Potassium ion currents in the crayfish giant axon. Dynamic characteristics.

The kinetics of the voltage-sensitive potassium channel in crayfish axon have been examined. The conductance increase after a step depolarization from rest can be described by a first-order kinetic process raised to the third power. When conditioning voltage levels preceded the test pulse, the steady-state conductance was found to be independent of initial conditions. Depolarizing conditioning voltages in general allowed superposition of test voltage potassium currents by a shift along the time axis. Hyperpolarizing conditioning voltages produced a delay in onset of conductance during the test pulse and changed the kinetics so that superposition was not possible. The delay increased during the hyperpolarization with a first-order lag having a time constant in the range of 1.5-3 ms. Return to the resting level caused recovery from the delayed state to follow a single exponential decay with a time constant of 1.9-2.2 ms. The steady state delay vs. voltage curves were not saturated at potentials as negative as -180 mV.     

Activation-inactivation coupling in Myxicola giant axons injected with tetraethylammonium.

Myxicola giant axons internally injected with tetraethylammonium chloride to block potassium currents were examined under voltage clamp. The sodium inactivation time constants obtained from the decline in INa during step depolarizations were substantially smaller than those obtained using conditioning prepulses to the same potentials and the ratios agreed with previous observations using TTX. Inactivation shifts were also measured and found to be comparable to previous results.     

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.     

Depletion and accumulation of potassium in the extracellular clefts of cardiac Purkinje fibers during voltage clamp hyperpolarization and depolarization: experiments in sodium-free bathing media

Voltage clamp hyperpolarization and depolarization result in currents consistent with depletion and accumulation of potassium in the extracellular clefts o cardiac Purkinje fibers exposed to sodium-free solutions. Upon hyperpolarization, an inward current that decreased with time (id) was observed. The time course of tail currents could not be explained by a conductance exhibiting voltage-dependent kinetics. The effect of exposure to cesium, changes in bathing media potassium concentration and osmolarity, and the behavior of membrane potential after hyperpolarizing pulses are all consistent with depletion of potassium upon hyperpolarization. A declining outward current was observed upon depolarization. Increasing the bathing media potassium concentration reduced the magnitude of this current. After voltage clamp depolarizations, membrane potential transiently became more positive. These findings suggest that accumulation of potassium occurs upon depolarization. The results indicate that changes in ionic driving force may be easily and rapidly induced. Consequently, conclusions based on the assumption that driving force remains constant during the course of a voltage step may be in error.     

Transmission in the squid giant synapse: a model based on voltage clamp studies

1. Voltage clamp studies were performed in squid giant synapse after blockage of the voltage-dependent sodium and potassium conductances. 2. Presynaptic depolarization under these conditions demonstrates the presence of voltage-dependent calcium conductance change for the duration of the voltage step, and a tail current at the break of the pulse. 2. This calcium current triggers a postsynaptic response which can be measured directly at the postsynaptic fiber. 4. These voltage clamp experiments have allowed the development of a mathematical model that describes the kinetics of the calcium current and the relationship between calcium current and transmitter release.     

Membrane currents in a calcium type muscle membrane under voltage clamp.

Four ionic current components were identified in the total membrane current recorded under voltage clamp conditions from the muscle membrane of the crayfish (Astacus fluviatilis). The early inward current component is dependent on the presence of Ca2+ ions, disappears in Ca2+ free solutions and is insensitive to variaton of external Na+ ions and to tetrodotoxin. The outward current consists of at least three components, an early, a late and a slow outward current. The outward currents are sensitive to TEA and their reversal potentials differ. The early potassium current may be separated in a proportion of fibres by a hump from the later potassium current. An insufficient space clamp as a cause of the hump was excluded by comparing the size of the clamped membrane area with the distribution of large membrane clefts in the fibre. The early outward current is critically dependent on the presence of Ca2+ ions and is relatively more sensitive to TEA ions and to conditioning depolarisation than the late outward current.     

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.     

