Potassium currents and conductance. Comparison between motor and sensory myelinated fibers.

The potassium conductance system of sensory and motor fibers from the frog Rana esculenta were studied and compared by means of the voltage clamp. The potassium ion accumulation was first estimated from the currents and reversal potentials within the framework of both a three-compartment model and diffusion-in-an-unstirred-layer model. The potassium conductance parameters were then computed using the measured currents and corrected ionic driving forces. It was found that the potassium accumulation is faster and more pronounced in sensory fibers, the voltage dependency of the potassium conductance is steeper in sensory fibers, the maximal potassium conductance, corrected for accumulation, is approximately 1.1 S/cm2 in sensory and 0.55 S/cm2 in motor fibers, and that the conductance time constants, tau n, are smaller in sensory than in motor fibers. These differences, which increase progressively with depolarization, are not detectable for depolarization of 50 mV or smaller. The interpretation of these findings in terms of different types of potassium channels as well as their implications with regard to the differences between the excitability phenomena in motor and sensory fibers are discussed.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. I. A study of the "cell-attached" configuration

A single channel current was studied in the membrane of the immature oocyte of the european frog (Rana esculenta) by using the "patch clamp" technique in the "cell attached" configuration. Single channel activity appeared as short outward currents when membrane potential was made positive inside; full activation required seconds to be complete, no inactivation being appreciable. Deactivation (or current block) upon membrane repolarization was so fast that no inward current could be detected in any case. The reversal potential, estimated by interpolating the I/V diagrams, was -30 mV using standard Ringer as electrode filling solution, and the elementary conductance was 95 pS. Neither reversal potential nor elementary conductance were affected by removal of external Ca2+ (Mg2+ or Ba2+ substitution) or external Cl- (methanesulphonate substitution). The reversal potential moved towards positive potentials by substituting external Na+ with K+, the magnitude of the shifts being consistent with a ratio PK/PNa = 6.4. A distinctive property of the current/voltage relation for this K-current is its anomalous bell-shape, the outward current displaying a maximum at membrane potentials around 75 mV with standard Ringer as electrode filling solution and tending to zero with more positive potentials.     

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue. Received: 5 May 1998 / Revised version: 27 October 1998 / Accepted: 19 January 1999     

Properties of membrane ionic currents in pituitary clone cells

Membrane ionic currents of the GH3 pituitary cell line have been studied using voltage clamp techniques. The inward current is completely blocked by cobalt (Co2+) ions and appeared to be carried by calcium ions. Three outward currents can be differentiated on the ground of kinetics and pharmacological studies: a transient current blocked by 4-aminopyridine (4 AP) and two delayed outward current which are voltage dependent. One is blocked by tetraethylammonium (TEA); the second is blocked by Co2+ and represents a calcium-activated potassium conductance.     

The ionic basis of oscillatory responses of skate electroreceptors

When physiological conditions are simulated, skate electroreceptors produce small maintained oscillatory currents. Larger damped oscillations of similar time-course are observed in voltage clamp. Subtraction of leakage in voltage clamp data shows that the oscillations involve no net outward current across the lumenal surface of the epithelium. The oscillations are much faster than the late outward current generated by the lumenal membranes of the receptor cells. Treatment of the basal surface of the epithelium with tetraethyl ammonium (TEA), high K, Co, or EGTA reversibly blocks the oscillations in voltage clamp, but has little or no effect on the epithelial action potential in current clamp or on the current-voltage relation. The TEA sensitivity of the oscillations indicates that they involve a potassium conductance in the basal membranes of the receptor cells. Treatment of the basal membranes with TEA and high calcium, with strontium, or with barium causes these membranes to produce large regenerative responses. Direct stimulation of the basal membranes then elicits a lumen-positive action potential whereas stimulation of the lumenal membranes elicits a diphasic action potential. Excitability of the basal membranes is abolished by extracellular Co, Mn, or La. Modulation of the lumenal membrane calcium conductance by the basal membrane conductances probably gives rise to the oscillatory receptor currents evoked by small voltage stimuli. The slower calcium-activated late conductance in the lumenal membranes may be involved in sensory accommodation.     

