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.     

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.     

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.     

Origin of Axon Membrane Hyperpolarization under Sucrose-Gap

One of the disadvantages of the sucrose-gap method for measuring membrane potentials with extracellular electrodes is a membrane hyperpolarization of the order of 30 to 60 mv, as compared with the resting potential obtained with intracellular microelectrodes in the absence of a sucrose-gap. In the present study the contribution of the sucrose-sea water junction potential to this hyperpolarization effect has been evaluated by comparing the effects on the resting potential of several anion and cation substitutions in the sea water bathing the lobster giant axon under sucrose-gap. Measurements with microelectrodes demonstrate a significant liquid junction potential between sucrose and standard artificial sea water. The value of this liquid junction potential as well as the measured resting membrane potential varies as a function of the anions and cations substituted in the sea water. Both the liquid junction potential and the sucrose-gap-induced hyperpolarization can be eliminated by using a low mobility anion to replace most of the chloride in sea water while the normal cation content remains unchanged. These data provide evidence that loop currents at the sucrose-sea water-axon junctions are at least partly responsible for membrane hyperpolarization under a sucrose gap.     

Current-Voltage Relations in the Lobster Giant Axon Membrane Under Voltage Clamp Conditions

The sucrose-gap method introduced by Stämpfli provides a means for the application of a voltage clamp to the lobster giant axon, which responds to a variety of different experimental procedures in ways quite similar to those reported for the squid axon and frog node. This is particularly true for the behavior of the peak initial current. However, the steady state current shows some differences. It has a variable slope conductance less than that of the peak initial current. The magnitude of the steady state slope conductance is related to the length of the repolarization phase of the action potential, which does not have an undershoot in the lobster. The steady state outward current is maintained for as long as 100 msec.; this is in contrast to a decline of about 50 per cent in the squid axon. Lowering the external calcium concentration produces shifts in the current-voltage relations qualitatively similar to those obtained from the squid axon. On the basis of the data available, there is no reason to doubt that the Hodgkin and Huxley analysis for the squid giant axon in sea water can be applied to the lobster giant axon.     

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.     

Tetrodotoxin Blockage of Sodium Conductance Increase in Lobster Giant Axons

Previous studies suggested that tetrodotoxin, a poison from the puffer fish, blocks conduction of nerve and muscle through its rather selective inhibition of the sodium-carrying mechanism. In order to verify this hypothesis, observations have been made of sodium and potassium currents in the lobster giant axons treated with tetrodotoxin by means of the sucrose-gap voltage-clamp technique. Tetrodotoxin at concentrations of 1 x 10^-7 to 5 x 10^-9 gm/ml blocked the action potential but had no effect on the resting potential. Partial or complete recovery might have occurred on washing with normal medium. The increase in sodium conductance normally occurring upon depolarization was very effectively suppressed when the action potential was blocked after tetrodotoxin, while the delayed increase in potassium conductance underwent no change. It is concluded that tetrodotoxin, at very low concentrations, blocks the action potential production through its selective inhibition of the sodium-carrying mechanism while keeping the potassium-carrying mechanism intact.     

Competitive Action of Calcium and Procaine on Lobster Axon : A study of the mechanism of action of certain local anesthetics

Voltage clamp studies with the squid giant axon have shown that changes in the external calcium concentration (Frankenhaeuser and Hodgkin, 1957) shift the sodium and potassium conductance versus membrane potential curves along the potential axis. Taylor (1959) found that procaine acts primarily by reducing the sodium and, to a lesser extent, the potassium conductances. Both procaine and increased calcium also delay the turning on of the sodium conductance mechanism. Calcium and procaine have similar effects on lobster giant axon. In addition, we have observed that the magnitude of the response to procaine is influenced by the external calcium concentration. Increasing external calcium tends to reduce the effectiveness of procaine in decreasing sodium conductance. Conversely, procaine is more effective in reducing the membrane conductance if external calcium is decreased. The amplitude of the nerve action potential reflects these conductance changes in that, for example, reductions in amplitude resulting from the addition of procaine to the medium are partially restored by increasing external calcium, as was first noted by Aceves and Machne (1963). These phenomena suggest that calcium and procaine compete with one another with respect to their actions on the membrane conductance mechanism. The fact that procaine and its analogues compete with calcium for binding to phospholipids in vitro (Feinstein, 1964) suggests that the concept of competitive binding to phospholipids may provide a useful model for interpreting these data.     

