Transient polarization currents in the squid giant axon.

It has been repeatedly noted that the change of conformation of the molecules that serve as the ion-selective channels for sodium and potassium conductance in the nerve membrane will be accompanied by a change in the dipole moment of the molecule. This time-dependent change of dipole moment will produce transient currents in the membrane. The canonical form for these currents is determined with conventional statistical mechanics formalism. It is pointed out that the voltage dependence of the conductance channel conductance determines the free energy of the system to within a factor that is an unknown function of the voltage. Since the dipole currents do not depend on this unknown function, they are completely determined 0y the observed properties of the conductance system. The predicted properties of these dipole currents, their time constants and strengths, are calculated. By using the observed properties of gating currents, the density of the sodium channels is computed. The predicted properties of the dipole currents are found to compare satisfactorily with the observed properties of gating currents.     

Temperature effects on gating currents in the squid giant axon.

The effects of temperature (3 degrees-26 degrees C) on the nonlinear components of the displacement current were measured in internally perfused, voltage clamped squid axons. Steps of potential were applied from a holding potential of -70mV (outside ground) to values from -130 to +70mV and either the current or its integral (charge) was recorded as a function of time. For that component of the charge movement not linearly related to voltage, the total charge moved in a few milliseconds (about 1,500 electronic charges/micron2) between saturation limits (e.g. -100mV to +50mV) showed an apparent increase of 13 +/- 5% for a 10 degrees C rise in temperature. Attempts to fit the falling phase of the gating current (or charge) with the sum of two exponentials showed temperature effects on both components but there was considerable scattering. At short times, records for current or charge made at 16 degrees C, expanded by a factor alpha, superimposed on those made at 6 degrees C for alpha about 1.6. For long times alpha was about 2.3.     

Induced capacitance in the squid giant axon. Lipophilic ion displacement currents

Voltage-clamped squid giant axons, perfused internally and externally with solutions containing 10(-5) M dipicrylamine (DpA-), show very large polarization currents (greater than or equal to 1 mA/cm2) in response to voltage steps. The induced polarization currents are shown in the frequency domain as a very large voltage-and frequency-dependent capacitance that can be fit by single Debye-type relaxations. In the time domain, the decay phase of the induced currents can be fit by single exponentials. The induced polarization currents can also be observed in the presence of large sodium and potassium currents. The presence of the DpA- molecules does not affect the resting potential of the axons, but the action potentials appear graded, with a much-reduced rate of rise. The data in the time domain as well as the frequency domain can be explained by a single-barrier model where the DpA- molecules translocate for an equivalent fraction of the electric field of 0.63, and the forward and backward rate constants are equal at -15 mV. When the induced polarization currents described here are added to the total ionic current expression given by Hodgkin and Huxley (1952), numerical solutions of the membrane action potential reproduce qualitatively our experimental data. Numerical solutions of the propagated action potential predict that large changes in the speed of conduction are possible when polarization currents are induced in the axonal membrane. We speculate that either naturally occurring substances or drugs could alter the cable properties of cells in a similar manner.     

Pressure dependence of the potassium currents of squid giant axon

Summary The effect of pressure upon the delayed, K, voltage-clamp currents of giant axons from the squidLoligo vulgaris was studied in axons treated with 300nm TTX to block the early, Na, currents. The effect of TTX remained unaltered by pressure. The major change produced by pressures up to 62 MPa is a slowing down of the rising phase of the K currents by a time scaling factor which depends on pressure according to an apparent activation volume, V, of 31 cm³/mole at 15°C; V increased to about 42 cm³/mole at 5°C.Pressure slightly increased the magnitude, but did not produce any obvious major change in the voltage dependence, of the steady-state K conductance estimated from the current jump at the end of step depolarizations of small amplitude (to membrane potentials,E, 20 mV) and relatively short duration. At higher depolarizations, pressure produced a more substantial increase of the late membrane conductance, associated with an apparent enhancement of a slow component of the K conductance which could not be described within the framework of the Hodgkin-Huxley (HH)n ⁴ kinetic scheme.The apparent V values that characterize the pressure dependence of the early component of the K conductance are very close to those that describe the effect of pressure on Na activation kinetics, and it is conceivable that they are related to activation volumes involved in the isomerization of the normal K channels. The enhancement of the slow component of membrane conductance by pressure implies either a large increase in the conductance of the ionic channels that are responsible for it or a strong relative hastening of their turn-on kinetics.     

