1. Individual cells were isolated from adult rats ventricular myocardium by a collagenase digestion procedure. 2. Steady membrane potentials recorded with conventional intracellular glass micro-electrodes from cells in a modified Krebs solution containing 3 . 8 mM-KCl and 0 . 5 mM-CaCl2 were less negative than -40 mV in most cells (-25 . 3 +/- 10 . 9 mV, mean +/- S.D., 211 cells). 3. After addition of the potassium selective ionophore valinomycin (60 nM) to the bathing solution all recorded membrane potentials were more negative than -60 mV (-74 . 8 +/- 7 . 0 mV, sixty-three cells). 4. The internal concentration of potassium in the cells was determined as 120 . 8 +/- 1 . 7 mM (+/- S.E., n = 24) by flame emission spectrometry after centrifugation through silicone oil, using tritiated water and D-[1-14C] mannitol to estimate total and extracellular water in the pellet. 5. In the majority of cells in the standard solution the membrane potential recorded within a few msec of penetration was more negative than -70 mV (-78 . 4 +/- 9 . 7 mV, seventy-three cells). In sixty-six cells penetration initiated an action potential which overshot zero by 31 . 3 +/- 7 . 1 mV. This overshoot was abolished by reducing the external sodium to 0 . 1 of the normal value, and reduced or abolished by addition of tetrodotoxin (30 microM). 6. Modifications of the standard bathing solution which increased the number of cells with steady recorded membrane potentials more negative than -60 mV were: isosmotic substitution of sucrose for NaCl; replacement of NaCl and KCl by sodium isethionate and potassium methyl sulphate; addition of 5 or 10 mM-CaCl2; addition of 10 mM-MnCl2. 7. For cells in solution containing 2 . 5 or 5 . 5 mM-CaCl2, input resistances estimated from the amplitude of hyperpolarizations evoked by 200 msec current pulses were approximately 40 M omega at a resting potential close to -80 mV and became much greater as cells were depolarized. Time constants measured at the resting potential were approximately 8 msec. 8. In certain conditions, repeated spontaneous action potentials were recorded from contracting cells, and in quiescent cells evoked action potentials could be initiated by applying brief depolarizing pulses through the micro-electrode. Action potentials were coincident with contractions. 9. It is concluded that the resting potential of these isolated cells is normally more negative than -70 mV, and that the cells retain the ionic mechanisms necessary for the generation of active currents.

SUMMARYRyanodine and cyclopiazonic acid were used to alter the ability of the sarcoplasmic reticulum (SR) to store calcium. Interventions of this kind significantly reduced the rate of spontaneous beating of guinea-pig sino-atrial node preparations. In addition, both of these drugs decreased the rate of rise and modified other characteristics of the action potentials recorded from this region. It is proposed that calcium released from the SR plays an important, previously unrecognized, role in pacemaking in the sino-atrial node, possibly through regulation of sarcolemmal ionic currents.

SUMMARY1. End-plate currents have been studied in glycerol-treated frog sartorius nerve-muscle preparations with the voltage-clamp technique.2. Adding the anticholinesterase prostigmine (3/gM) to the solution bathing the muscle caused a 2-7 (mean 3.3) times increase in the time constant of decay of end-plate currents. The anticholinesterase edrophonium (15 /tM) also prolonged the time course of end-plate currents.3. Pre-treatment of the preparation with collagenase, which leads to the removal of acetylcholinesterase in the synaptic cleft, prolongs the time course of end-plate currents.4. Curare (1-2 /tM), cobratoxin (0.13 ,sm), or oc-bungarotoxin (0.13-0*26 /SM) decreased the time constant of decay of end-plate currents in the presence of prostigmine.5. These observations are consistent with the suggestion that repeated binding of acetylcholine (ACh) molecules to receptors as the ACh escapes from the synaptic cleft can contribute to the prolongation of end-plate currents which occurs when acetylcholinesterase activity is eliminated.6. Increasing the amount of transmitter released from the presynaptic nerve terminal leads to a prolongation of end-plate currents in the presence of prostigmine.7. In the presence of prostigmine, the second of two end-plate currents (interval 2-10 msec) decays more slowly than the first.8. ACh (1-40 AM) or carbachol (40 AM) applied in the solution bathing the muscle prolongs end-plate currents in the presence of prostigmine.