Axon voltage-clamp simulations. II. Double sucrose-gap method.

This is the second in a series of four papers on the simulation of the voltage clamp of cylindrical excitable cells. In this paper we evaluate the double sucrose-gap voltage-clamp technique for the squid and lobster giant axons. Using the Crank-Nicolson method of solution of the cable equations and differential equations representing the voltage clamp circuit we studied the effect of length of the sucrose gap "node" on the voltage profile along an excitable cell during a simulated voltage clamp. The voltage gradients along the region of the cell within the node produce "notches" in the current recording as well as changes in the magnitude of the sodium and potassium current for a given voltage step. Our results show that good voltage clamp control requires node lengths less than one-half the axon diameter.     

Reexamination of the double sucrose gap technique for the study of lobster giant axons. Theory and experiments

The double sucrose gap technique for the study of lobster giant axons has been reexamined. The leakage behavior of the system cannot be successfully modeled by conventional sucrose gap theory, but is accounted for by the McGuigan-Tsien model that takes into account the cable properties of membrane under sucrose. The facts of high-leakage conductance and the ability to maintain large resting potentials in the face of low sucrose gap resistance lead to a hypothesis that membrane resistance under sucrose is very low because of a large negative surface potential. Computer simulations of the leakage behavior of the conventional gap model and the McGuigan-Tsien model were compared with experimental measurements on lobster axons using normal sucrose or sucrose doped with Na+, Ca2+ or La3+ ions. As the concentration of doping ion increased, the leakage rose, but the species of doping ion had more influence on leakage than gap resistance. At equal gap resistance, leakage decreased with an increase in valence of the doping species. Leakage was even lower in La-doped sucrose at 20 M omega gap resistance than in normal sucrose at 200 M omega gap resistance. Resting potentials decreased with decreasing gap resistance and increasing valence of the doping species. Resting potential behavior was successfully simulated with a hybrid model consisting of a point node flanked by infinite cables and a shunt between ground and the voltage-measuring pool. The data support the hypothesis that the membrane resistance under sucrose is low and that it can be raised by doping the sucrose with multivalent cations, with La3+ being particularly effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Some Inhibitors on the Temperature-Dependent Component of Resting Potential in Lobster Axon

The resting membrane potential of the lobster axon becomes 5–8 mv more negative when the temperature of the perfusion solution is increased 10°C. This potential change is about twice that predicted if the axon membrane potential followed that expected for a potassium ion electrode potential. When the inhibitors, 2, 4-dinitrophenol, sodium cyanide, and sodium azide, were added separately to the perfusion medium the potential change was reduced to about 1.4 times that predicted for a potassium ion electrode potential. Assays of axons exposed to these inhibitors showed that ATP levels were reduced to about one-fourth that obtained for control axons. Ouabain added to the perfusion medium reduced the potential change to that expected for a potassium ion electrode potential. These results suggest that the resting potential changes with temperature as a result of the activity of an electrogenic ion pump.     

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.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Effects of thiamine antagonists on nerve conduction. I. Actions of antimetabolites and fern extract on propagated action potentials

To assess the hypothesis that thiamine is directly involved in the permeability changes at the sodium channel during nerve conduction, the effects of thiamine antagonists on lobster giant axon resting and action potentials were determined. Thiamine antimetabolites, in millimolar concentrations, reversibly decreased the maximum rate of rise and amplitude of the action potential while increasing its duration. In particular, thiamine tert-butyl disulfide (TTBD) elicited the formation of pronounced shoulders during repolarization, lengthening the action potential by 2–50 times, depending on dose. Antimetabolites also depolarized the resting membrane, but this change was poorly reversible and may indicate a dual mechanism for antimetabolite action. An extract of the fern, Pteris aquilina, reversibly decreased the maximum rate of rise of the action potential and depolarized the resting potential. It also elevated and prolonged the action potential after-depolarization, sometimes causing repetitive activity. The strength of these actions was correlated with the antithiamine potency of the extract, and was diminished by addition of thiamine to the extract.     