Pressure dependence of sodium gating currents in the squid giant axon

Asymmetric displacement currents, I_g, were measured in squid axons at different hydrostatic pressures, P, up to 60 MPa. Potassium and sodium currents were abolished by intracellular Cs⁺ and TEA⁺, by extracellular Tetrodotoxin (TTX), and by Na⁺ substitution with Tris⁺. The time course of Ig became progressively slower with increasing pressure, and the amplitude decreased. With appropriate scaling in time and amplitude, I_g records at any given P could be made to superimpose very well with those obtained at atmospheric pressure. The same scaling factors yielded a good superposition of all records obtained for voltage steps to membrane potentials in the range-30 to +42 mV. The ratio between the amplitude and time factors was larger than unity and increased with P, indicating a progressive decrease (up to 35% at 60 MPa) of the total charge displaced, Q, with no significant change in its voltage dependence. The time-scaling factor increased exponentially with P, as expected if all the steps involved in the opening of a sodium channel, and producing a major charge redistribution, have the same activation volume, V _g 17 cm³/mol. This value is roughly one-half of that characterizing the pressure dependence of sodium current activation, suggesting that some late, rate-limiting step in the opening of sodium channels has a large activation volume without being accompanied by an easily detected charge movement.Part of the decrease of Q with pressure could be attributed to an increase in sodium inactivation. However, we cannot exclude the possibility that there is a reversible reduction in the number of fast activating sodium channels, similar to the phenomenon that has been reported to occur at low temperatures (Matteson and Armstrong 1982).     

Effects of fatty acids on membrane currents in the squid giant axon

Summary The effects of fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular and extracellular application of the test substances. Fatty acids mainly suppress the Na current but have little effect on the K current. These effects are completely reversed after washing with control solution. The concentrations required to suppress the peak inward current by 50% and Hill number were determined for each fatty acid. ED₅₀ decreased about 1/3 for each increase of one carbon atom. The standard free energy was –3.05 kJ mole^–1 for CH₂. The Hill number was 1.58 for 2-decenoic acid. The suppression effect of the fatty acids depends on the number of carbon atoms in the compounds and their chemical structure. Suppression of the Na current was clearly observed when the number of carbon atoms exceeded eight. When fatty acids of the same chain length were compared, 2-decenoic acid had strong inhibitory activity, but sebacic acid had no effect at all on the Na channel. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (J. Physiol. (London) 117:500–544, 1952) and the changes in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ({ie113-1}) were shifted 20 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ({ie113-2}) was almost no change by application of the fatty acids. The time constant for activation ({ie113-3}) of K current was shifted 20 mV in a depolarizing direction by the application of the fatty acids.     

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.     

Presynaptic calcium currents in squid giant synapse.

A voltage clamp study has been performed in the presynaptic terminal of the squid stellate ganglion. After blockage of the voltage-dependent sodium and potassium conductances, an inward calcium current is demonstrated. Given a step-depolarization pulse, this voltage- and time-dependent conductance has an S-shaped onset. At the "break" of the voltage step, a rapid tail current is observed. From these results a kinetic model is generated which accounts for the experimental results and predicts for the time course and amplitude a possible calcium entry during presynaptic action potentials.     

Incomplete inactivation of sodium currents in nonperfused squid axon.

Perfused squid axons in which K-conductance is blocked show, under voltage clamp, incomplete inactivation of the sodium conductance. The presence of this phenomenon in nonperfused axons was found by comparing membrane current records before and after tetrodotoxin addition to the bathing solution. Sodium currents in nonperfused axons are comparable in behavior at positive potentials to those seen in Cs-perfused axons.     

Asymmetry currents in intracellularly perfused squid giant axons.

Asymmetry currents were recorded from intracellularly perfused squid axons subjected to exactly equal positive and negative voltage clamp pulses at a temperature close to 0 degrees C. The voltage and time dependence of the asymmetry currents was studied at a holding potential of minus 80 to minus 100 mV. The effect of varying the holding potential was investigated. The latter experiments showed that the voltage dependence of the asymmetrical charge movement is different from the voltage dependence of the m system.     

On the relation between displacement currents and activation of the sodium conductance in the squid giant axon.

The early time course of the current passing across the membrane in squid giant axons in which the ionic currents have been blocked reveals substantial asymmetries during and after the application of hyperpolarizing and depolarizing voltage-clamp pulses of identical size. Since the integral of the 'on' and 'off' current transients is zero, these currents must result from charge movements confined to the membrane and, therefore, they are nonlinear displacement currents. The steady state rearrangement of the charges as a consequence of sudden displacements of the membrane potential is consistent with a Boltzmann distribution of charges between two states characterized by different energy levels. Following changes in membrane potential the charges undergo a first order transition between these states. The relaxation time constant for the transition at a given temperature is a function of membrane potential. We propose that these displacement currents arise from a redistribution of the charges involved in the sodium gating system.     