Plasmalemmal,voltage-dependent ionic currents from excitable pulvinar motor cells ofMimosa pudica

Summary Plasmalemmal ionic currents from excitable motor cells of the primary pulvinus ofMimosa pudica were investigated by patch-clamp techniques. In almost all of the enzymatically isolated protoplasts, a delayed rectifier potassium current was activated by depolarization, while no currents were detected upon hyperpolarization. This sustained outward current was reversibly blocked by Ba and TEA and serves to repolarize the membrane potential. Outward single channel currents that very likely underly the macroscopic outward potassium current had an elementary conductance of 20 pS. In addition, in a few protoplasts held at hyperpolarized potentials, depolarization-activated transient inward currents were observed, and under current clamp, action potential-like responses were triggered by depolarizing current injections or by mechanical perturbations. The activation characteristics of both inward currents and spikes showed striking similarities compared to those of action potentialsin situ.     

The effect of pentylenetetrazol on the metacerebral neuron of Helix pomatia

The effects of Pentylenetetrazol (PTZ) on the metacerebral giant cell (MCC) of the snail, Helix pomatia were studied. Actions on membrane resistance, time constant, resting and action potentials, outward and inward ionic currents were examined. Superfusion with PTZ in concentrations of 25 to 50 mmol/l, induced a gradually evolving convulsive state, which could be studied by intracellular recording from the MCCs. In the pre-convulsive state an acceleration of the spontaneous activity developed and was followed by paroxysmal depolarization shifts (PDSs), in the convulsive phase. PTZ prolonged the membrane time constant by about 10 percent, but this could not be traced back to alterations in membrane resistance or capacity. The resting membrane potential was not significantly altered; the action potentials were prolonged by slowing down of both the rising and decaying phases. The outward potassium currents were repressed by PTZ in a voltage dependent manner. The decrease of the IA current became more pronounced at increasingly positive command pulses, while IK was relieved from depression especially at longer pulse durations. Inward currents were isolated with the aid of suppression of outward currents by 50 mmol/l TEA. Under these conditions sodium currents, measured in calcium deficient Ringer solution were moderately depressed, while the calcium currents, examined during sodium-free superfusion, were mildly enhanced by PTZ. It is concluded that PTZ effects on ionic conductances, on membrane parameters, on the resting potential and ionic currents explain only modifications of spike potentials occurring in the convulsive state and do not account for the PDS, the central phenomenon of the convulsive electrographic activity, at least in this thoroughly examined type of neuron.     

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.     

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.     

Ionic currents in secondary sensory hair cells isolated from the statocysts of squid and cuttlefish

Mechanosensitive hair cells in the statocysts of cephalopods underlie a sophisticated detection system for linear and angular accelerations. To investigate the operation of this system, secondary sensory hair cells were dissociated from the sensory epithelia of these statocysts and their voltage sensitive ionic conductances identified and characterized under whole cell voltage clamp.All secondary hair cells showed two outward potassium conductances; first, a current similar to the previously described delayed rectifier, I_K and second, a current similar to the molluscan A current, I_A. A small number of hair cells (15%) also showed an inward sodium current; the presence of this current was correlated with the presence of small membrane extensions at the base of the cell. The sodium current could be blocked by TTX and was abolished by substituting choline for sodium in the external medium. An inward L-type, calcium current was also identified. This current showed rapid activation, with little inactivation, could be carried by barium ions, and was blocked by Nifedipine in the external solution.These data provide the first information on the ionic conductances in the basolateral membranes of invertebrate secondary sensory hair cells and form a basis for comparison with analogous vertebrate hair cells.     

Role of calcium and protein kinase C in development of the delayed rectifier potassium current in Xenopus spinal neurons.

The delayed rectifier current of embryonic Xenopus spinal neurons plays the central role in developmental conversion of calcium-dependent action potentials to sodium-dependent spikes. During its maturation, this potassium current undergoes a pronounced increase in rate of activation. The mechanism underlying the change in kinetics was analyzed with whole-cell voltage clamp of neurons cultured under various conditions. Calcium is necessary at an early stage of development, to permit influx that triggers subsequent release of calcium from intracellular stores. Its action is prevented by depletion of protein kinase C and mimicked by stimulation of the kinase. Calcium influx through voltage-dependent channels at early stages of development regulates the differentiation of potassium current kinetics and modulation of the ionic dependence of action potentials.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

On the voltage-dependent action of tetrodotoxin.

The use of the maximum rate-of-rise of the action potential (Vmax) as a measure of the sodium conductance in excitable membranes is invalid. In the case of membrane action potentials, Vmax depends on the total ionic current across the membrane; drugs or conditions that alter the potassium or leak conductances will also affect Vmax. Likewise, long-term depolarization of the membrane lessens the fraction of total ionic current that passes through the sodium channels by increasing potassium conductance and inactivating the sodium conductance, and thereby reduces the effect of Vmax of drugs that specifically block sodium channels. The resultant artifact, an apparent voltage-dependent potency of such drugs, is theoretically simulated for the effects of tetrodotoxin on the Hodgkin-Huxley squid axon.     

