Time-dependent Outward Current Research Articles

Effects of enprofylline, theophylline and terbutaline on second inward currents in papillary muscles from ferrets and guinea-pigs.

In ferret and guinea pig papillary muscles enprofylline (10 microM-10 mM) and theophylline (0.1-2 mM) alone or in combination with terbutaline (0.05 microM-0.1 microM) decreased the action potential duration and increased the plateau height, increased the peak force of contraction and facilitated the depolarization-induced automaticity. In voltage clamp, the xanthines alone or in combination with terbutaline increased second inward currents, ICa,f and ICa,2, but had relatively less effect on the time-dependent outward current. No qualitative differences between enprofylline and theophylline could be detected but the former was about 5 times more potent in increasing ICa,f. In clinically relevant concentrations, enprofylline and theophylline alone, or in combination with terbutaline caused a small (2-5%) shortening of the action potential.

Effects of goniopora toxin on the action potential and membrane currents of guinea-pig single ventricular cells

The effects of Goniopora toxin (GPT), a polypeptide isolated from a coral, Goniopora spp., on action potential and membrane currents were studied in single ventricular cells of the guinea-pig using the whole-cell clamp technique with a single patch electrode. GPT at a concentration of 10 nmol/l prolonged the duration of the action potential without significant change in the resting membrane potential and action potential amplitudes. This prolongation became more evident at lower stimulus frequencies and persisted after washing with toxin-free solution. Tetrodotoxin (TTX, 1 mumol/l), but not Co2+ (2 mmol/l), abolished the prolonged action potential. Under voltage-clamp conditions, a sustained inward current, not present in the control, followed the transient inward current during depolarizing pulses in the GPT-treated cells. The current-voltage relationship for the sustained inward current was much the same as that for the fast sodium current reported in rat single ventricular cells (Brown et al. 1981). Both the sustained and transient currents were abolished by the shift of holding potential in the direction of depolarization and reappeared after repolarization; the reappearance of the sustained current was much slower than that of the transient current. TTX but not Co2+ abolished both the sustained and transient inward currents. Calcium current and time-independent current were not affected by GPT. Time-dependent outward current induced by large depolarizing pulses was attenuated by GPT.(ABSTRACT TRUNCATED AT 250 WORDS)

A time-dependent and voltage-sensitive K+ current in single cells from frog atrium.

A quantitative description of the time-dependent and voltage-sensitive outward currents in heart has been hampered by the complications inherent to the multicellular preparations previously used. We have used the whole-cell patch-clamp technique to record the delayed outward K+ current, IK, in single cells dissociated from frog atrium. Na+ currents were blocked with tetrodotoxin and Ca2+ currents with Mn2+ or Cd2+. After depolarizations from -50 mV to potentials positive to -30 mV, a time-dependent outward current was observed. This current has been characterized according to its steady state activation, kinetics, and ion transfer function. The current is well described as a single Hodgkin-Huxley conductance. The deactivation of the current is a single exponential. Activation of the current is sigmoid and is fitted well by raising the activation variable to the second power. The reversal potential of IK is near EK and shifts by 57 mV/10-fold change in [K+]o. This suggests that the current is carried selectively by K ions. The threshold for activation is near -30 mV. IK is maximally activated positive to +20 mV and shows no inactivation. The fully activated current-voltage relationship is linear between -110 and +50 mV. Neither Ba2+ (250 microM) nor Cd2+ (100 microM) affects IK.

Repolarization currents in embryonic chick atrial heart cell aggregates

Outward membrane currents in aggregates of atrial cells prepared from 7-12-d-old chick embryonic hearts were measured with the two microelectrode voltage-clamp technique. Two outward current components, Ix1 and Ix2, were found in the plateau potential range of the action potential. The Ix1 component is activated between -50 and -20 mV; the Ix2 component is activated between -15 and +20 mV. The Ix1 component inwardly rectifies, whereas Ix2 has an approximately linear current-voltage relation. These preparations lack a time-dependent pacemaker current component, even though they beat spontaneously with an interbeat interval of approximately 1 s. A mathematical model of electrical activity is described based on our measurements of time-dependent outward current, and measurements in the literature of inward current components.