Effects of thiamine antagonists on nerve conduction. I. Actions of antimetabolites and fern extract on propagated action potentials.

To assess the hypothesis that thiamine is directly involved in the permeability changes at the sodium channel during nerve conduction, the effects of thiamine antagonists on lobster giant axon resting and action potentials were determined. Thiamine antimetabolites, in millimolar concentrations, reversibly decreased the maximum rate of rise and amplitude of the action potential while increasing its duration. In particular, thiamine tert-butyl disulfide (TTBD) elicited the formation of pronounced shoulders during repolarization, lengthening the action potential by 2-50 times, depending on dose. Antimetabolites also depolarized the resting membrane, but this change was poorly reversible and may indicate a dual mechanism for antimetabolite action. An extract of the fern, Pteris aquilina, reversibly decreased the maximum rate of rise of the action potential and depolarized the resting potential. It also elevated and prolonged the action potential after-depolarization, sometimes causing repetitive activity. The strength of these actions was correlated with the antithiamine potency of the extract, and was diminished by addition of thiamine to the extract.     

Ionic permeability of K, Na, and Cl in potassium-depolarized nerve. Dependency on pH, cooperative effects, and action of tetrodotoxin.

The passive ionic membrane conductances (gj) and permeabilities (Pj) of K, Na, and Cl of crayfish (Procambarus clarkii) medial giant axons were determined in the potassium-depolarized axon and compared with that of the resting axon. Passive ionic conductances and permeabilities were found to be potassium dependent with a major conductance transition occurring around an external K concentration of 12-15 mM (Vm = -60 to -65 mV). The results showed that K, Na, and Cl conductances increased by 6.2, 6.9, and 27-fold, respectively, when external K was elevated from 5.4 to 40 mM. Permeability measurements indicated that K changed minimally with K depolarization while Na and Cl underwent an order increase in permeability. In the resting axon (K0 = 5.4 mM, pH = 7.0) PK = 1.33 X 10(-5), PCl = 1.99 X 10(-6), PNa = 1.92 X 10(-8) while in elevated potassium (K0 = 40 mM, pH 7.0), PK = 1.9 X 10(-5), PCl = 1.2 X 10(-5), and PNa = 2.7 X 10(-7) cm/s. When membrane potential is reduced to 40 mV by changes in internal ions, the conductance changes are initially small. This suggests that resting channel conductances depend also on ion environments seen by each membrane surface in addition to membrane potential. In elevated potassium, K, Na, and Cl conductances and permeabilities were measured from pH 3.8 to 11 in 0.2 pH increments. Here a cooperative transition in membrane conductance or permeability occurs when pH is altered through the imidazole pK (approximately pH 6.3) region. This cooperative conductance transition involves changes in Na and Cl but not K permeabilities. A Hill coefficient n of near 4 was found for the cooperative conductance transition of both the Na and Cl ionic channel which could be interpreted as resulting from 4 protein molecules forming each of the Na and Cl ionic channels. Tetrodotoxin reduces the Hill coefficient n to near 2 for the Na channel but does not affect the Cl channel. In the resting or depolarized axon, crosslinking membrane amino groups with DIDS reduces Cl and Na permeability. Following potassium depolarization, buried amino groups appear to be uncovered. The data here suggest that potassium depolarization produces a membrane conformation change in these ionic permeability regulatory components. A model is proposed where membrane protein, which forms the membrane ionic channels, is oriented with an accessible amino terminal group on the axon exterior. In this model the ionizable groups on protein and phospholipid have varied associations with the different ionic channel access sites for K, Na, and Cl, and these groups exert considerable control over ion permeation through their surface potentials.     