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.     

4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers

We have studied the effects of the potassium-blocking agent 4-aminopyridine (4-AP) on the action potential and membrane currents of the sheep cardiac Purkinje fiber. 4-AP slowed the rate of phase 1 repolarization and shifted the plateau of the action potential to less negative potentials. In the presence of 4-AP, the substitution of sodium methylsulfate or methanesulfonate for the NaCl of Tyrode's solution further slowed the rate of phase 1 repolarization, even though chloride replacement has no effect on the untreated preparation. In voltage clamp experiments, 4-AP rapidly and reversibly reduced the early peak of outward current that is seen when the Purkinje fiber membrane is voltage-clamped to potentials positive to -20 mV. In addition, 4-AP reduced the steady outward current seen at the end of clamp steps positive to -40 mV. 4-AP did not appear to change the slow inward current observed over the range of -60 to -40 mV, nor did it greatly change the current tails that have been used as a measure of the slow inward conductance at more positive potentials. 4-AP did not block the inward rectifying potassium currents, IK1 and IK2. A phasic outward current component that was insensitive to 4-AP was reduced by chloride replacement. We conclude that the early outward current has two components: a chloride-sensitive component plus a 4-AP-sensitive component. Since a portion of the steady-state current was sensitive to 4-AP, the early outward current either does not fully inactivate or 4-AP blocks a component of time-independent background current.     

Potassium ion accumulation in a periaxonal space and its effect on the measurement of membrane potassium ion conductance

Summary Potassium currents of various durations were obtained from squid giant axons voltage-clamped in artificial seawater solutions containing sufficient tetrodotoxin to block the sodium conductance completely. From instantaneous potassium current-voltage relations, the reversal potentials immediately at the end of these currents were determined. On the basis of these reversal potential measurements, the potassium ion concentration gradient across the membrane was shown to decrease as the potassium current duration increased. The kinetics of this change was shown to vary monotonically with the potassium ion efflux across the membrane estimated from the integral over time of the potassium current divided by the Faraday, and to be independent of both the external sodium ion concentration and the presence or absence of membrane series resistance compensation. It was assumed that during outward potassium current flow, potassium ions accumulated in a periaxonal space bounded by the membrane and an external diffusion barrier. A model system was used to describe this accumulation as a continuous function of the membrane currents. On this basis, the mean periaxonal space thickness and the permeability of the external barrier to K⁺ were found to be 357 Å and 3.21×10^–4 cm/sec, respectively. In hyperosmotic seawater, the value of the space thickness increased significantly even though the potassium currents were not changed significantly. Values of the resistance in series with the membrane were calculated from the values of the permeability of the external barrier and these values were shown to be roughly equivalent to series resistance values determined by current clamp measurements. Membrane potassium ion conductances were determined as a function of time and voltage. When these were determined from data corrected for the potassium current reversal potential changes, larger maximal potassium conductances were obtained than were obtained using a constant reversal potential. In addition, the potassium conductance turn-on with time at a variety of membrane potentials was shown to be slower when potassium conductance values were obtained using a variable reversal potential than when using a constant reversal potential.     

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.     

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.     

Two types of outward K+ channel currents in early embryonic chick ventricular myocytes

Outward K+ currents were recorded from 3-day-old embryonic chick ventricular myocytes using the patch clamp method. Two types of macroscopic outward currents were observed, one with rapid activation and de-activation time courses, and the other displaying a slower activation and long-duration tail currents. A time-dependent inactivation at positive potentials was a feature of the rapidly-activating current, allowing resolution of an early outward current. Single K+ channel currents were recorded using the outside-out patch technique. Two classes of K+ channels, which may contribute to the macroscopic currents, were differentiated on the basis of their conductances and kinetics. One class (ca 20 pS conductance) showed a rapid activation upon depolarization, and the other class (ca 60 pS) had a more delayed activation. A time-dependent inactivation of the rapid-activating, single-channel K+ current was also recorded. The two types of K+ channels contribute outward current during the plateau and promote the repolarization of the action potential, and the slowly de-activating K+ current may also be involved in the electrogenesis of automaticity observed in some of these cells.