Ca2+-channel current and its modification by the dihydropyridine agonist BAY k 8644 in isolated smooth muscle cells

The electrophysiological properties of single smooth muscle cells isolated from the longitudinal layer of the guinea-pig ileum were studied with the whole-cell patch-clamp technique. The finding of resting potentials between -45 and -50 mV and the occurrence of spontaneous electrical activity when K+ was the predominant intracellular cation indicated that the cells were not leaky or hyperpermeable. The existence of an inward Ca2+ current overlapping in time with an outward rectifying K+ current was demonstrated. The latter could be selectively blocked by replacing internal K+ with Cs+ and external Ca2+ with Ba2+. Depolarizations to potentials between -40 and +50 mV evoked time-dependent inward currents, with a maximum peak value between -20 and 0 mV. For depolarizations beyond +50 mV time-dependent outward currents appeared. These currents were inhibited by 0.1 mM CdCl2. The activation of the inward current showed a sigmoidal time course, and the rate of onset of the current increased at more positive potentials. Inactivation could be described by two exponentials. The threshold for activation was about -40 mV, and full activation was reached at 0 mV. Inactivation was complete near 0 mV, whereas the channels were fully available at -80 mV. The fully-activated Ca2+-channel current was strongly voltage dependent. The conductance decreased for potentials close to the reversal potential, and showed rectification for hyperpolarizing potentials. The Ca2+ agonist BAY k 8644 enhanced the Ca2+-channel current without a significant effect on its kinetics. The fully-activated current and the steady-state activation were enhanced in a rather voltage-independent way.

Intracellular recordings from pineal cells in tissue culture: membrane properties and response to norepinephrine

Membrane properties and responsiveness to norepinephrine were studied in pinealocytes in tissue culture using both conventional and whole-cell patch-clamp techniques. Action potentials could be recorded in normal and tetrodotoxin-containing saline. Both outward and inward rectification were prominent. Under voltage-clamp, depolarizing voltage steps evoked time-dependent outward currents showing no inactivation over time. Hyperpolarizing steps evoked slowly developing inward currents which did not inactivate. If the holding potential was sufficiently negative, depolarizing steps also elicited a transient outward current. Iontophoresis of norepinephrine produced a hyperpolarization in 30% of cells tested.

P-94 - Effects of goniopora toxin on the action potential and membrane currents of guinea-pig single ventricular cells

The effects of Goniopora toxin (GPT), a polypeptide isolated from a coral, Goniopora spp., on action potential and membrane currents were studied in single ventricular cells of the guinea-pig using the whole-cell clamp technique with a single patch electrode. GPT at a concentration of 10 nmol/l prolonged the duration of the action potential without significant change in the resting membrane potential and action potential amplitudes. This prolongation became more evident at lower stimulus frequencies and persisted after washing with toxin-free solution. Tetrodotoxin (TTX, 1 μmol/l), but not Co2+ (2 mmol/1), abolished the prolonged action potential. Under voltage-clamp conditions, a sustained inward current, not present in the control, followed the transient inward current during depolarizing pulses in the GPT-treated cells. The current-voltage relationship for the sustained inward current was much the same as that for the fast sodium current reported in rat single ventricular cells (Brown et al. 1981). Both the sustained and transient currents were abolished by the shift of holding potential in the direction of depolarization and reappeared after repolarization; the reappearance of the sustained current was much slower than that of the transient current. TTX but not Co2+ abolished both the sustained and transient inward currents. Calcium current and time-independent current were not affected by GPT. Time-dependent outward current induced by large depolarizing pulses was attenuated by GPT. From these results, it is suggested that GPT predominantly acts on the sodium channels in cardiac muscle, to give rise to a sustained sodium current which is responsible for the prolongation of action potential, and that the sodium channels modified by this toxin may reveal slow inactivation and slow recovery from inactivation as compared with those of unmodified sodium channels.

Effects of intracellular acidification on membrane currents in ventricular cells of the guinea pig.

