Response of delayed (K+) channels to the time-dependent clamping function in squid giant axon. I. Ascending ramps

Squid giant axons are voltage-clamped with ascending potential ramps whose slopes range from 0.5 mV/msec to 60 mV/msec and delayed (K+) currents are observed. The parametric current-voltage curves exhibit a delay period of minimal current followed by a rapid increase of current toward a final steady state. Both the initial delay and the slope of the subsequent rising phase increase with increasing ramp slope. When the Hodgkin-Huxley equations are used to generate theoretical current-voltage curves, the sharp difference between the delay and rising phases is muted and the ramp slope must be increased to produce an adequate representation of the data. A muted biphasic response is also observed when the current-voltage curves are generated using modified Hodgkin-Huxley parameters and a correction for K+ accumulation in the periaxonal space. These modified equations provide an accurate fit for step-potential clamp current data. Since the ramp experiments include all relevant clamping potentials, the experiments provide a sensitive test for kinetic models of K+ on flow in the delayed (K+) channels of squid giant axon.     

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.     

Ionic conductances of squid giant fiber lobe neurons

The cell bodies of the neurons in the giant fiber lobe (GFL) of the squid stellate ganglion give rise to axons that fuse and thereby form the third-order giant axon, whose initial portion functions as the postsynaptic element of the squid giant synapse. We have developed a preparation of dissociated, cultured cells from this lobe and have studied the voltage-dependent conductances using patch-clamp techniques. This system offers a unique opportunity for comparing the properties and regional differentiation of ionic channels in somatic and axonal membranes within the same cell. Some of these cells contain a small inward Na current which resembles that found in axon with respect to tetrodotoxin sensitivity, voltage dependence, and inactivation. More prominent is a macroscopic inward current, carried by Ca2+, which is likely to be the result of at least two kinetically distinct types of channels. These Ca channels differ in their closing kinetics, voltage range and time course of activation, and the extent to which their conductance inactivates. The dominant current in these GFL neurons is outward and is carried by K+. It can be accounted for by a single type of voltage-dependent channel. This conductance resembles the K conductance of the axon, except that it partially inactivates during relatively short depolarizations. Ensemble fluctuation analysis of K currents obtained from excised outside-out patches is consistent with a single type of K channel and yields estimates for the single channel conductance of approximately 13 pS, independently of membrane potential. A preliminary analysis of single channel data supports the conclusion that there is a single type of voltage-dependent, inactivating K channel in the GFL neurons.     

Surface Charge of Giant Axons of Squid and Lobster

A method is described for the determination of the electrophoretic mobility of single, isolated, intact, giant axons of squid and lobster. In normal physiological solutions, the surface of hydrodynamic shear of these axons is negatively charged. The lower limit of the estimated surface charge density is -1.9 × 10^-8 coul cm^-2 for squid axons, -4.2 × 10^-8 coul cm^-2 for lobster axons. The electrophoretic mobility of squid axons decreases greatly when the applied transaxial electric field is made sufficiently intense; action potential propagation is blocked irreversibly by transaxial electric fields of the same intensity. The squid axon recovers its mobility hours later and is then less affected by transaxial fields. Eventually, a state is reached in which the transaxial field irreversibly reverses the sign of the surface charge. In contrast, there is no obvious effect of electric field on the mobility of lobster axons. The mobility of lobster axons becomes undetectable in the presence of Th⁴⁺ at a concentration which blocks the action potential, and in the presence of La³⁺ at a concentration which does not affect propagation. Quinine does not alter lobster axon mobility at a concentration which blocks action potential conduction. Replacement of extracellular Na⁺ by K⁺ is without effect upon lobster axon mobility. The electrophysiological implications of the results are discussed.     

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.     

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.     

Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).     

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.     

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.     

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.