potential and Resting Potential of the Lobster Giant Axon

Experiments were performed to determine the quantitative relation existing between action potential and resting potential of the lobster giant axon. Resting potential changes were induced by either increasing the external potassium concentration or by reducing the external calcium concentration. For either treatment the action potential amplitude is proportional to the logarithm of the resting potential minus a constant. This constant is equivalent to the minimum resting potential at which a propagated spike is possible, and is larger for depolarization in low calcium than in high potassium. Thus the change in action potential per unit change in resting potential is greater in low external calcium than in high external potassium. Analog computer solutions to the Hodgkin-Huxley equations for squid axon membrane potentials show that, if the initial conditions are properly specified, the action potential is proportional to the logarithm of the potassium potential minus a constant. The experimental results and the analog computations suggest that reducing external calcium produces changes in the invertebrate axon that cannot be accounted for solely on the basis of alterations in the potassium potential.     

Action of External Divalent Ion Reduction on Sodium Movement in the Squid Giant Axon

Voltage clamp measurements of the sodium potential have been made on the resting squid giant axon to study the effect of variations in external divalent ion concentration upon net sodium flux. From these measurements the intracellular sodium concentration and the net sodium inflow were calculated using the Nernst relation and constant activity coefficients. While an axon bathed in artificial sea water shows a slow increase in internal sodium concentration, the rate of sodium accumulation is increased about two times by reducing external calcium and magnesium concentrations to 0.1 times their normal values. The mean inward net sodium flux increases from a mean control value of 97 pmole/cm² sec. to 186 pmole/cm² sec. in low divalent solution. Associated with these effects of external divalent ion reduction are a marked decrease in action potential amplitude, little or no change in resting potential, and a shift along the voltage axis of the curve relating peak sodium conductance to membrane potential similar to that obtained by Frankenhaeuser and Hodgkin (1957). These results implicate divalent ions in long term (minutes to hours) sodium permeability.     

Effects of external ions on membrane potentials of a lobster giant axon

The effects of varying external concentrations of normally occurring cations on membrane potentials in the lobster giant axon have been studied and compared with data presently available from the squid giant axon. A decrease in the external concentration of sodium ions causes a reversible reduction in the amplitude of the action potential and its rate of rise. No effect on the resting potential was detected. The changes are of the same order of magnitude, but greater than would be predicted for an ideal sodium electrode. Increase in external potassium causes a decrease in resting potential, and a decrease in potassium causes an increase in potential. The data so obtained are similar to those which have been reported for the squid giant axon, and cannot be exactly fitted to the Goldman constant field equation. Lowering external calcium below 25 mM causes a reduction in resting and action potentials, and the occasional occurrence of repetitive activity. The decrease in action potential is not solely attributable to a decrease in resting potential. Increase of external calcium from 25 to 50 mM causes no change in transmembrane potentials. Variations of external magnesium concentration between zero and 50 mM had no measurable effect on membrane potentials. These studies on membrane potentials do not indicate a clear choice between the use of sea water and Cole's perfusion solution as the better external medium for studies on lobster nerve.     

Effects of external ions on membrane potentials of a crayfish giant axon

Transmembrane potentials in the crayfish giant axon have been investigated as a function of the concentration of normally occurring external cations. Results have been compared with data already available for the lobster and squid giant axons. The magnitude of the action potential was shown to be a linear function of the log of the external sodium concentration, as would be predicted for an ideal sodium electrode. The resting potential is an inverse function of the external potassium concentration, but behaves as an ideal potassium electrode only at the higher external concentrations of potassium. Decrease in external calcium results in a decrease in both resting potential and action potential; an increase in external calcium above normal has no effect on magnitude of transmembrane potentials. Magnesium can partially substitute for calcium in the maintenance of normal action potential magnitude, but appears to have very little effect on resting potential. All ionic effects studied are completely reversible. The results are in generally good agreement with data presently available for the lobster giant axon and for the squid giant axon.     