The membrane currents of single ventricular cells were measured under whole cell voltage clamp using a giga-sealed patch electrode, and the effects of intracellular acidification were examined by perfusing the electrode pipette with different pH solutions. The plateau of the action potential was shortened when the pH of the pipette solution was lowered from the control of 7.2 to 6, and finally to 5. The pH 6 pipette solution evoked a time-independent outward current at positive potentials and increased the slope conductance near the resting potential. These changes were suppressed by removal of both intra- and extracellular potassium ion, indicating that these currents were carried by potassium ions, but not by protons. Increasing the calcium concentration in the pipette from pCa 8 to pCa 6 induced a time-dependent outward current which had a reversal potential of about -13 mV. This result clearly differed from the changes induced by the acidic pipette solution, suggesting that the calcium-mediated conductance was not involved in the genesis of the acidic effects. The calcium current was not significantly affected by perfusion at pH 6, but was decreased by the more acidic (pH 5) solution. When the calcium current was recorded in sodium- and potassium-free external solution but with a cesium-rich internal solution, however, the calcium current was suppressed even with a weak acidic (pH 6.8) pipette solution. This effect was attributed not to an increased sensitivity of the calcium channel to protons, but to a more extensive intracellular acidification, which might have been caused by a depressed extrusion of proton via a sodium-hydrogen exchange mechanism on the surface membrane.

A comparative electrophysiological study of enzymatically isolated single cells and strips of frog ventricle

Single heart cells were obtained from frog ventricle using an enzymatic dispersion technique. The whole cell variation of the patch clamp technique was used to monitor action potential and cell membrane currents. The clamp circuit could be switched electronically between voltage and current clamp modes. The effects of seal leakage currents were to depolarize the cell, reduce the amplitude of the plateau, and lengthen the action potential duration. A scheme to compensate for these currents is presented. The membrane currents obtained from the single cell under voltage clamp conditions were compared to those obtained from multicellular preparations using the single sucrose gap technique. Hyperpolarizing clamps showed time-dependent, depletion-related K+ currents for the multicellular preparation, whereas for the single cell no such currents were observed. The absence of extracellular accumulation or depletion of K+ in the single cell was confirmed by the lack of post-clamp afterpotentials or changes in resting potential following a train of frequently elicited action potentials. The TTX-insensitive inward current was relatively faster in the single cell, compared to that measured in the multicellular preparation. A delayed time-dependent outward current was observed in the positive potential range for both single and multicellular preparations. The isochronal current-voltage (I-V) relations obtained at 400 ms were N-shaped for both preparations, but was more negative for the single cell at potentials positive to -20 mV. The results indicate a strong similarity between membrane currents obtained in single and multicellular preparations. The differences in the currents in the two preparations are due in large part to accumulation or depletion of K+ in the extracellular space.

Do catecholamines directly modulate the delayed plateau potassium current in frog atrium?

An important electrophysiological action of catecholamines in the heart is the modulation of the second inward current (iCa). Catecholamine augmentation of iCa has been attributed to an increase in the number of functional channels, as well as an increase in the probability of channel opening [1, 4, 12]. Another important electrophysiological action of catecholamines in the heart is the modulation of time-dependent outward currents. In the Purkinje fibre, epinephrine has been shown to shift the voltage-dependence of the pacemaker current, iK2 [13]. The delayed outward current(s) which is activated over the plateau range of potentials (ix) is also augmented by catecholamines in Purkinje fibers [14] as well as frog atrium [3]. Several possible mechanisms for enhancement of the outward plateau current by catecholamines have been proposed [2]: (i) a direct increase in the maximal conductance of the channel; (ii) an indirect enhancement of outward current mediated by stimulation of Na+-K+ exchange (which may reduce potassium concentration in restricted extracellular spaces and hence produce an increase in the driving force for K+) or (iii) an indirect augmentation of outward current via enhancement of intracellular Ca2+ (calcium-activated potassium conductance) [10]. To distinguish between these possible mechanisms, the influence of epinephrine and isoproterenol on the delayed outward K+ current in single isolated bullfrog atrial cells was examined. Evidence is presented that in single frog atrial cells, concentrations of epinephrine or isoproterenol which produce large increases in the second inward current have no detectable effect upon the delayed plateau K+ current.