Selective modification of sodium channel gating in lobster axons by 2, 4, 6-trinitrophenol: Evidence for two inactivation mechanisms

Trinitrophernol (TNP) selectively alters the sodium conductance system of lobster giant axons as measured in current clamp and voltage clamp experiments using the double sucrose gap technique. TNP has no measurable effect on potassium currents but reversibly prolongs the time-course of sodium currents during maintained depolarizations over the full voltage range of observable currents. Action potential durations are increased also. Tm of the Hodgkin-Huxley model is not markedly altered during activation of the sodium conductance but is prolonged during removal of activation by repolarization, as observed in sodium tail experiments. The sodium inactivation versus voltage curve is shifted in the hyperpolarizing direction as is the inactivation time constant curve, measured with conditioning voltage steps. This shift speeds the kinetics of inactivation over part of the same voltage range in which sodium currents are prolonged, a contradiction incompatible with the Hodgkin-Huxley model. These results are interpreted as support for a hypothesis of two inactivation processes, one proceeding directly from the resting state and the other coupled to the active state of sodium conductance.     

Sodium-activated potassium current in mouse diaphragm

The mouse diaphragm muscle fiber was studied using the loose patch clamp technique. The voltage gated sodium currents were evoked by step pulses from a holding potential of about −70 mV. Following the activation of the sodium current, a very large and fast outward current was evoked. The sensitivity of this current to 4-aminopyridine and tetraethylammonium indicates the potassium ion as the possible carrier for the channel. Furthermore, the sensitivity to tetrodotoxin and extracellular sodium demonstrated the sodium dependence of this current.     

Conditioning hyperpolarization-induced delays in the potassium channels of myelinated nerve.

Hyperpolarizing conditioning pulses delay the onset of potassium channel current in voltage-clamped myelinated nerve fibers. Both the development of and recovery from this conditioning are approximately exponential functions of time: the time constants are functions of the conditioning voltage. The delay is larger and develops faster for more hyperpolarized conditioning pulses. The magnitude of the delay (but not the rate of development or recovery) depends upon the test potential-small test depolarizations produce larger delays than large depolarizations. The currents with and without the conditioning pulse cannot be made to superimpose by a simple time translation.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

Effects of conditioning polarization on the membrane ionic currents in rat myometrium

Summary Membrane ionic currents were measured in pregnant rat uterine smooth muscle under voltage clamp conditions by utilizing the double sucrose gap method, and the effects of conditioning pre-pulses on these currents were investigated. With depolarizing pulses, the early inward current was followed by a late outward current. Cobalt (1mm) abolished the inward current and did not affect the late outward currentper se, but produced changes in the current pattern, suggesting that the inward current overlaps with the initial part of the late outward current. After correction for this overlap, the inward current reached its maximum at about +10 mV and its reversal potential was estimated to be +62 mV. Tetraethylammonium (TEA) suppressed the outward currents and increased the apparent inward current. The increase in the inward current by TEA thus could be due to a suppression of the outward current. The reversal potential for the outward current was estimated to be –87 mV. Conditioning depolarization and hyperpolarization both produced a decrease in the inward current. Complete depolarization block occurred at a membrane potential of –20 mV. Conditioning hyperpolarization experiments in the presence of cobalt and/or TEA revealed that the decrease in the inward current caused by conditioning hyperpolarization was a result of an increase in the outward current overlapping with the inward current. It appears that a part of the potassium channel population is inactivated at the resting membrane potential and that this inactivation is removed by hyperpolarization.     

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.     

Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.     

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.     

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.     

Effects of physostigmine on the voltage dependent ionic conductances of skeletal muscle fibres

The voltage dependent ionic conductances were studied by analysing the phase plane trajectories of action potentials evoked by electrical stimulation of the sartorius muscles of the frog (Rana esculenta). The delayed outward potassium current was measured also under voltage clamp conditions on muscle fibres of either the frog (Rana esculenta) or Xenopus laevis. On analysing the effect of physostigmine decreasing the peak amplitude, the rate of both the rising and falling phases of the action potentials, it was revealed that the alkaloid at a concentration of 1 mmol/l reduced significantly both the delayed potassium conductance and the outward ionic current values during the action potentials. The inhibition of sodium conductance and inward ionic current was less expressed. The maximum value of delayed potassium conductance measured under voltage clamp conditions was decreased by 1 mmol/l physostigmine. The time constant determined from the development of delayed potassium conductance was increased at a given membrane potential. The voltage vs. n relationship describing the membrane potential dependence of the delayed rectifier was not influenced by physostigmine. It has been concluded that physostigmine changes the time course of the action potentials by decreasing the value of both voltage dependent ionic conductances and by slowing down their kinetics. It is discussed that results obtained from the phase plane analysis of complex pharmacological effects can only be accepted with some restrictions.     

