Block Of Potassium Current Research Articles

ANG II blocks potassium currents in zona glomerulosa cells from rat, bovine, and human adrenals

Angiotensin II (ANG II) is a principal secretagogue of adrenal zona glomerulosa (ZG) cells. The transduction process includes a depolarization of the plasma membrane and the activation of calcium influx. The ANG II-induced depolarization is associated with an increase in total membrane resistance. To directly address the mechanism underlying these observations, we examined the effect of ANG II on K+ currents of rat, bovine, and human ZG cells, using whole cell patch clamp. Although some differences were seen in the characteristics of K+ currents between species, ANG II consistently blocked outward currents in ZG cells [rat: 47.1 +/- 4.5% (SE), n = 17; bovine: 38.6 +/- 3.3%, n = 21; and human: 13-63%, n = 3]. With the use of the cell-attached mode, single-channel recordings in bovine ZG cells demonstrated K+ channels that were reversibly blocked when ANG II was added to the bath solution. This indicates that the block of K+ channels by ANG II involves a diffusible intracellular messenger rather than a direct receptor-channel interaction. The decreased conductance of K+ can account for the ANG II-induced membrane depolarization.

On the active form of 4‐aminopyridine: block of K+ currents in rabbit Schwann cells.

1. The blocking action of 4-aminopyridine (4-AP) on the outward potassium currents evoked by depolarization of rabbit Schwann cells in short-term primary culture was studied with the whole-cell configuration of the patch-clamp method. 2. We have determined the apparent equilibrium dissociation constant, K, for the action of 4-AP to block potassium currents at a series of different extracellular and intracellular pH values. 4-Aminopyridine is an organic base and exists in both charged and uncharged forms in aqueous solution. Changes in the pH of the extracellular and intracellular solutions therefore also change the extracellular and intracellular proportions of these two forms, and the values of K that were obtained were found to depend in a consistent way on both the extracellular and the intracellular pH. 3. At alkaline extracellular pH, K was decreased. At acidic extracellular pH, K was increased. In contrast, increasing the intracellular pH from 7.2 to 8.1 reduced the apparent potency of extracellularly applied 4-AP (i.e. increased K), and decreasing the intracellular pH (to 6.4) increased this apparent potency (i.e. decreased K). 4. The 4-AP analogues, 2-aminopyridine, 3-aminopyridine and 3,4-diaminopyridine, were also tested. At half-block of the potassium current, the intracellular concentration of the cationic form of the various aminopyridines (applied extracellularly at pH 7.2) varied by a factor of less than five, whereas that of the uncharged form varied by a factor of over 700. 5. The results are inconsistent with the hypothesis that the cationic form of the aminopyridines, acting from the extracellular solution, contributes in any substantial way to potassium channel block. It also seems unlikely that the uncharged form, acting either extracellularly or intracellularly, is solely responsible for the block. However, the results as a whole are consistent with the idea that it is the cation acting from the intracellular side that blocks the 4-AP-sensitive potassium channel and that the affinity with which 4-AP blocks the channel depends on the intracellular pH. The results would be explained if the cation competes with protons for a binding site that has an apparent pKa of about 7.0. 6. The results, nevertheless, are not inconsistent with the possibility that both the uncharged form and the intracellular charged form of 4-AP are active in blocking the 4-AP-sensitive potassium channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic stimulation activates a non‐selective cation current in canine pyloric circular muscle cells.

1. Cholinergic stimulation of circular muscle from the canine pyloric sphincter results in excitatory junction potentials and an increase in slow-wave frequency. Experiments were performed on isolated pyloric muscle cells to determine the effects of acetylcholine on membrane conductance and voltage-dependent ionic currents. 2. Acetylcholine depolarized circular muscle cells and increased membrane conductance. Under voltage clamp, these effects were associated with the development of an inward current. 3. The ACh-dependent current (IACh) reversed at about -20 mV and was about equally selective for potassium and sodium. Changes in the chloride gradient had no effect on the reversal potential of IACh. 4. The response to ACh was blocked by atropine suggesting that the response was mediated by muscarinic receptors. IACh could not be elicited in the presence of ions normally used to block potassium currents (e.g. bath-applied TEA+ and replacement of Ki+ with Csi+. 5. In some cells single-channel openings could be resolved in response to ACh. These channels had a slope conductance of 30 pS, and open probability increased with depolarization. 6. Acetylcholine had little or no effect on voltage-dependent Ca2+ currents, and increased voltage-dependent outward currents. The latter effect may have been due to increased release of Ca2+ from internal stores. 7. The non-selective cationic current elicited by ACh can explain the excitatory junction potentials in pyloric muscle cells that are generated by transmural nerve stimulation and may also explain the chronotropic effects of ACh on slow waves.

Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers.

Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are compatible with the idea that after depolarization charge 1 is transformed into charge 2. D-600 appears to favor the conversion of charge 1 into charge 2. Since D-600 also favors contractile inactivation, charge 2 could represent the state of the voltage sensor for excitation-contraction coupling in the inactivated state.

