The role of slow potassium current in phenomena of voltage-dependent action of 4-aminopyridine in amphibian myelinated nerve fibres

The mechanism underlying the voltage-dependent action of 4-aminopyridine (4-AP) is investigated in experiments on amphibian myelinated nerve fibres (Rana ridibunda Pallas) by way of extracellular recording of electrical activity and using activators of potassium current (potassium-free solution and nitric oxide NO) and inhibitors of sodium current (tetrodotoxin). Measurement of action potential (AP) areas was used to evaluate the extent of general membrane depolarization during the activity of nerve fibres. Tetrodotoxin-induced decrease in general membrane depolarization (when the action potential amplitude was reduced by less than 20%) leads to an increase in the duration of depolarizing after-potential (DAP). This supports the dependence of time course of DAP in the presence of 4-AP on ratio of fast and slow potassium channels. In the absence of 4-AP, potassium-free solution and NO increase the potassium current through fast potassium channels (decreasing AP duration, reducing DAP and sometimes producing fast hyperpolarizing after-potential (HAP) after shortened AP), and in the presence of 4-AP these activators increase potassium current through unblocked slow potassium channels (making the development of slow HAP induced by 4-AP more rapid). The increase of slow HAP induced by 4-AP under the influence of potassium-free solution with NO supports the idea that slow HAP is due to activation of slow potassium channels and argues against the notion of removal of block of fast potassium channels. All analyzed phenomena of voltage-dependent action of 4-AP in amphibian myelinated nerve fibers can be accounted for by the activation of slow potassium current produced by membrane depolarization and a decrease of the amount of fast potassium channels involved in the membrane repolarization.     

Electrotropic acetylcholine action on the mammalian auricle by means of the "phase plane" trajectories of the action potential

A single sucrose gap techniques has been used to study action potentials and phase plane trajectories of them in atrial trabeculae of the rabbit. Using polynomial representations of current-voltage relationships a model of membrane action potential of atrial myocardial fibres is described and allows an interpretation of recording data from the phase plane trajectories. Our findings show: 1. Increasing extracellular calcium concentration increases a potassium conductivity of the atrial membrane. 2. An anomalous rectification concerning repolarizing currents in atrial fibres decreases with increasing extracellular calcium. 3. Acetylcholine (3.10(-4) g.cm-3) abolishes the anomalous rectification. These results are discussed in relation to previous electrophysiological studies of negative electrotropic effects of acetylcholine in cardiac muscle.     

Enhancement of inward current by serotonin in neurons of Aplysia

In RB cells of Aplysia, serotonin, in the presence of TEA, 4AP and Ba, elicits a voltage-dependent inward current. In Ba-TEA-4AP seawater, RB cells showed a negative slope region (NSR) in their current-voltage (I-V) relationship when measured at the end of 2-s commands from a holding potential of -60 mV. Addition of serotonin to the bathing solution enhanced the NSR. When holding potential was lowered to -10 mV, the NSR as well as the effects of serotonin were greatly reduced. Addition of 20 mM cobalt to the bathing solution blocked both the NSR and the inward current produced by serotonin. Changes in potassium concentration produced no consistent shift in voltage sensitivity nor change in amplitude of the current elicited by serotonin. Intracellular injection of cesium sufficient to broaden action potentials did not block the enhancement of NSR by serotonin. These results support the conclusion that in RB cells, serotonin produces a voltage-dependent current carried by calcium ions.     

Electrical properties of skeletal muscle fibres of the flour moth larva Ephestia kuehniella

The electrical properties of the ventral longitudinal muscle fibres in the flour moth larva Ephestia kuehniella were investigated at rest and during electrical activity. The membrane resting potential was only partially dependent on the K-concentration gradient across the muscle membrane. The electrical constants λ, τ, R_m, R_i, and C_m were determined according to the equations for ‘short cables’ (Table 1). Current-voltage relationships of the muscle membrane were measured: they revealed anomalous as well as delayed rectification of the membrane. Stimulation of the muscle fibres with intracellular current pulses elicited graded action potentials in most fibres; in some fibres ‘all-or-none’ action potentials were generated. In contrast to graded action potentials these ‘all-or-none’ action potentials were propagated without decrement along the muscle fibre. Indirect stimulation of the muscle fibres resulted in large excitatory junction potentials which generally gave rise to action potentials.     