Interactions of Calcium with Sodium and Potassium in Membrane Potentials of the Lobster Giant Axon

Experiments were performed on the lobster giant axon to determine the relation between intracellular spike amplitude and external calcium ion concentration. Action potential decline in low external calcium is greatly accelerated by simultaneous removal of external sodium ion. Correlation of the time course of spike decline in low calcium-low sodium solution with the time courses of spike decline in low calcium alone and in low sodium alone indicates that the effect of simultaneous removal of both ions is significantly greater than the sum of the individual effects. For a given time of treatment, spike amplitude was a function of external calcium concentration. While spike height is proportional to the log of the external calcium concentration over the range 2.5 to 50 millimolar, the proportionality constant is dependent upon the sodium concentration. Under the conditions of low external sodium (50 per cent reduction) the slope of the linear relationship between the spike height and the log of the external calcium concentration is about 5 times greater than in normal external sodium. Decreasing external calcium concentration and simultaneously increasing external potassium concentration produce a greater spike reduction than the arithmetic sum of spike reductions in low calcium alone and in high potassium alone. It is suggested that calcium interacts strongly with sodium and potassium in the spike-generating mechanism. A theoretical basis for these results is discussed.     

Blockage of Sodium Conductance Increase in Lobster Giant Axon by Tarichatoxin (Tetrodotoxin)

Tarichatoxin, isolated from California newt eggs, has been found to selectively block the increase of sodium conductance associated with excitation in lobster giant axons at nanomolar concentrations. This resulted from a reduction in the amplitude of the conductance increase rather than a change in its temporal characteristics. The normal potassium conductance increase with depolarization is not altered. A high concentration of calcium applied concomitantly with the toxin significantly improves the reversibility of the sodium blocking. This toxin has recently been identified as chemically identical with tetrodotoxin from the puffer fish. Toxins from the two sources are equally effective and are shown to have an action which is distinctly different from that of procaine.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.     

The Effects of Mechanical Stimulation on Some Electrical Properties of Axons

Rapid, short duration mechanical compression of lobster giant axons by a crystal-driven stylus produces a depolarization and an increase in membrane conductance which develop immediately with compression but take several seconds to recover. The conductance increase occurs even when the depolarization is prevented electrically. If sodium is removed from the external medium or if procaine is added to it, compression produces almost no depolarization. Small bundles of myelinated frog fibers are depolarized by rapid compression but recover very rapidly (milliseconds); "off" responses are occasionally seen. The results are discussed in terms of the mechanoelectric transducer behavior of an axon membrane.     

Effects of the dipolar form of phloretin on potassium conductance in squid giant axons.

The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK(Em) and greater reductions maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; on postulates a phloretin "receptor" near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.     

Membrane Potentials of the Lobster Giant Axon Obtained by Use of the Sucrose-Gap Technique

A method similar to the sucrose-gap technique introduced be Stäpfli is described for measuring membrane potential and current in singly lobster giant axons (diameter about 100 micra). The isotonic sucrose solution used to perfuse the gaps raises the external leakage resistance so that the recorded potential is only about 5 per cent less than the actual membrane potential. However, the resting potential of an axon in the sucrose-gap arrangement is increased 20 to 60 mv over that recorded by a conventional micropipette electrode when the entire axon is bathed in sea water. A complete explanation for this effect has not been discovered. The relation between resting potential and external potassium and sodium ion concentrations shows that potassium carries most of the current in a depolarized axon in the sucrose-gap arrangement, but that near the resting potential other ions make significant contributions. Lowering the external chloride concentration decreases the resting potential. Varying the concentration of the sucrose solution has little effect. A study of the impedance changes associated with the action potential shows that the membrane resistance decreases to a minimum at the peak of the spike and returns to near its initial value before repolarization is complete (a normal lobster giant axon action potential does not have an undershoot). Action potentials recorded simultaneously by the sucrose-gap technique and by micropipette electrodes are practically superposable.     

The Effects of Several Alcohols on the Properties of the Squid Giant Axon

The effects of several alcohols on the resting potential, action potential, and voltage-clamp currents of the squid giant axon have been measured. All the alcohols employed are similar in that they depress maximum sodium conductance much more than maximum potassium conductance. Octyl alcohol differs from the others (C₂ through C₅) in that it has less tendency to depolarize the axon. Depolarization is always accompanied by a decrease of g_K near the resting potential, such that the ratio g_K/g_leak is decreased. Steady-state inactivation of the sodium ion current is unaffected by alcohols, as is membrane capacity. Resting membrane conductance is usually decreased by alcohols. The findings are discussed in relation to work on monomolecular films.     