Evaluation of antiarrhythmic drugs on defibrillation energy requirements in dogs. Sodium channel block and action potential prolongation.

Antiarrhythmic drugs have been reported to produce variable effects on defibrillation energy requirements. However, the relation between the in vitro electrophysiologic effects of these agents and the changes in defibrillation energy requirements have not been systematically examined. Therefore, we evaluated the effects of the sodium channel blocking drugs lidocaine and procainamide, the action potential prolonging drugs N-acetyl procainamide and clofilium, and the potassium current blocker cesium in acute canine models with the same internal spring and epicardial patch electrodes used in humans for ventricular defibrillation testing. Ten series of experiments were performed in 78 dogs. Nonlinear regression was used to derive curves of energy dose versus percent successful defibrillation attempts and the 50% and 90% effective energy dose for each experimental condition. Saline control experiments indicated that the preparation was stable throughout the 6-hour duration of the experiments. Lidocaine doubled the defibrillation energy requirement (p less than 0.001) at a mean plasma concentration of 8.2 micrograms/ml. The effect of lidocaine on defibrillation energy was reversible, present at therapeutic plasma concentrations, linearly related to plasma concentration (r = 0.69, p less than 0.002), and present even after only 5-second episodes of ventricular fibrillation. In contrast, procainamide had no effect on defibrillation energy at mean plasma concentrations of 8.5 and 13 micrograms/ml, even after prolonged (30-second) episodes of ventricular fibrillation, whereas N-acetyl procainamide, clofilium, and cesium all decreased the energy requirement for defibrillation by 13-27%. Moreover, with the addition of N-acetyl procainamide, there was a trend toward diminishing the increase in defibrillation energy requirement caused by lidocaine. All agents prolonged the mean ventricular fibrillation cycle length. Lidocaine shortened the QT interval, whereas all other agents increased the QT (p less than 0.05). The major electrophysiologic effect of lidocaine is of sodium channel blockade, whereas, N-acetyl procainamide, clofilium, and cesium predominantly increase the action potential duration, and procainamide exerts both effects. Thus, these data indicate that sodium channel block and action potential prolongation exert significant and antagonistic modulating effects on defibrillation energy requirements.

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in nerve fibers

We have examined the effects of venom from the scorpion Pandinus imperator on the membrane currents of voltage-clamped frog myelinated nerve fibers using the Vaseline-gap method. Crude venom, applied externally in concentrations from 50 to 500 micrograms/ml, selectively blocked the voltage-gated potassium currents without affecting nodal sodium currents or resting conductances. Block of potassium channels by Pandinus venom was highly dependent on the membrane voltage, being greater at negative potentials than at positive potentials. The blocking effects of Pandinus venom were irreversible on the time scale of our experiments; however, even high concentrations of venom failed to block potassium currents completely at positive potentials. These results suggest that Pandinus venom contains a component(s) that interacts specifically and strongly with a subpopulation of axonal potassium channels.

Histamine decreases calcium-mediated potassium current in guinea pig hippocampal CA1 pyramidal cells.

The action of histamine on CA1 pyramidal cells was studied in a hippocampal slice preparation. In the presence of tetrodotoxin (TTX) and tetraethylammonium (TEA), histamine had little effect on the calcium spikes. Using the single-electrode voltage-clamp technique, the actions of histamine on membrane currents were tested. In TTX, histamine (1 microM) decreased outward current only at potentials more depolarized than approximately -50 mV, where calcium-mediated potassium current is predominant. In the presence of manganese, histamine was without effect. Histamine (10 microM) did not affect the transient outward potassium current (A-current), the inward M-current resulting from small hyperpolarizing steps, or the inward Q-current elicited by larger hyperpolarizing steps. Blocking potassium currents with TEA or replacing calcium with barium revealed a slow inward current normally carried by calcium. With TTX present to block sodium currents, histamine (10 microM) did not reduce the inward current. The outward current reduced by a maximally effective concentration of histamine (10 microM) can be further decreased by manganese. The results support the conclusion that histamine selectively decreases the calcium-mediated potassium conductance in CA1 pyramidal cells of hippocampus. The possibility is raised that there is a component of calcium-mediated potassium current that is insensitive to histamine.

Effects of dimethylsulfoxide on membrane currents of neuroblastoma × glioma hybrid cell

Giga-ohm seal whole cell recording technique was used to examine ionic currents changes induced by dimethylsulfoxide (DMSO) in neuroblastoma × glioma hybrid NG 108-15 cells. DMSO (0.5–1%) reversible blocks sodium, potassium and calcium currents and shifts by about 6 mV the sodium inactivation curve towards more negative voltages.