Membrane current through adenosine‐triphosphate‐regulated potassium channels in guinea‐pig ventricular cells.

The question whether activation of the ATP-regulated K channel is responsible for macroscopic anoxia-induced outward currents was examined in ventricular cells isolated enzymatically from guinea-pig heart. Gigaseal patch-clamp electrodes were used for a whole-cell voltage clamp. Membrane currents were compared in the same cell while the cell interior was dialysed by perfusing the electrode with different solutions. When the cell was dialysed with various ATP-deficient (less than or equal to 2 mM) internal solutions, the Ca current decreased in a dose-dependent manner to less than 10% of control at 0.5 mM-ATP. A slight (ca. 25%) decrease of the slope conductance for hyperpolarizing current was observed. When a delayed rectification on depolarization followed by a marked outward current tail on repolarization was present under control conditions, this time-dependent outward current was also depressed. An increase in a time-independent outward current was observed accompanied by marked current fluctuations. The outward current showed a reversal potential near the K equilibrium potential, inward rectification, and no relaxation on voltage jumps. The power density spectrum of the current fluctuations showed a pattern similar to the spectrum calculated from the single-channel currents of ATP-regulated K channels. The amplitude of the single-channel current, estimated from the fluctuations, was almost equal to that of the single-channel current. The total number of channels within one cell was estimated as 2000-3000. It is concluded that the ATP-regulated K channels are responsible for the increase in the outward current and the shortening of the action potential duration under various anoxic conditions.

Computer analysis of prolongation of cardiac action potential duration

Using Beeler and Reuter's mathematical model of action potential in myocardial fibers, a similar extent of prolongation of action potential duration at −60 mV (APD −60) was produced at the infinite interval of stimulation by altering parameter values for slow inward current ( i s) time-independent ( i k 1 ) and time-dependent ( i x 1 ) outward currents. Among five types of prolongation of APD −60 thus produced, the plateau height is high in two types at this interval, but in three at a shorter interval. The degree of prolongation varies between these five types at this shorter interval. The stimulus interval-action potential duration (interval-duration) relation curves reflect changes in time-dependent parameters of these currents. Thus various types of APD prolongation may be distinguished from each other by the different behavior of action potentials at low and high frequencies of stimulation. i x 1 increases by 40-fold at the moment of the maximum rate of depolarization at an interval of 330 msec, but is still negligibly small as compared with sodium current.

The effects of Ni2+ on ionic currents and tension generation in frog ventricular muscle

The effect of Ni2+ on E-C coupling events of the frog ventricular muscle were studied using a single sucrose gap voltage clamp technique. The results showed that Ni2+ increased the overshoot potential and depressed and prolonged the plateau of the action potential. Ni2+ also increased the dependence of the overshoot potential on [Na]0 from 18 to 58 mV per decade. In the presence of Ni2+, TTX blocked both the upstroke and the plateau of the action potential. The combination of TTX and Ni2+ suppressed the tension-voltage relation, the time-dependent outward currents and K+ efflux. While suppression of the tension-voltage relation by Ni2+ alone was reversed by increasing [Ca]0, the effects of Ni2+ plus TTX are not reversed by addition of Ca2+. The results suggest that Ni2+ may alter the action potential by slowing the inactivation of the Na+ current and blocking the inward Ca2+ current. Although the tension-suppressant effects of Ni2+ could be attributed to the inhibition of a slowly inactivating Ca2+ current, the effects of Ni2+ in the presence of TTX were less readily explained. Several possible mechanisms are considered which are all consistent with the hypothesis that development of tension in ventricular strips is mediated by both a Ca2+ current and a Ca2+ counter-transport system.

Phenytoin reduces calcium current in the cardiac Purkinje fiber.

