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Elementary Ca2+ and Ba2+ currents were recorded from cell-attached membrane patches of ventricular myocytes from adult guinea pig hearts using the improved patch-clamp technique (Hamill et al. 1981). High concentrations of Ba2+ or Ca2+ (50 or 90 mM) were used in the pipettes to increase the signal-to-noise ratio. All data were derived from elementary current analyses in patches containing only one channel.1)In response to voltage steps, channel openings occurred singly or in bursts of closely spaced unitary current pulses separated by wider shut intervals. During depolarizations of small amplitude from the resting potential, channel openings occurred almost randomly, whereas during larger depolarizations the events were grouped preferentially at the beginning.2)Channel openings became more probable with increased depolarization; simultaneously, unitary current amplitudes declined in an ohmic manner. Elementary current amplitudes were slightly larger, when 50 mM Ba2+ replaced 50 mM Ca2+ in the pipettes (slope conductances 9 and 10 pS, respectively), but more than doubled, when Ba2+ was increased to 90 mM (slope conductance 18 pS). Clear outward currents through Ca2+ channels were not observed under these conditions.3)Peak amplitudes of reconstructed mean currents doubled when 50 mM Ba2+ replaced 50 mM Ca2+ and were larger still when 90 mM Ba2+ was used in the pipettes. The current-volrage relations of the reconstructed mean currents showed a positive shift along the voltage axis as Ba2+ was increased or substituted equimolarly by Ca2+. Correspondingly, the open state probability-voltage relations (activation curves) showed a parallel shift as Ba2+ was increased, which was less pronounced when Ba2+ was replaced equimolarly by Ca2+.4)Determination of Ca2+ channel inactivation using 90 mM Ba2+ in the pipettes indicated an overlap with channel activation in a limited voltage range, resulting in a steadystate “window” current. Inactivation can occur without divalent cation influx.5)Formation of an inside-out patch resulted in a fast rundown of elementary Ca2+ channel currents.6)Channel openings were often grouped in bursts. The lifetimes of the open state, the bursts, and the closed states were estimated for Ba2+ and Ca2+ as permeating ions. At least two exponentials were needed to fit the histogram of the lifetimes of all closed states. The lifetimes of the individual openings and bursts were mono-exponentially distributed. The kinetics of the Ca+ channel depended on the voltage and the permeating ion. During +30 mV depolarizations, no significant effect of the permeating ion on channel gating could be detected. The significant increase in burst length (tb) during +50 mV depolarizations, however, seemed to be only due to an increase in the lifetime of the open state (to) for Ba2+, whereas for Ca2+,to was only moderately prolonged but simultaneously, the number of openings per burst increased.7)A three-state sequential scheme is peoposed to model the activation pathway of Ca2+ channels. The latency-to-first-event histogram is also consistent with a process in which multiple closed states precede the open state.

1. Propagated action potentials were recorded in right ventricular papillary muscles from guinea‐pig heart while exposed to Na‐free, Ca‐free and Mg‐free solutions containing Mn. 2. When Na was totally replaced by 95 mM‐Mn the overshoot was about 45 mV while the resting potential was about ‐90mV. 3. The overshoot of action potentials was increased by about 20‐30 mV per tenfold increase of Mn concentration over the range of 2‐50 mM. 4. Similar increases of overshoots with increasing of Mn concentration also occurred in the presence of 0‐6 mM‐Ca. Increasing of Ca from 5 to 20 mM had little influence on the overshoot but shortened the duration of the Mn‐dependent action potential in the presence of 5 mM‐MN. 5 Mn‐dependent action potentials were not depressed by 3 X 10(5) M tetrodotoxin but by La. 6. These results suggest that Mn passes through the slow inward current channel to generate the action potential seen under the Na‐free condition. 7. The overshoot and duration of the Mn‐dependent action potential decreased with stimulation. At stimulus frequencies (Hz) of 0‐5, 0‐2, 0‐1, 0‐017 and 0‐0033 the overshoot of action potential in 5 mM‐Mn Tyrode decreased by 0‐5‐1 mV per an action potential. This depression of the action potential is explained by assuming intracellular accumulation of Mn.

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.

1. The mechanism of Ca2+ channel block by nitrendipine was studied by recording single‐channel activity from cell‐attached patches on guinea‐pig ventricular cells using patch pipettes containing 50 mM‐Ba2+. Test depolarization pulses to around 10 mV with a duration of 100 ms were applied repetitively at 2 Hz. 2. The percentage of non‐blank sweeps was maximal (about 40%) at a holding potential between ‐65 and ‐130 mV and decreased sigmoidally with its depolarization. Nitrendipine shifted the availability‐voltage relationship in a hyperpolarizing direction. 3. From the number of consecutive non‐blank sweeps and that of blank sweeps, the duration of the available state and that of the unavailable state were estimated. 4. The histogram of the duration of the available state showed a single‐exponential distribution. Its mean duration was about 1.5 s and was shortened by nitrendipine. Correspondingly, the decay of the mean current during the depolarization step was accelerated by nitrendipine. 5. In the presence of 100 nM‐nitrendipine the histogram of the duration of the unavailable state at large negative holding potentials was simulated as the sum of two exponential components, one with a time constant similar to that in the control and the other with a time constant of 6‐7 s. 6. The histogram of the duration of the unavailable state at depolarized holding potentials was simulated by a double‐exponential curve also in the control. The duration of the slow component was prolonged by nitrendipine. 7. The prolongation of the unavailable states initiated by drug binding during depolarization steps and maintained during depolarized holding potentials is the mechanism of the blockade. The rate constants of the state transitions between an available state and two unavailable states were estimated.

Anthopleurin-A (AP-A) prolonged the duration of action potential and increased the developed tension of isolated guinea-pig and canine ventricular muscle. Voltage clamp experiments by using a single sucrose gap method were performed to investigate the ionic mechanism of the prolongation of the action potential in guinea-pig ventricular muscle. The prolongation of action potential by AP-A was accompanied by a decreased net outward current in a quasi-steady state. The slow inward current and the delayed potassium current were not changed by AP-A. The prolongation of action potential by AP-A was reversed by tetrodotoxin, thus it was concluded that AP-A induced tetrodotoxin-sensitive inward current which lasted long after the initial fast Na current. Modification of the AP-A-induced positive inotropic effect was also examined by using the canine blood-perfused ventricular muscle and was consistent with the above electrophysiological effects; tetrodotoxin, not nifedipine, suppressed percent changes produced by AP-A and also that by veratrine.