An outwardly rectifying anionic background current in atrial myocytes from the human heart

This report describes a hitherto unreported anionic background current from human atrial cardiomyocytes. Under whole-cell patch-clamp with anion-selective conditions, an outwardly rectifying anion current (I(ANION)) was observed, which was larger with iodide than nitrate, and with nitrate than chloride as charge carrier. In contrast with a previously identified background anionic current from small mammal cardiomyocytes, I(ANION) was not augmented by the pyrethroid tefluthrin (10 microM); neither was it inhibited by hyperosmolar external solution nor by DIDS (200 microM); thus I(ANION) was not due to basal activity of volume-sensitive anion channels. I(ANION) was partially inhibited by the Cl(-) channel blockers NPPB (50 microM) and Gly H-101 (30 microM). Incorporation of I(ANION) into a human atrial action potential (AP) simulation led to depression of the AP plateau, accompanied by alterations to plateau inward calcium current, and to AP shortening at 50% but not 90% of complete repolarization, demonstrating that I(ANION) can influence the human atrial AP profile.     

Aminopyridine block of transient potassium current

The blocking action of 4-aminopyridine (4-AP) and 3, 4-diaminopyridine (Di-AP) on transient potassium current (IA) in molluscan central neurons was studied in internal perfusion voltage-clamp experiments. Identical blocking effects were seen when the drugs were applied either externally or internally. It was found that aminopyridines have two kinds of effects on IA channels. The first involves block of open channels during depolarizing pulses and results in a shortening of the time to peak current and an increase in the initial rate of decay of current. This effect of the drug is similar to the block of delayed potassium current by tetraethylammonium (TEA). The other effect is a steady block that increases in strength during hyperpolarization, is removed by depolarization, and is dependent on the frequency of stimulation. The voltage dependence of steady state block approximates the voltage dependence of inactivation gating a changes e-fold in approximately 10 mV. These data suggest that the strength of block may depend on the state of IA gating such that the resting state of the channel with open inactivation gate is more susceptible to block than are the open or inactivated states. A multistate sequential model for IA gating and voltage-dependent AP block is developed.     

Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina

A sustained high voltage-activated (HVA), nifedipine- and cadmium- sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na- acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout- dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium- dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis.     

Ranolazine inhibits shear sensitivity of endogenous Na+ current and spontaneous action potentials in HL-1 cells

Na_V1.5 is a mechanosensitive voltage-gated Na⁺ channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na⁺ current and delayed rectifier (I_Kr) currents. Recently, ranolazine was also shown to be an inhibitor of Na_V1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na⁺ current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na⁺ current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.     

Aminopyridines block an inactivating potassium current having slow recovery kinetics.

The blocking action of aminopyridines on an inactivating K current (lKi) in GH3 pituitary cells was studied before and after altering the macroscopic decay of the current with N-bromoacetamide (NBA). The first depolarizing pulse delivered either seconds or minutes after beginning 4-aminopyridine (4AP) application, elicited a current with both a more rapid decay and a reduced peak amplitude. The rapid decay (or time-dependent block) was especially prominent in NBA-treated cells. With continued drug application, subsequent test pulses revealed a stable block of peak current, greater in NBA-treated than control cells. Recovery from block was enhanced by hyperpolarizing holding potentials and by the first depolarizing pulse delivered after prolonged recovery intervals. Unlike aminopyridine block of other K currents, there was no convincing evidence for voltage shifts in activation or inactivation, or for voltage and frequency-dependent unblock. Increasing the open probability of the channels did, however, facilitate the block. Although the behavior of currents in 4AP was suggestive of "open channel block," the block was not produced by 4-aminopyridine methiodide, a positively charged aminopyridine. Moreover, because partial block and recovery occurred without opening the channels we suggest that aminopyridines bind to, or near, this K channel, that this binding is enhanced by opening the channel, and that a conformational change is induced which mimics inactivation. Because recovery from block is enhanced by negative potentials, we suggest that aminopyridine molecules may become "trapped" by inactivation awaiting the slow process of reactivation to escape their binding sites.     

Changes in the electrical activity of myocardial fibers following heart transplantation to under the skin of the ear in mice]

Intracellular rest and action potentials (RP and AP, respectively) of mouse heart graft were reduced on the 3rd--4th days after the transplantation as compared with the intracellular potentials of newborn mice. Beginning from the 5th--6th days there occurred a gradual increase of the intracellular potentials of amplitude. The firt 7--8 days, both in the case of allograft and heterograft, the changes in the intracellular activity were the same. Then in heterograft there occurred a repeated reduction of the RP and AP amplitude of the graft myocardial fibers, this apparently being connected with the development of the rejection reaction. In the allograft samples the amplitude of the intracellular potentials approached by the end of the first month after the graft with RP and AP values of the recipient's myocardial fibers.     

Effect of ryanodine on the electrical and mechanical activity of frog atrial fibers

The effect of ryanodine on the action potential, slow inward current and mechanical activities of frog atrial fibres was studied by means of the double sucrose gap technique. Ryanodine was shown to reduce the amplitude of the slow inward current, to cause an intracellular Ca accumulation and to decrease the tonic component of the tension.     