We have investigated the effects of phenytoin on the electrical and mechanical activity of isolated calf and dog cardiac Purkinje fibers. Phenytoin (5-100 microM) lowers and shortens the plateau phase of the Purkinje fiber action potential and reduces twitch tension. We studied the ionic basis of these effects by combining a conventional two-microelectrode voltage clamp procedure with pharmacological techniques that allow separation of time-dependent plateau membrane currents. We find that phenytoin reduces voltage-dependent calcium current in the Purkinje fiber. In addition, this drug slightly reduces two time-dependent outward currents. These effects of phenytoin on membrane current account, at least in part, for its influence on the action potential and twitch tension.

Inhibition of the slow inward current and the time-dependent outward current of mammalian ventricular muscle by gentamicin.

The aminoglycoside antibiotic, gentamicin (GM), depressed the plateau phase and shortened the duration of the action potential in guinea pig papillary muscle. Its effect on the membrane currents was studied by a single sucrose gap voltage clamp method. The slow inward current (is) was remarkably diminished by GM with little change in its time course, in the voltage-dependency of the steady-state inactivation and activation or in its reversal potential. The maximal amplitude of is, obtained by subtracting the Co2+-resistant current, was reduced to 57% by 0.1 mmol/l GM and almost reduced to zero by 1 mmol/l GM. The efficacy of GM in inhibiting (is) was reduced by increasing the external Ca2+ concentration from 1.8 to 5.4 or 10.8 mmol/l, but not by the application of adrenaline. The time-dependent outward current (ik) was also decreased by GM but only at higher concentrations. It is proposed that the depressant action of GM on is was due to a blockade of slow channels, whereby GM may have dislocated Ca from the binding sites at slow channels on the external surface of the membrane.

Effect of alpha- and beta-sympathomimetic agonists on calcium-dependent slow action potential and force of contraction in the rabbit papillary muscle.

The influence of sympathomimetic α- and β-agonists on calcium-dependent slow action potential (SAP) and force of contraction were analyzed in the partially depolarized rabbit papillary muscle. 1. The effects of phenylephrine (3×10−6 mol/l) on SAP were compared with those of isoprenaline (5×10−8 mol/l) since the contractions that could be elicited at these concentrations were of similar amplitude. The maximum rate of rise (Vmax) of the slow action potential, which was taken to reflect the slow calcium inward current (Isi), was evidently smaller when elicited by phenylephrine than by isoprenaline. 2. Vmax of SAP induced by phenylephrine (3×10−6 mol/l) or isoprenaline (5×10−8 mol/l) was significantly increased in either case by additional application of isoprenaline. The positive inotropic effect induced by additional isoprenaline was associated with an increase in the rate of force development. 3. Vmax of SAP evoked by isoprenaline (5×10−8 mol/l) remained unchanged by additional application of phenylephrine (3×10−6 mol/l) whereas the duration of SAP was significantly prolonged. Additional phenylephrine markedly increased the time to peak force but left the rate of force development unchanged. These effects of phenylephrine on slow action potential and force of contraction were completely blocked by prazosin (10−8 mol/l). 4. The duration of SAP induced by dopamine was shortened by prazosin (10−8 mol/l) without alteration of Vmax, whereas force of contraction was reduced. 5. For further analysis of the mechanisms responsible for the prolongation of SAP by the α-mimetic effect of phenylephrine, additional experiments have been performed. In normal Tyrode solution containing MnCl2 (10 mmol/l), phenylephrine (3×10−6 mol/l) prolonged the duration of action potential without reestablishing force development. Prazosin (10−8 mol/l) completely antagonized this effect of phenylephrine. 6. These findings indicate that an increase in the slow calcium inward current induced by α-stimulation is much smaller than that induced by β-stimulation. The prolongation of the action potential evoked by α-stimulation seems to be induced by a suppression of the time-dependent outward current. This current may play an important role in the α-adrenoceptor-mediated positive inotropic effect.

Voltage-clamp investigations of membrane currents underlying pace-maker activity in rabbit sino-atrial node.