Catecholamine inhibition of calcium action potentials in rat locus coeruleus neurones

Intracellular recordings were made from neurones in the nucleus locus coeruleus in a slice of tissue cut from the rat pons. Clonidine (100nM–10μM), noradrenaline (10μM–1 mM) and adrenaline (10μM–1 mM) all reduced the duration of the spontaneously occurring action potential of the neurones. This effect was also observed on the action potential in the presence of tetrodotoxin, which results from calcium entering the cell. These concentrations of clonidine, noradrenaline and adrenaline always hyperpolarized the membrane. This hyperpolarization was prevented by two procedures which block potassium currents—intracellular caesium and extracellular barium. In conditions of potassium current blockade, noradrenaline (100 μM–1 mM) and adrenaline (20μM–1 mM) shortened the calcium action potential but clonidine was ineffective even at 10 μM. Adrenaline and noradrenaline also suppressed inward calcium and barium currents measured under voltage clamp. This action of noradrenaline and adrenaline was not prevented by yohimbine (10 μM), propranolol (20 μM) or prazosin (1 μM); it was reduced by a concentration of phentolamine about 100 times higher than its K e for α 2-adrenoceptors on locus coeruleus neurones. It is concluded that noradrenaline and adrenaline can directly inhibit calcium action potentials in locus coeruleus neurones when applied in high concentrations, but that this does not involve an α 2-adrenoceptor.

Role of presynaptic calcium ions and channels in synaptic facilitation and depression at the squid giant synapse.

1. The roles of presynaptic calcium influx and calcium accumulation in synaptic facilitation and depression were explored at the giant synapse in the stellate ganglion of the squid. 2. Calcium currents were recorded in the presynaptic terminal, using a three-electrode voltage clamp and blocking sodium and potassium currents pharmacologically. The calcium influx was constant during pairs or trains of brief depolarizing pulses that elicited facilitating or depressing excitatory post-synaptic potentials (e.p.s.p.s). 3. The relationship between calcium influx and transmitter release during brief depolarizing pulses of varying amplitude resembled a power function with exponent of about 2. 4. Presynaptic calcium concentration transients were measured by injecting the dye arsenazo III and detecting absorbance changes microspectrophotometrically. Increments in intracellular free calcium accompanying single action potentials appeared constant for repeated action potentials that elicited facilitating e.p.s.p.s. 5. The presynaptic calcium concentration remains elevated for several seconds following action potentials. 6. Presynaptic injection of calcium ions by interbarrel ionophoresis evokes a postsynaptic depolarization, apparently reflecting a large increase in miniature e.p.s.p. frequency. Presynaptic action potentials remain unaffected by this treatment, but e.p.s.p.s triggered by them are facilitated for several seconds, and then depressed. 7. The results are consistent with the hypothesis that synaptic facilitation is due to the action of residual calcium or a calcium complex remaining in the presynaptic terminal after electrical activity. The late depression of release during calcium injection may be a result of the continual release of transmitter and consequent depletion of a presynaptic store.

Reconstruction of sino-atrial node pacemaker potential based on the voltage clamp experiments.

The pacemaker activity of the S-A node cell was explained by reconstructing the pacemaker potential using a Hodgkin-Huxley type mathematical model which was based on the reported voltage clamp data. In this model four dynamic currents, the sodium current iNa, the slow inward current, is, the potassium current, iK, and the hyperpolarization-activated current, ih, in addition to a time-dependent leak current, i1 were included. The model simulated the spontaneous action potential the current voltage relationship, and the voltage clamp experiment in normal Tyrode solution of the rabbit S-A node. Furthermore, the changes of activity induced by the potassium current blocker Ba2+, by applying constant current, acetylcholine, and epinephrine were also reconstructed. It was strongly suggested that the pacemaker depolarization in the S-A node cell is mainly due to a gradual increase of iS during diastole, and that the contribution of iK is much less compared to the potassium current iK2 in the Purkinje fiber pacemaker depolarization. The rising phase of the action potential was due to iS and the plateau phase is determined by both the inactivation of iS and activation of iK.

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.

Inward calcium current in twitch muscle fibres of the frog.

1. Voltage clamp experiments using the three micro-electrode voltage clamp technique were performed on sartorius muscles of the frog. 2. By blocking potassium currents with tetraethylammonium and replacing chloride ions with sulphate a slow inward current was detected. 3. The slow inward current is mainly carried by calcium, since it is abolished by cobalt and D-600, it depends on external calcium, and is not affected by removing external sodium or by tetrodotoxin (TTX). 4. The slow inward current has a mean threshold of -40 mV, reaches a mean maximum value at ca. 0 mV of 81 microamperemetercm-2 and has a mean reversal potential of +38 mV. 5. The calcium current is inactivated by the application of 2 sec conditioning prepulses according to a sigmoid curve with V(h) = -42 mV and k = 6.2 mV. 6. The slow time course of this calcium current makes it rather unlikely that it participates in contraction during a twitch, but it might be activated during long depolarizations as potassium contractures.

Voltage-clamp analysis of the sodium and potassium currents in skeletal muscle fibres treated with 4-aminopyridine

External application of low concentrations of 4-aminopyridine blocks potassium currents without affecting sodium currents in pieces of single frog skeletal muscle fibres. The blockade of potassium currents was voltage-dependent, being partially relieved on depolarization.

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.