Effect of conditioning potential on potassium current kinetics in the frog node.

The kinetics of potassium conductance changes were determined in the voltage clamped frog node (Rana esculenta), as a function of conditioning prepotential. The conditioning potential duration varied from 1 to 50 ms and the amplitude between -60 and +130 mV (relative to rest). The conductance kinetics were determined at a single test potential of +20 mV (depolarization) by means of the slope of log [ninfinity - nt] vs. time relationship which defines the time constant of the process (tau). The values of tau, after conditioning hyperpolarizations, were around 5 ms, up to 10 times greater than values obtained following a strong depolarization. The tau vs. pre-potential curve was sigmoid in shape. These differences were only slightly dependent on [K+]0 or conditioning pulse duration. The steady-state current values were also found to be a function of conditioning potential. After conditioning hyperpolarizations, the log [ninfinity - nt] vs. time curve could not be fitted by a single exponent regardless of the power of n chosen. The prepotential dependency of potassium current kinetics is inconsistent with the Hodgkin-Huxley axon model where the conductance parameters are assumed to be in either one of two possible states, and where the rate of transfer from one state to the other follows first order kinetics. In contrast the described kinetics may be consistent with complex multistate potassium "channel" models or membranes consisting of a number of types of channels.     

Increased voltage-gated potassium conductance during interleukin 2- stimulated proliferation of a mouse helper T lymphocyte clone

Recent work has demonstrated the presence of voltage-gated potassium channels in human peripheral blood T lymphocytes (Matteson, R., and C. Deutsch, 1984, Nature (Lond.), 307:468-471; DeCoursey T. E., T. G. Chandy, S. Gupta, and M. D. Cahalan, 1984, Nature (Lond.), 307:465-468) and a murine cytolytic T-cell clone (Fukushima, Y., S. Hagiwara, and M. Henkart, 1984, J. Physiol., 351:645-656). Using the whole cell patch clamp, we have found a potassium conductance with similar properties in a murine noncytolytic T lymphocyte clone, L2. Under voltage clamp, a step from a holding potential of -70 mV to +50 mV produces an average outward current of 100-150 pA in "quiescent" L2 cells at the end of their weekly maintenance cycle. When these cells are stimulated with human recombinant interleukin 2 (rIL2, 100 U/ml), they grow in size and initiate DNA synthesis at approximately 24 h. Potassium conductance is increased as early as 8 h after stimulation with rIL2 and rises to a level 3-4 times that of excipient controls by 24 h. The level remains elevated through 72 h, but as the cells begin to leave the cell cycle at 72-96 h, the conductance decreases quickly to a value only slightly higher than the initial one. Quinine, a blocker of this conductance, markedly reduces the rate at which L2 cells traverse the cell cycle, while also reducing the rate of stimulated protein synthesis. The regulation of potassium conductance in L2 cells during rIL2-stimulated proliferation suggests that potassium channel function may play a role in support of the proliferative response.     

Adaptation and accommodation in the squid axon

Current clamp data of the squid axon indicate that there is a qualitative change in the adaptive response as the magnitude of the current step is increased. Large stimulus currents have a strong inhibitory effect on spike generation and on active responses in general. Such currents always lead to only one action-potential and to the elimination of post-spike subthreshold oscillation. In view of a direct connection between stimulus current and potassium current I _K, the potassium channel of the Hodgkin-Huxley model is reinterpreted in a natural way such that the K⁺ conductance is directly dependent on I _K in addition to a voltage dependence. The I-_Kdependence seems to dominate whenever the stimulus current is greater than approximately 35 μA/cm². For current ramps, and large current steps, such a current formulation leads to good agreement with the data.