Intracellular pH electrode : Experiments on the giant squid axon

A new, easy method to produce and calibrate a 1-μm tip intracellular pH electrode is described. This antimony electrode and a micro-calomel electrode were inserted into the giant axon of Loligo pealii. The potential obtained when the axon was bathed in seawater corresponded to a pH of 7.0 ± 0.2. It was found that acidification of the external perfusate induced a drop in axoplasmatic pH leading to changes in the membrane electrical properties. Changes of resting or action potentials did not influence intracellular pH.     

Squid giant axons. A model for the neuron soma?

Insertion of electrically floating wires along the axis of a squid giant axon produces an apparent increase in diameter in the region where the wire surface has been treated to give it a low resistance. The shape of action potentials propagating into this region depend upon the surface resistance (and the length) of the wire. As this segment's internal resistance is lowered by reducing the wire's surface resistance, the following characteristic sequence of changes in the action potential is seen at the transition region: (a) the duration increases; (b) two peaks develop, the first one generated in the normal axon region and the second one generated later in the axial wire region, and; (c) blockage occurs (for a very low resistance wire). Action potentials recorded at the membrane region near the tip of the axial wire in (b) resemble those recorded at the initial segment of neurons upon antidromic invasions. Squid axon action potentials propagated from a normal region into that containing the low resistance wire also resemble antidromic invasions recorded in neuron somas. Hyperpolarizing current pulses applied through the wire act as if the wire surface resistance was momentarily reduced. For example, the two components of the action potential recorded at the axial wire membrane region noted in (b) can be sequentially blocked by the application of increasing hyperpolarizing current through the wire. Similar effects are seen when hyperpolarizing currents are injected into motoneuron somas. It is concluded that the geometrical properties of the junction of a neuron axon with its soma may be in themselves sufficient to determine the shape of the action potentials usually recorded by microelectrodes.     

Effects of Replacement of External Sodium Chloride with Sucrose on Membrane Currents of the Squid Giant Axon

It was observed that a reduction of the sodium chloride concentration in the external solution bathing a squid giant axon by replacement with sucrose resulted in marked decreases in the peak inward and steady-state outward currents through the axon membrane following a step decrease in membrane potential. These effects are quantitatively acounted for by the increase in series resistance resulting from the decreased conductivity of the sea water and the assumption that the sodium current obeys a relation of the form I = k₁C₁ - k₂C₂ where C₁, C₂ are internal and external ion activities and k₁, k₂ are independent of concentration. It is concluded that the potassium ion current is independent of the sodium concentration. That the inward current is carried by sodium ions has been confirmed. The electrical potential (or barrier height) profile in the membrane which drives sodium ions appears to be independent of sodium ion concentration or current. A specific effect of the sucrose on hyperpolarizing currents was observed and noted but not investigated in detail.     

Demonstration of two stable potential states in the squid giant axon under tetraethylammonium chloride

1. Intracellular injection of tetraethylammonium chloride (TEA) into a giant axon of the squid prolongs the duration of the action potential without changing the resting potential (Fig. 3). The prolongation is sometimes 100-fold or more. 2. The action potential of a giant axon treated with TEA has an initial peak followed by a plateau (Fig. 3). The membrane resistance during the plateau is practically normal (Fig. 4). Near the end of the action potential, there is an apparent increase in the membrane resistance (Fig. 5D and Fig. 6, right). 3. The phenomenon of abolition of action potentials was demonstrated in the squid giant axon treated with TEA (Fig. 7). Following an action potential abolished in its early phase, there is no refractoriness (Fig. 8). 4. By the method of voltage clamp, the voltage-current relation was investigated on normal squid axons as well as on axons treated with TEA (Figs. 9 and 10). 5. The presence of stable states of the membrane was demonstrated by clamping the membrane potential with two voltage steps (Fig. 11). Experimental evidence was presented showing that, in an "unstable" state, the membrane conductance is not uniquely determined by the membrane potential. 6. The effect of low sodium water was investigated in the axon treated with TEA (Fig. 12). 7. The similarity between the action potential of a squid axon under TEA and that of the vertebrate cardiac muscle was stressed. The experimental results were interpreted as supporting the view that there are two stable states in the membrane. Initiation and abolition of an action potential were explained as transitions between the two states.