Gating currents in th intact crayfish giant axon.

Both single-sweep and signal-averaged asymmetry current are measured from intact crayfish axons after ionic currents are blocked with tetrodotoxin and 4-aminopyridine. The ON asymmetry charge saturates at about 0 mV and no ON charge movement is detectable at voltages negative to -140 mV. The areas of ON and OFF asymmetry charge are equal for short depolarizations but the ratio QOFF/QON decreases for longer depolarizing pulses. Sodium and asymmetry current magnitudes can be changed in parallel by lowering the hold potential or by imposing conditioning prepulses. Our results are consistent with the concept that asymmetry current in generated by movement of trapped charge in association with Na channel gating.     

Modulation of aminopyridine block of potassium currents in squid axon.

Aminopyridines are known to block potassium (K) currents in excitable membranes in a manner dependent upon membrane potential, such that the block is relieved by depolarization and restored upon repolarization. In the present study, the effects of aminopyridines on voltage-dependent potassium (K) channels were examined in internally perfused, voltage-clamped squid giant axons. The time course of block restoration after conditioning depolarization was found to be modulated by membrane electric field, K-channel gating, and external cations. Depolarized holding potentials accelerated block restoration without altering steady-state block levels, suggesting that the voltage dependence of block restoration may be related to K channel gating rather than drug binding per se. In support of this notion, low external calcium concentration, which shifts the voltage dependence of K-channel gating to more negative potentials, also accelerated block restoration. Conversely, the relationship between the rate of block restoration and membrane holding potential was shifted in the depolarizing direction by phloretin, an agent that shifts the dependence of K-channel opening on membrane potential in a similar manner. Modification of K-channel gating also was found to alter the rate of block restoration. Addition of internal zinc or internal treatment with glutaraldehyde slowed the time course of both K-channel activation and aminopyridine block restoration. Aminopyridines also were found to interact in the K channel with external Cs+, NH4+, and Rb+, each of which slowed aminopyridine block restoration. Our results suggest that aminopyridines enter and occlude K channels, and that the availability of the binding site may be modulated by channel gating such that access is limited by the probability of the channel reaching an intermediate closed state at the resting potential.     

Electrical phenomena in nerve; squid giant axon

The action of a number of agents, which may be classified as "stabilizers" and "unstabilizers" on the electrical oscillations and after-potentials in the squid giant axon has been examined. The effects on the spike, "positive overshoot," and "potassium potential" were also observed, but where possible concentrations were employed which left these phenomena unaltered. Veratrine augmented the oscillations and the negative after-potential, particularly with repetitive stimulation. Yohimbine caused a small long lasting positive after-potential and depressed the oscillations, effects also enhanced with repetitive activity. Cocaine and procaine suppressed the oscillations and the negative after-potential but DDT was completely inert. An elevation in the medium calcium depressed the oscillations and the naturally occurring negative after-potential; negative after-potentials induced with veratrine were increased by calcium. A decrease in the potassium augmented the oscillations and the negative after-potential. A hypothesis is presented in which these effects are interpreted in terms of potassium concentration at the fiber surface as regulated by a labile permeability and metabolism. This is discussed in relation to the available evidence for these factors. It is a pleasure to acknowledge the author's indebtedness to Dr. D. E. S. Brown, Director, and to his staff at the Bermuda Biological Station for Research for the cooperation and special facilities provided during the initiation of this work. Dr. T. Baylor of Princeton University very kindly provided the camera and film used in Bermuda.     

Anion conductances of the giant axon of squid Sepioteuthis.

Anion conductances of giant axons of squid, Sepioteuthis, were measured. The axons were internally perfused with a 100-mM tetraethylammonium-phosphate solution and immersed in a 100-mM Ca-salt solution (or Mg-salt solution) containing 0.3 microns tetrodotoxin. The external anion composition was changed. The membrane currents had a large amount of outward rectification due to anion influx across Cl- channels of the membrane (Inoue, 1985). The amount of outward rectification depended on the species of anion used and was strongly influenced by temperature and internal pH. In contrast to the anion conductances themselves, the conductance relative to Cl- (gA/gCl) was found to be quite stable against changes in the membrane potential, temperature, and pH. It is therefore suggested that each gA/gCl is an intrinsic quantity of the Cl- channel of the squid axon membrane. The sequence and values of gA/gCl obtained in this study were NO3- (1.80) greater than I- (1.40) greater than Br- (1.07) greater than Cl- (1.00) greater than MeSO3- (0.46) greater than H2PO2- (0.33) greater than CH3COO- (0.29) greater than SO4(2-) (0.06).     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.