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).     

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.     

Calcium currents in squid giant axon.

Voltage-clamp experiments were carried out on intracellularly perfused squid giant axons in a Na-free solution of 100 mM CaCl2+sucrose. The internal solution was 25 mM CsF+sucrose or 100 mM RbF+50mM tetraethylammonium chloride+sucrose. Depolarizing voltage clamp steps produced small inward currents; at large depolarizations the inward current reversed into an outward current. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward current was seen with 100 mM MgCl2+sucrose as internal solution. It is concluded that the inward current is carried by Ca ions moving through the sodium channel. The reversal potential of the tetrodotoxin-sensitive current was +54mV with 25 mM CsF+sucrose inside and +10 mV with 100 mM RbF+50 mM tetraethylammonium chloride+sucrose inside. From the reversal potentials measured with varying external and internal solutions the relative permeabilities of the sodium channel for Ca, Cs and Na were calculated by means of the constant field equations. The results of the voltage-clamp experiments are compared with measurements of the Ca entry in intact axons.     

Interaction of internal anions with potassium channels of the squid giant axon

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.     

The influence of external potassium on the inactivation of sodium currents in the giant axon of the squid, Loligo pealei

Isolated giant axons were voltage-clamped in seawater solutions having constant sodium concentrations of 230 mM and variable potassium concentrations of from zero to 210 mM. The inactivation of the initial transient membrane current normally carried by Na⁺ was studied by measuring the Hodgkin-Huxley h parameter as a function of time. It was found that h reaches a steady-state value within 30 msec in all solutions. The values of h _∞, τ_h, α_h,and β_h as functions of membrane potential were determined for various [K _o]. The steady-state values of the h parameter were found to be inversely related, while the time constant, τ_h, was directly related to external K⁺ concentration. While the absolute magnitude as well as the slopes of the h _∞ vs. membrane potential curves were altered by varying external K⁺, only the magnitude and not the shape of the corresponding τ_h curves was altered. Values of the two rate constants, α_h and β_h, were calculated from h_∞ and τ_h values. α_h is inversely related to [K_o] while β_h is directly related to [K_o] for hyperpolarizing membrane potentials and is independent of [K_o] for depolarizing membrane potentials. Hodgkin-Huxley equations relating α_h and β_h to E_m were rewritten so as to account for the observed effects of [K_o]. It is concluded that external potassium ions have an inactivating effect on the initial transient membrane conductance which cannot be explained solely on the basis of potassium membrane depolarization.     

The effect of sodium and potassium ions on the impedance change accompanying the spike in the squid giant axon

Decrease of the sodium concentration of the medium depresses both the spike and the associated impedance change in almost identical fashion. Elevation of the potassium level also depresses both phenomena, but affects the impedance change more than the spike; it slows the return to the initial impedance level. The effects on the threshold to brief square waves are also described. These results appear largely accounted for by the observations of Hodgkin and Huxley with the voltage clamp technique and by their recent hypothesis as to nature of the spike processes.     

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.     

Dynamics of potassium ion currents in squid axon membrane. A re-examination.

The original experiments of Cole and Moore (1960. Biophys. J. 1:161-202.), using conditioning and test membrane potentials to examine the dynamics of the potassium channel conductance in the squid axon, have been extended to test voltage levels by the use of tetrodotoxin to block the sodium conductance. The potassium currents for test voltage levels from -20 to +85 mV were superposable by translation along the time axis for all conditions tested: (a) with depolarizing conditioning voltages; (b) with hyperpolarizing conditioning voltages; and (c) in normal and in high potassium external media. The only deviations from superposition seen were when the internal sodium concentration was abnormally high and the potassium currents showed saturation at high levels of depolarization. Some restoration toward normal kinetics could be obtained by rapidly repeated depolarizations.     

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.     

Kinetics of ion movement in the squid giant axon

The loss of Na²², K⁴², and Cl³⁶ from single giant axons of the squid, Loligo pealii, following exposure to an artificial sea water containing these radioisotopes, occurs in two stages, an initial rapid one followed by an exponential decline. The time constants of the latter stage for the 3 ion species are, respectively, 290, 200, and 175 minutes. The outflux of sodium is depressed while that of potassium is accelerated in the absence of oxygen; the emergence of potassium is slowed by cocaine, while that of sodium is unaffected. One cm. ends of the axons take up about twice as much radiosodium as the central segment; this difference in activity is largely preserved during exposure to inactive solution. Such marked differences are not observed with radiopotassium. From the experimental data estimates are given of the influxes and outfluxes of the individual ions. The kinetics of outflux suggests a cortical layer of measureable thickness which contains the ions in different proportions from those in the medium and which governs the rate of emergence of these ions from the axon as though it contained very few but large (relative to ion dimensions) pores.     

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.     

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.     

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.     

The effect of tetrodotoxin on the sodium gating current in the squid giant axon.

The effect of tetrodotoxin (TTX) on the sodium gating current in the squid giant axon was examined by recording the current that flowed at the pulse potential at which the ionic current fell to zero, first in the absence and then in the presence of TTX. The addition of 1 microM TTX to the bathing solution had no consistent effect on the size of the initial peak of the gating current, but resulted in small changes in the timecourse of its subsequent relaxation which were mainly caused by a reduction of about one quarter in the component that has a delayed onset and may possibly arise from changes in the state of ionization of groups in the channel wall when the lumen fills with water. Our findings suggest that the binding of TTX at the outer face of the sodium channel does not interfere with the mechanisms of activation and inactivation by the voltage sensors, but has an allosteric effect on the access of internal cations to the inside of the channel.     

The effect of temperature,potassium, and sodium on the conductance change accompanying the action potential in the squid giant axon

Conductance changes associated with the response of the squid giant axon have been studied at two temperature ranges (26–27°C.; 9–10°C.) and with modified concentrations of sodium and potassium in the medium. The phase of "initial after-conductance," during which the membrane resistance increases above the resting value, is smaller at the lower temperature. At both temperature ranges it is diminished by doubling K⁺ in the medium and enhanced by removal of K⁺. Halving the Na⁺ of the medium also enhances this phase when K⁺ is absent, but not otherwise. The time course of the conductance changes alters in form with changes of the external medium. These changes indicate independent changes in the complex of ionic events associated with the response. The experiments therefore confirm the reality of the phase of increased membrane resistance. The magnitude of this change appears to be considerable and requires a transient decrease in the mobility and/or concentration of ions in the membrane. The possible cause of this decrease is discussed.