Slow Changes of Potassium Permeability in the Squid Giant Axon

A slow potassium inactivation i.e. decrease of conductance when the inside of the membrane is made more positive with respect to the outside, has been observed for the squid axon. The conductance-potential curve is sigmoid shaped, and the ratio between maximum and minimum potassium conductance is at least 3. The time constant for the change of potassium conductance with potential is independent of the concentration of potassium in the external solution, but dependent upon potential and temperature. At 9 degrees C and at the normal sea water resting potential, the time constant is 11 sec. For lower temperature or more depolarizing potentials, the time constant is greater. The inactivation can be described by modifying the Hodgkin-Huxley equation for potassium current, using one additional parameter. The modified equation is similar in form to the Hodgkin-Huxley equation for sodium current, suggesting that the mechanism for the passive transport of potassium through the axon membrane is similar to that for sodium.     

A study of the effects of externally applied sodium-ions and detection of spatial non-uniformity of the squid axon membrane under internal perfusion

Electrical properties of the axon membrane were examined under internal perfusion of squid giant axons with a dilute solution of NaF or CsF. The rate of propagation of the action potential was markedly enhanced when NaCl was added to the external CaCl₂ solution. The membrane conductance both at rest and during the action potential was increased with increasing Na-concentration in the external medium. In the perfusion zone of these axons, the action potentials in different parts of the membrane were found to terminate in a more-or-less spatially random and temporally irregular fashion. When the electric field outside the axon membrane was examined with hyperfine glass-pipette electrodes, small rectangular potential changes of uniform amplitude were observed. The small potential changes, which resemble those obtained by Bean in EIM-treated lipid bilayer, were interpreted as indicating spatial non-uniformity of the axon membrane during excitation. The importance of long-range electric interaction between different parts of the axon membrane is emphasized.     

Grayanotoxin, veratrine, and tetrodotoxin-sensitive sodium pathways in the Schwann cell membrane of squid nerve fibers

The actions of grayanotoxin I, veratrine, and tetrodotoxin on the membrane potential of the Schwann cell were studied in the giant nerve fiber of the squid Sepioteuthis sepioidea. Schwann cells of intact nerve fibers and Schwann cells attached to axons cut lengthwise over several millimeters were utilized. The axon membrane potential in the intact nerve fibers was also monitored. The effects of grayanotoxin I and veratrine on the membrane potential of the Schwann cell were found to be similar to those they produce on the resting membrane potential of the giant axon. Thus, grayanotoxin I (1-30 muM) and veratrine (5-50 mug-jl-1), externally applied to the intact nerve fiber or to axon-free nerve fiber sheaths, produce a Schwann cell depolarization which can be reversed by decreasing the external sodium concentration or by external application of tetrodotoxin. The magnitude of these membrane potential changes is related to the concentrations of the drugs in the external medium. These results indicate the existence of sodium pathways in the electrically unexcitable Schwann cell membrane of S. sepioidea, which can be opened up by grayanotoxin I and veratrine, and afterwards are blocked by tetrodotoxin. The sodium pathways of the Schwann cell membrane appear to be different from those of the axolemma which show a voltage-dependent conductance.     

Interactions of aminopyridines with potassium channels of squid axon membranes.

The effects of aminopyridines on ionic conductances of the squid giant axon membrane were examined using voltage clamp and internal perfusion techniques. 4-Aminopyridine (4-AP) reduced potassium currents, but had no effect upon transient sodium currents. The block of potassium channels by 4-AP was substantially less with (a) strong depolarization to positive membrane potentials, (b) increasing the duration of a given depolarizing step, and (c) increasing the frequency of step depolarizations. Experiments with high external potassium concentrations revealed that the effect of 4-AP was independent of the direction of potassium ion movement. Both 3- and 2-aminopyridine were indistinguishable from 4-AP except in potency. It is concluded that aminopyrimidines may be used as tools to block the potassium conductance in excitable membranes, but only within certain specific voltage and frequency limits.     

Inhibition of potassium conductance with external tetraethylammonium ion in Myxicola giant axons.