Action potentials were recorded from single cells isolated from rat and guinea‐pig ventricular muscle. In rat cells the repolarization showed two distinct phases, referred to as the early and late phases. In guinea‐pig cells there was a maintained plateau. Reducing external sodium by replacement with lithium or choline suppressed the late phase of the action potential in rat cells, and shortened the plateau of the action potential in guinea‐pig cells. Intracellular EGTA abolished contraction while suppressing the late phase of the action potential in rat cells, and shortening the plateau in guinea‐pig cells. Ryanodine (1 μm), which is thought to inhibit the release of calcium from internal stores, suppressed contraction and the late phase of the action potential in rat cells. In guinea‐pig cells, there was no substantial effect of ryanodine (1 μm) on either contraction or the time course of the action potential. The late phase of the action potential in rat cells was suppressed by increasing the external potassium concentration to 12 mm, and enhanced by reducing external potassium to 1.2 mm It is concluded that an inward current activated by internal calcium contributes to the late phase of the action potential in rat cells, and to the plateau in guinea‐pig cells. Two possibilities are a current arising from electrogenic sodium‐calcium exchange, and a current through ion channels activated by calcium. The effects of reducing external sodium would be consistent with either mechanism. The contribution of such an inward current would be expected to be modified by outward currents through a rectifying potassium conductance which varies with external potassium concentration. In the rat, but not the guinea‐pig, the rise in internal calcium which activates the inward current seems to be largely dependent on ryanodine‐sensitive release of calcium from internal stores.

The rapidly activating delayed rectifier potassium current, IKr, was studied in guinea‐pig ventricular myocytes in the presence of thiopentone, which blocks the more slowly activating component of the delayed rectifier potassium current, IKs, and using whole cell perforated patch clamp or switched voltage clamp with sharp electrodes to minimise intracellular dialysis. Activation of protein kinase A (PKA) by isoprenaline or forskolin caused an increase in IKr tail currents. Following a 300 ms depolarising step to +20 mV, mean tail current amplitude was increased 47 ± 12% by isoprenaline, and 73 ± 13% by forskolin. No increase in IKr was observed when IKr was studied using whole cell ruptured patch clamp and there was no change in the reversal potential of IKr in the presence of isoprenaline. The rectification of the current sensitive to E4031, a selective IKr blocker, was markedly reduced in the presence of isoprenaline and the region of negative slope was absent. This is consistent with a reduction in the inactivation of IKr and was supported by the finding that IKr, in the presence of isoprenaline, was somewhat less sensitive to block. E4031 (5 μm) blocked only 81 ± 5% of IKr in the presence of isoprenaline compared to 100 ± 0% in control. The forskolin‐ and isoprenaline‐induced increases in IKr were inhibited by staurosporine and by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide I. Direct activation of PKC by phorbol dibutyrate increased IKr tail currents by 24 ± 5%. Both the isoprenaline‐ and forskolin‐induced increases in IKr were inhibited when calcium entry was reduced by block of ICa with nifedipine or when myocytes were pre‐incubated in BAPTA‐AM. The selective PKA inhibitor KT5720 prevented the isoprenaline‐induced increase in IKr only when the increase in ICa was also suppressed. These data show a novel mechanism of regulation of IKr by PKC and this kinase was activated by β‐adrenoceptor stimulation. IKr seems to be enhanced through a reduction in the C‐type inactivation which underlies the rectification of the channel and such a mechanism may occur in other channels with this type of inactivation.