1. Small preparations of spontaneously beating rabbit sino-atrial node have been investigated using the two micro-electrode voltage-clamp technique. 2. Hyperpolarizing clamp pulses given from holding potentials of about -45 mV reveal a time-dependent change of a membrane current, if, which is shown to overlap the pace-maker range (-65 mV to -45 mV) for these preparations. 3. The changes in if are shown to be quite distinct from the de-activation of the time-dependent outward current, iK. 4. The time-dependent changes of the if system increase during adrenaline application and therefore contribute to the chronotropic action of adrenaline on the heart. 5. Evidence for a link between slow inward current (iSi) and time-dependent outward current (iK) in rabbit sino-atrial node is presented and assessed.

Ionic currents during hypoxia in voltage-clamped cat ventricular muscle.

To explore the mechanisms underlying the shortening of the cardiac action potential in hypoxia, we studied the effect of hypoxia on the ionic currents in cat papillary and trabecular muscles using the single sucrose gap-voltage clamp technique. For potentials positive to -70 mV, hypoxia induces an increase in time-independent outward current. The changes in the tail current suggest that time-dependent outward current is not increased but, rather, reduced. Because the time course of ik remains unchanged, we concluded that the shortening of the action potential is not a result of a change in the time-dependent outward current. In the potential range of the plateau, the amplitude of the slow inward current is not affected by hpoxia. Its time constant of inactivation appears slightly decreased. The prolongation of the action potential by epinephrine during hypoxia is accompanied by an increase in the slow inward current. As a result of these studies, we conclude that the shortening of the cardiac action potential in the early stage of hypoxia results from an increase in K+ outward background current.

Effect of acetylcholine on membrane currents in guinea-pig papillary muscle.

1. The effect of acetylcholine (ACh) on the membrane current components in guinea-pig papillary muscle was studied by the single sucrose gap voltage clamp method. 2. The slow inward current (is) elicited by various depolarizations from the holding potential of -40 mV was evidently diminished by ACh greater than 5 x 10(-7) M. The amplitude of is, estimated as the difference between the peak of is and the current at 150 msec, was reduced to 56.8 +/- 12.9% (mean +/- S.D., n = 34) by 2 x 10(-8) M-ACh when estimated at the membrane potential where the amplitude was maximal. 3. is was suppressed almost completely by 2 mM-Co2+, is obtained by subtracting the current remaining in 2 mM-Co2+ was also diminished to the same extent by 2 x 10(-6) M-Ach with little change in its time course; the degree of decrease was not dependent on the membrane potential. 4. The time-dependent outward current during the depolarizing pulse was also diminished by 2 x 10(-6) M-Ach. Correspondingly, the positive tail current obtained when the membrane was repolarized to the holding potential of -40 mV after various 1 sec depolarizations was reduced in a proportionate manner to 68.6 +/- 7.3% (n = 10). 5. The time-independent inward rectifying outward current was not affected by 2 x 10(-6) M-Ach. 6. Atropine (10(-6) M) restored is and the time-dependent outward current which had been previously depressed by 2 x 10(-6) M-ACh. 7. ACh may decrease tension development by depressing is, thereby preserving the duration of the action potential with relatively little change, since, different from atrial muscle, ACh does not increase the outward current in mammalian ventricular muscle.

Calcium and Cardiac Electrophysiology: Some Experimental Considerations

Electrophysiologic experiments in cardiac tissue suggest that Ca2+ is involved in generation of the action potential, the pacemaker potential, and conduction of the slow wave of depolarization. For instance, removal of Ca2+ inhibits the slow inward current and prolongs the action potential and suppresses the slow diastolic depolarization. Divalant cations Mn2+, Co2+, Cd2+, Mg2+, block the slow inward current and suppress pacemaker activity, but shorten the action potential. Ni2+ specifically blocks the slow inward current and prolongs the action potential. Ca2+ also plays a central role in generation of diastolic depolarizaittn. Cd2+ inhibits the diastolic depolarizaton and the upstoke of the action potential in SA nodal cells, while blocking the time-dependent inward current in the pacemaker potential range and the time-dependent outward current. A variety of molecular transport systems ranging from the Ca-channel to a Ca2+-Na+ or Ca2+-K+ exchanges to Ca2+-induced activation of the K+ current have been postulated to explain the effects of Ca2+ on cardiac electrophysiologic processes.