Hiroshi Irisawa

Inward current activated during hyperpolarization in the rabbit sinoatrial node cell.

Inward current activated by hyperpolarizationih) was dissected from the K-current by the difference in its activation voltage range and the selective blocking effect of Ba2+ on the K-current. Theih shows little specificity to any particular ion, and its reversal potential was −25 mV. The current system can be expressed well by Hodgkin-Huxley type kinetics. The time constant ofih ranged from 2–4 s at about −70 mV, but it became shorter at about −10 mV. Theih began to activate at −50 mV and fully saturated at about −100 mV. The fully activated current-voltage relation shows no rectifying property. Activation and deactivation time courses were fitted by a single exponential with the same time constant at a given membrane potential. Althoughih plays only a small role during the normal action potential in the isolated preparation, it plays a significant role in keeping the pacemaker cell at a low membrane potential.

Membrane currents in the rabbit sinoatrial node cell as studied by the double microelectrode method

SummaryWhen a strand of the rabbit sinoatrial node tissue was shortened by ligation, the spatial decay of electrotonic potential decreased and the input impedance increased. In a piece of the tissue 0.2–0.3 mm in diameter apparently uniform current spread was obtained. Action potentials recorded from three different sites in this small piece occurred simultaneously and were superimposable.In voltage clamp experiments using the double microelectrode method, the membrane potential was usually held at −30 to −40 mV, where no net current flowed. When membrane potential was suddenly changed from the holding potential, the sign and the time course of the ionic current varied with membrane potential. Hyperpolarization gave an inward current which increased with time. Depolarization gave a transient inward current followed by sustained outward current, and repolarization gave an outward current tail which exponentially subsided with a time constant of 0.37 s.The membrane time constant was 12.0 ms. When the specific membrane capacitance was assumed to be 1 μF/cm2, the specific membrane resistance at the resting potential was 12 kΩcm2. The peak of the transient inward current on depolarization was 1.3×10−5 A/cm2.

Slow inward current and its role mediating the chronotropic effect of epinephrine in the rabbit sinoatrfal node

The ionic mechanism underlying the chronotropic effect of epinephrine on the rabbit sinoatrial (S-A) node has been studied. Epinephrine (5.5×10−6 M) increased the spontaneous rate from 206±25 min−1 to 242±39 min−1. The effect of epinephrine was reproducible on repetitive applications.Voltage clamp experiments using the two microelectrode technique revealed the following changes in the membrane current: epinephrine (5.5×10−7 M) increased the limiting conductance for the slow inward current (is) by approximately 30% and the potassium current (ik) by about 10%, keeping the kinetics ofis andik constant. From the holding potential of −70 mV the activation ofis was observed on step depolarization positive to −60 or −55 mV in both control and epinephrine solution. The hyperpolarization-activated current (ih) was also increased by about 20% at −70 mV, and its time course was slightly accelerated.Participation ofis for the chronotropic effect of epinephrine was strongly suggested by the findings thatis was partially available positive to −60 mV and that epinephrine could not increase the slope of diastolic depolarization whenis was blocked by D 600.

Transient depolarization and spontaneous voltage fluctuations in isolated single cells from guinea pig ventricles. Calcium-mediated membrane potential fluctuations.

Under the influence of cardiotonic steroids, single ventricular cells exhibit transient depolarization after a train of driven action potentials or, in voltage clamp experiments, transient inward current after a depolarizing clamp pulse. Transient depolarization or transient inward current was abolished by an intracellular injection of ethyleneglycol-bis β-aminoethyl ether)-JV,N′-tetraacetic acid (EGTA) or by superfusion of 5 mM caffeine. Transient depolarization was elicited even in the control Tyrodes solution by an intracellular injection of CaCl2 or augmented by an injection of adenosine 3′,5′-cyclic monophosphoric acid (cAMP). Along with transient depolarization or transient inward current, digitalis intoxication promoted spontaneous oscillatory fluctuations in membrane potential or in membrane current. Their power spectra showed peaks at frequencies ranging from 2 to 7 Hz, which coincided well with the frequency of repetitive transient depolarization or transient inward current. The fluctuations were eliminated by intracellular injections of EGTA and decreased in amplitude by 5 mM caffeine with a shift toward higher frequencies. Depolarization of the membrane caused a shift of the spectrum peak toward higher frequencies. These results suggest that an oscillatory release of Ca from intracellular storage sites is the common basis underlying both the transient events (depolarization or inward current) and the spontaneous miniature fluctuations in membrane potential or current.

Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea‐pig.

Effects of ATP and of cyclic AMP on membrane current systems were investigated in isolated single ventricular cells from guinea‐pig hearts by applying the suction electrode method. The intracellular milieu was dialysed with various solutions which were perfused continuously through the suction pipette. The presence of ATP, cyclic AMP and EGTA in the perfusion solution kept the plateau phase of the action potential almost intact for as long as 30 min. With depolarizing voltage‐clamp pulses from holding potentials between ‐30 and ‐40 mV, the slow inward current (isi) was activated at potentials positive to ‐20 mV. The inactivation time course of isi was fitted by two exponential components in the potential range between ‐10 mV and +30 mV. By increasing ATP from 2 to 9.5 mM in the solution, the amplitude of isi was increased and the slow component of inactivation was accelerated. The steady‐state current‐voltage relationship (I‐V curve), exhibited a negative slope that became steeper after increasing the ATP concentration. The current was shifted towards the outward direction between ‐40 mV and ‐10 mV and became more inward between ‐10 mV and +40 mV. Increase of the cyclic AMP concentration from 30 to 60 microM also enhanced the amplitude of isi, but the negative slope in the steady‐state I‐V curve was unaffected. Assuming that the concentration of free Ca2+ in the cell was well buffered at a low level by the EGTA‐Ca buffer solution in the pipette, it was concluded that [ATP]i and [cyclic AMP]i exert a direct influence on membrane current systems of the ventricular cell.

Action potential and membrane currents of single pacemaker cells of the rabbit heart

Single, viable pacemaker cells were isolated from sinoatrial (S-A) and atrioventricular (A-V) nodes by treating with collagenase. In normal Tyrode solution containing 1.8 mM Ca2+, these pacemaker cells had a round configuration and contracted rhythmically at a frequency of about 150–260/min. The amplitude, duration, and maximum rate of rise of the spontaneous action potentials recorded using patch clamp electrodes were similar to those obtained from multicellular preparations. Amplitudes of the recorded membrane current were normalized with reference to the surface area of the cell by assuming the cell shape as a plane oblate spheroid. The membrane resistance of the isolated nodal cells was 14.9±4.0 kΩ·cm2 (n=12) at about −35 mV and the membrane capacitance was 1.30±0/24 μF/cm2 (n=18). The inactivation time course of the slow inward current,isi, was fitted with a sum of two exponentials with time constants of 6.7±0.6 ms and 46.6±15.3 ms (n=4) at +10 mV. The amplitude ofisi peaked at 0∼+10 mV in the current-voltage relationship and was 18.2±8.4 μA/cm2. The potassium current,iKwas activated in the voltage range positive to −50 mV and was saturated at about +20 mV. The amplitude of the fully-activatediKat −40 mV was 3.3±1.4 μA/cm2 (n=10) and showed an inward-going rectification. The activation of the hyperpolarization-activated current was observed at potentials negative to −70 mV in seven of 14 experiments. The current density and membrane capacitance calculated could be overestimated and the membrane resistance underestimated, because of the presence of caveolae on the cell surface. However, these data give the nearest possible estimates of the electrical constants in the nodal cells, which cannot be measured accurately in the conventional multicellular preparations.

A time- and voltage-dependent potassium current in the rabbit sinoatrial node cell

SummaryVoltage clamp experiments were conducted in the rabbit sinoatrial node (S-A node) using the double microelectrode technique. When the membrane was repolarized to the resting potential after depolarizing test pulses, the outward current slowly decayed to the steady level (outward current tail). The magnitude of the outward current tail was a sigmoid function of the amplitude of the preceding depolarization. The degree of activation of this current varied from 0 at about − 50 mV to 1 at about +20 mV. The time course of the current change was a simple exponential and was independent of the preceding depolarization. The reciprocal time constant appeared to be a U-shaped function of the membrane potential with a minimum value of about 3 s−1 at −40 mV. The instantaneous current voltage relation was an inward-going rectifier, but showed no detectable negative slope. The reversal potential, obtained between 10 and 50 mM [K]o, decreased with a slope of 58 mV for a 10-fold increase in [K]o. These findings indicate that the outward current tail in the S-A node cell is attributable to a single component of K current (pacemaker current component). The pacemaker current component is mainly responsible for the slow diastolic depolarization.