Effect of stretching on the action potentials of the human and rabbit ventricular myocardium

The effect of stretching from L0 to Lmax on the electrical activity was studied on human myocardial preparations from patients with heart disease and on strips of rabbit ventricular myocardium. Muscular deformation was shown to decrease the amplitude and velocity of depolarization in slow action potentials. The action potentials (AP) possessing a fast depolarization phase were not sensitive to physiological stretching. Antiarrhythmic drugs--ethmozin (2 X 10(-5) M) and ethacizin (2 X 10(-6) M)--caused a decrease in the rate of AP depolarization, thus increasing AP sensitivity to deformation. It is suggested that stretching under the action of ethmozin and ethacizin reduced cardiomyocyte excitability due to suppression of slow Ca-current.     

Different effects of blocked potassium channels on action potentials, accommodation, adaptation and anode break excitation in human motor and sensory myelinated nerve fibres: computer simulations

 Action potentials and electrotonic responses to 300-ms depolarizing and hyperpolarizing currents for human motor and sensory myelinated nerve fibres have been simulated on the basis of double cable models. The effects of blocked nodal or internodal potassium (fast or slow) channels on the fibre action potentials, early and late adaptations to 30-ms suprathreshold slowly increasing depolarizing stimuli have been examined. The effects of the same channels on accommodation after the termination of a prolonged (100 ms) hyperpolarizing current pulse have also been investigated. By removing the nodal fast potassium conductance the action potentials of the sensory fibres are considerably broader than those of the motor neurons. For both types of fibres, the blocked nodal slow potassium channels have a substantially smaller effect on the action potential repolarization. When the suprathreshold depolarizing current intensity is increased, the onset of the spike burst occurs sooner, which is common in the behaviour of the fibres. The most striking differences in the burst activity during early adaptation have been found between the fibres when the nodal fast potassium channels are blocked. The results obtained confirm the fact that the motor fibres adapt more quickly to sustained depolarizing current pulses than the sensory ones. The results also show that normal human motor and sensory fibres cannot be excited by a 100-ms hyperpolarizing current pulse, even at the threshold level. When removing the potassium channels in the nodal or internodal axolemma, the posthyperpolarization increase in excitability is small, which is common in the behaviour of the fibres. However, anode break excitation can be simulated in the fibres with simultaneous removal of the potassium channels under the myelin sheath, and this is more pronounced in the human sensory fibres than in motor fibres. This phenomenon can also be found when the internodal and some of the nodal (fast or slow) potassium channels are simultaneously blocked. Received: 8 November 1999 / Accepted in revised form: 29 February 2000     

Ranolazine inhibits shear sensitivity of endogenous Na+ current and spontaneous action potentials in HL-1 cells

Na_V1.5 is a mechanosensitive voltage-gated Na⁺ channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na⁺ current and delayed rectifier (I_Kr) currents. Recently, ranolazine was also shown to be an inhibitor of Na_V1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na⁺ current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na⁺ current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.     

Ionic mechanism of the Woodwors staircase

Analysis of single-chamber model of electromechanical coupling in the myocardial cell has shown that Woodwors staircase can be imitated in two cases: 1) stationary input current Ca2+ strongly exceeds the potential-dependent uptake of Ca2+ into the cell through the sarcolemma; 2) the action potential (AP) is shortened abruptly with an increase of the myocardium stimulation frequency. The experiments performed on a fragment of the frog heart ventricle supported the conclusions of the model. Blocking of Ca-channels with nifedipine (10(-6) g/mol) at the background of isotonic substitution of 70% of NaCl resulted in the development of "negative staircase" with an increase of stimulation rhythm. An abrupt shortening of AP after rest at joint action of adrenaline (10(-6) g/ml) and blocker of Ca-channels D-600 (10(-6) g/ml) was accompanied by Woodwors staircase.     

Investigations of the Electrical Properties of Cardiac Muscle Fibres with the Aid of Intracellular Double-Barrelled Electrodes

Current has been passed through the cell membrane of muscle fibres of the isolated rabbit right ventricle with the aid of intracellular double-barrelled microelectrodes. Two types of muscle fibres were distinguished which are called P and V fibres. The relation between the intensity of a hyperpolarising current applied during the rising phase and the maximum amplitude of the action potential was different in these fibres. For P fibres the relation was essentially linear over most of the range of currents used. For V fibres the change in maximum action potential amplitude was either negligible or did not appear until a certain value of hyperpolarising current was reached. This behaviour of V fibres can be understood if a drop in polarisation resistance occurs during the rising phase and is of such short duration that the polarisation resistance has returned to its resting value before the crest of the action potential is reached. P fibres have an estimated mean resting polarisation resistance of (106 ± 13) K ohms, and a rheobase current strength of (0.08 ± 0.02) µa. In V fibres the resting polarisation resistance was (47 ± 29) K ohms and the rheobase current strength (0.47 ± 0.28) µa.     