In voltage clamp experiments, externally applied tetraethylammonium ion (TEA) was found to have minimal effects on transient sodium currents and to suppress steady-state potassium currents of Myxicola giant axons by causing a specific decrease in the maximum potassium conductance gK. The dose-response curve suggests a one-to-one stoichiometry for TEA-receptor binding with an apparent dissociation constant on 24 mM. The suppression of IK is essentially reversible. Experiments performed on high external potassium ion concentrations indicate that both outward and inward IK were blocked by external TEA. The results thus suggest the presence of TEA receptors on the outer surface of Myxicola axonal membrane similar to those reported in the frog node.     

Magnitude and location of surface charges on Myxicola giant axons

The effects of changes in the concentration of calcium in solutions bathing Myxicola giant axons on the voltage dependence of sodium and potassium conductance and on the instantaneous sodium and potassium current-voltage relations have been measured. The sodium conductance-voltage relation is shifted along the voltage axis by 13 mV in the hyperpolarizing direction for a fourfold decrease in calcium concentration. The potassium conductance-voltage relation is shifted only half as much as that for sodium. There is no effect on the shape of the sodium and potassium instantaneous current-voltage curves: the normal constant-field rectification of potassium currents is maintained and the normal linear relationship of sodium currents is maintained. Considering that shifts in conductances would reflect the presence of surface charges near the gating machinery and that shape changes of instantaneous current-voltage curves would reflect the presence of surface charges near the ionic pores, these results indicate a negative surface charge density of about 1 electronic charge per 120 A2 near the sodium gating machinery, about 1 e/300 A2 for the potassium gating machinery, and much less surface charge near the sodium or potassium pores. There may be some specific binding of calcium to these surface charges with an upper limit on the binding constant of about 0.2 M-1. The differences in surface charge density suggest a spatial separation for these four membrane components.     

Single sodium channels from the squid giant axon

Since the work of A. L. Hodgkin and A. F. Huxley (1952. J. Physiol. [Lond.].117:500-544) the squid giant axon has been considered the classical preparation for the study of voltage-dependent sodium and potassium channels. In this preparation much data have been gathered on macroscopic and gating currents but no single sodium channel data have been available. This paper reports patch clamp recording of single sodium channel events from the cut-open squid axon. It is shown that the single channel conductance in the absence of external divalent ions is approximately 14 pS, similar to sodium channels recorded from other preparations, and that their kinetic properties are consistent with previous results on gating and macroscopic currents obtained from the perfused squid axon preparation.     

Barbiturates Block Sodium and Potassium Conductance Increases in Voltage-Clamped Lobster Axons

Sodium pentobarbital and sodium thiopental decrease both the peak initial (Na) and late steady-state (K) currents and reduce the maximum sodium and potassium conductance increases in voltage-clamped lobster giant axons. These barbiturates also slow the rate at which the sodium conductance turns on, and shift the normalized sodium conductance vs. voltage curves in the direction of depolarization along the voltage axis. Since pentobarbital (pK_a = 8.0) blocks the action potential more effectively at pH 8.5 than at pH 6.7, the anionic form of the drug appears to be active. The data suggest that these drugs affect the axon membrane directly, rather than secondarily through effects on intermediary metabolism. It is suggested that penetration of the lipid layer of the membrane by the nonpolar portion of the barbiturate molecules may cause the decrease in membrane conductances, while electrostatic interactions involving the anionic group on the barbiturate, divalent cations, and "fixed charges" in the membrane could account for the slowing of the rate of sodium conductance turn-on and the shift of the normalized conductance curves along the voltage axis.     

Action of Certain Tropine Esters on Voltage-Clamped Lobster Axon

Tropine p-tolylacetate (TPTA) and its quaternary analogue, tropine p-tolylacetate methiodide (TPTA MeI) decrease the early transient (Na) and late (K) currents in the voltage-clamped lobster giant axon. These agents, which block the nerve action potential, reduce the maximum Na and K conductance increases associated with membrane depolarization. They also slow the rate at which the sodium conductance is increased and shift the (normalized) membrane conductance vs. voltage curves in the direction of depolarization along the voltage axis. All these effects are qualitatively similar to those resulting from the action of procaine on the voltage-clamped axon. One unusual effect of the tropine esters, noticeable particularly at large depolarization steps, is that they cause the late, K current to reach a peak and then fall off with increasing pulse duration. This effect has not been reported to occur as a result of procaine action. Tropine p-chlorophenyl acetate (TPClA), which differs from TPTA only by the substitution of a p-Cl for a p-CH₃ group on the benzene ring, had a negligible effect on axonal excitability.