Does the “pacemaker current” generate the diastolic depolarization in the rabbit SA node cells?

Small preparations of spontaneously beating rabbit sino-atrial node (SA node) were voltage clamped with the two-microelectrode technique. The effects of 0.25–5 mM Cs+ on the spontaneous pacing rate and the time-dependent inward “pacemaker” current,ih, were studied. In the presence of 2 mM Cs+, the spontaneous pacing rate decreased only slightly even thoughih was strongly depressed at potentials negative to −60 mV Cs+ had little or no effect on other time-dependent currents observed with clamp pulses less negative than −50 mV. Since no voltage-dependence to the Cs+ effect onih could be measured (between −90 mV and −20 mV), it was considered unlikely that the lack of Cs+ effect on the rate of diastolic depolarization results from a voltage-dependent effect of Cs+ on theih channel.Adrenaline produced a marked positive chronotropic effect in Cs+-treated SA node cells. This effect was accompanied by marked enhancement of the slow inward current (isi) with no change in the Cs+-blockedih current. These results are consistent with the idea thatih plays a minor role in generation of pacemaker depolarization, and suggest a more prominent role ofisi in the generation of diastolic depolarization in SA nodal cells.

Membrane currents in the rabbit atrioventricular node cell.

The rabbit A-V node was dissected into pieces (0.2×0.2×0.2 mm) smaller than its space constant of 692±96 μm (n=5). These small specimens showed spontaneous action potentials whose configurations were similar to those of large specimens before dissection. The membrane time constant was 21.5±1.5 ms (n=5).Voltage clamp experiments were performed on the above specimens using the two-microelectrode technique. On depolarization from the holding potential of −40 mV to various potential levels a transient inward current and delayed outward current were recorded. On repolarization an outward current tail was observed. The transient inward current was blocked by application of D600 (2×10−7 g/ml) but was insensitive to TTX (1×10−7 g/ml). The inward current was decreased by superfusion with Na- or Ca-free Tyrode solution. Thus, this current was classified as the slow inward current (is). When the K concentration in the Tyrode solution was varied, the reversal potential of the outward current tail changed as expected for a K electrode, indicating that the outward current was carried by K ions. On hyperpolarization slow activation of inward current was recorded. The reversal potential of this current was between −20 and −30 mV, which was analogous to hyperpolarization activated current,ih, in the S-A node. A contribution of sodium current (iNa) to the action potential was obviously demonstrated from an inhibitory effect of TTX on the upstroke of the anodal break excitation. The ionic selectivity of each current system is compared with analogous current systems in other cardiac tissues and a possible mechanism for the slow conduction in the A-V node is discussed.

Modification of the cardiac action potential by intracellular injection of adenosine triphosphate and related substances in guinea pig single ventricular cells.

Effects of varying the intracellular adenosine triphosphate level on both the action potential and the membrane current were studied in single ventricular cells isolated from the guinea pig heart, using collagenase. Intracellular injection of adenosine triphosphate elevated the plateau potential level and prolonged the action potential duration. Similar results were obtained by injecting adenosine diphosphate, adenosine monophosphate, or creatine phosphate, i.e., substances considered to increase the intracellular concentration of adenosine triphosphate. In contrast, the action potential was depressed by procedures which could reduce the intracellular adenosine triphosphate level, such as an injection of creatine, superfusion of glucose-free Tyrodes solution containing 5.4 mM cyanide ion, or an injection of adenosine monophosphate into the cyanide-superfused cell. When the membrane current was recorded under the voltage clamp, it was found that the injection of adenosine triphosphate increased the amplitude of the slow inward current, whereas the superfusion of cyanide ion did not significantly decrease the slow inward current, although the action potential became considerably shorter. It was also found that the adenosine monophosphate injection decreased the amplitude of the net outward membrane current at the plateau level and increased it at around —40 mV, and thus intensified the N-shape of the isochronal 0.3-second current-voltage curve. The cyanide ion superfusion produced the opposite effect; in response to depolarizing clamp pulses more positive to the plateau level, the membrane current increased significantly with cyanide ion, but increased only slightly with adenosine triphosphate. These results suggest that intracellular adenosine triphosphate modifies the membrane currents at the plateau potential range, thus altering the action potential duration.