A model for 4-aminopyridine action on K channels: similarities to tetraethylammonium ion action.

We present a model for the action of 4-aminopyridine (4AP) on K channels. The model is closely based on the gating current studies of the preceding paper and has been extended to account for ionic current data in the literature. We propose that 4AP, like tetraethylammonium ion and other quaternary ammonium ions, enters and leaves the channel only when the activation gate is open, a proposal that is strongly supported by the literature. Once in the open channel, 4AP's major action is to bias the activation gate toward the closed conformation by approximately the energy of a hydrogen bond. S4 segment movement, as reflected in gating currents, is almost normal for a 4AP-occupied channel; only the final opening transition is affected. The model is qualitatively the same as the one used for many years to explain the action of quaternary ammonium ions.     

Effects of ruthenium red on excitation and contraction in muscle fibres with Ca2+ electrogenesis.

The effect of ruthenium red (RR) on the electrical and contractile responses, membrane Ca currents, staining patterns of the external and internal membrane system were tested in intact and mechanically skinned muscle fibres of the crayfish Astacus fluviatilis. The following results were obtained: 1. Depression of the contractile responses following membrane depolarization (twitch, tetanus, potassium contractures). 2. Caffeine contractures were unaffected in intact (100 mumol/l - 1 mmol/l RR) and blocked in skinned fibres (30 mumol/l RR). 3. Mechanical threshold and mechanical latency were increased and/or prolonged. 4. The rate of depolarization of the action potentials (AP) was decreased and decremental spread of AP was recorded. 5. Both fast and slowly inactivating Ca ionic currents were decreased and the time constants of activation (tau(m] and inactivation (tau(h] were prolonged after RR (100 mumol/l) pretreatment. 6. The penetration of RR into the T-system was inversely related to its binding to the sarcolemma. The depression of depolarization-induced contractions was most pronounced in fibres with unstained sarcolemma and stained T-tubules. In intact fibres, neither terminal cisternae nor other elements of SR were stained. On the contrary, all internal membrane structures were stained in skinned fibres. There was a gradient of staining intensity from surface toward the interior.     

B-type natriuretic peptide (BNP) attenuates the L-type calcium current and regulates ventricular myocyte function

A fundamental question in physiology is how hormones regulate the functioning of a cell or organ. It was therefore the aim of this study to investigate the effect(s) of BNP-32 on calcium handling by ventricular myocytes obtained from the rat left ventricle. We specifically tested the hypothesis that BNP-32 decreased the L-type calcium current (I(Ca,L)). Perforated patch clamp technique was used to record I(Ca,L) and action potential (AP) in voltage and current clamp mode, respectively. Myocyte shortening was measured using a photodiode array edge-detection system and intracellular calcium transients were measured by fluorescence photometry. Western blotting was used to determine the relative change in the expression of proteins. At the concentrations tested, BNP-32 significantly decreased cell shortening in a dose-dependent manner; increased the phase II slope of the AP by 53.0%; increased the APD(50) by 16.9%; reduced the I(Ca,L) amplitude with a 22.9% decrease in the peak amplitude and reduced Ca(2+)-dependent inactivation; increased the V(1/2) activation of the L-type calcium channel by 51.1% and decreased V(1/2) inactivation by 31.8%; and, intracellular calcium transient amplitude was significantly decreased by 32.0%, whereas the time to peak amplitude and T(1/2) were both significantly increased by 38.7% and 89.4% respectively. Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) protein expression was reduced by BNP-32. These data suggest that BNP-32 regulates ventricular myocyte function by attenuating I(Ca,L), altering the AP and reducing SERCA2a activity and/or expression. This study suggests a novel constitutive mechanism for the autocrine action of BNP on the L-type calcium channel in ventricular myocytes.     

The induction of periodic and chaotic activity in a molluscan neurone

During prolonged exposure to extracellular 4-aminopyridine (4 AP) the periodic activity of the somatic membrane of an identified molluscan neurone passes from a repetitive regular discharge of >90 mV amplitude action potentials, through double discharges to <50 mV amplitude oscillations. Return to standard saline causes the growth of parabolic amplitude-modulated oscillations that develop, through chaotic amplitude-modulated oscillations, into regular oscillations. These effects are interpreted in terms of the actions of 4 AP on the dynamics of the membrane excitation equations.Emma and Leslie Reid scholar