Faculty Bibliography

We compared the maximal upstroke velocity of action potentials in short rabbit Purkinje fibers with sodium currents measured with a two-microelectrode voltage clamp. The number of sodium channels available to open during a sudden depolarization was varied either by blockade with tetrodotoxin or by inactivation with steady depolarizations. In both cases, the maximal upstroke velocity was found to be a very nonlinear measure of the number of available sodium channels. For example, 3 microM tetrodotoxin blocks 85% of the sodium channels, but reduces the maximal upstroke velocity by only 33%. Voltage clamp and upstroke velocity experiments were reconstructed with a computer model of the rabbit Purkinje fiber preparation that was closely based on experimental measurements of passive cable properties and sodium channel characteristics. The simulations indicate that our voltage clamp measurements of sodium current accurately report changes in channel availability, but they also show that the maximal upstroke velocity is a strongly nonlinear index of available sodium conductance. Most of the nonlinearity arises from the activation kinetics of the sodium channels: as the pool of available channels decreases, a greater percentage of those channels activate and contribute inward current at the time of the maximal upstroke velocity. Simulations predict that the maximal upstroke velocity-available sodium conductance relationship would still remain nonlinear at 37 degrees C or under different stimulus conditions that give uniform or continuously propagated action potentials. The nonlinearity may invalidate inferences based on earlier maximal upstroke velocity experiments: the existence of two types of sodium channels with different tetrodotoxin sensitivity, steady state voltage dependence of tetrodotoxin block, voltage range over which sodium channels inactivate, and rapid, then slow recovery of sodium channel availability following a sudden repolarization. All of these conclusions need to be reevaluated

Calcium channels carry out vital functions in a wide variety of excitable cells but they also face special challenges. In the medium outside the channel, Ca2+ ions are vastly outnumbered by other ions. Thus, the calcium channel must be extremely selective if it is to allow Ca2+ influx rather than a general cation influx. In fact, calcium channels show a much greater selectivity for Ca2+ than sodium channels do for Na+ despite the high flux that open Ca channels can support. Relatively little is known about the mechanism of ion permeation through Ca channels. Earlier models assumed ion independence or single-ion occupancy. Here we present evidence for a novel hypothesis of ion movement through Ca channels, based on measurements of Ca channel activity at the level of single cells or single channels. Our results indicate that under physiological conditions, the channel is occupied almost continually by one or more Ca2+ ions which, by electrostatic repulsion, guard the channel against permeation by other ions. On the other hand, repulsion between Ca2+ ions allows high throughput rates and tends to prevent saturation with calcium

Adrenergic modulation of calcium channels profoundly influences cardiac function, and has served as a prime example of neurohormonal regulation of voltage-gated ion channels. Channel modulation and increased Ca influx are mediated by elevation of intracellular cyclic AMP and protein phosphorylation. The molecular mechanism of the augmented membrane Ca conductance has attracted considerable interest. An increase in the density of functional channels has often been proposed, but there has previously been no direct evidence. Single-channel recordings show that isoprenaline or 8-bromocyclic AMP increase the proportion of time individual channels spend open by prolonging openings and shortening the closed periods between openings. To look for an additional contribution of changes in the number of functional channels, we applied ensemble fluctuation analysis to whole-cell recordings of cardiac Ca channel activity. Here we present evidence that in frog ventricular heart cells beta-adrenergic stimulation increases NF, the average number of functional Ca channels per cell. We also find that isoprenaline slows the time course of both activation and inactivation, and that the enhancement of peak current decreases gradually with greater membrane depolarization

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 muM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 muM, at a negative holding potential where inactivation was completely removed, to approximately 10 muM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (tauless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (tau approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 muM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 muM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the 'modulated-receptor hypothesis' of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle

Organic inhibitors of calcium influx prevent outward as well as inward current through cardiac calcium channels but do not slow current activation. Although block is antagonized by raising external calcium or barium concentrations, the competitive effect of permeant cations does not occur at the same cation binding site at which inorganic blockers act. Organic drugs show varying degrees of use-dependent block, due in part to blockade of open channels. Nitrendipine blockade of calcium currents requires doses greater than 100-fold higher than expected from radioligand binding to isolated membranes

1. Calf cardiac Purkinje fibres were exposed briefly to the ionophore nystatin to promote exchange of caesium for intracellular potassium. The effects of Cs loading were stable for at least 30 min, but they could be reversed by nystatin-mediated K loading.2. After Cs loading, the resting potential shifted to about -20 mV and the current-voltage relationship showed a strong inhibition of inwardly rectifying K channels.3. Anodal break stimulation evoked normal action potential upstrokes and twitch contractions. The early repolarization (phase 1) was markedly slowed.4. Cs loading simplified the pattern of current changes evoked by step depolarizations over the plateau range. Membrane current reached an inward peak and then declined monotonically.5. The current signal showed no hint of the transient outward current found in untreated or K-loaded preparations. Furthermore, Cs loading abolished the outward tails associated with deactivation of transient outward current, and occluded the blocking effect of the K-channel inhibitor 4-aminopyridine.6. Inhibition of transient outward current revealed a maximal inward current of about 5 muA/muF in 5.4 mM-Ca(o), which is considerably larger than the net inward current without Cs loading.7. The inward current was attributed to Ca channels on the basis of its sensitivity to membrane potential, extracellular Ca, D600, Mn and Cd.8. Cs loading also reduced slow current changes associated with delayed rectification and pace-maker depolarization.9. The results support the hypothesis that the transient outward current is carried by K(+) ions, while providing a method for unmasking inward Ca current

1. Effects of digitalis compounds on slow inward Ca current I(si)) and contractile force were examined in ferret ventricular muscle (single sucrose-gap voltage clamp) and calf Purkinje fibres (two micro-electrode voltage clamp).2. In ventricular muscle, ouabain increased I(si) and inward current tails associated with I(si) conductance. The enhancement of I(si) followed a time course similar to the development of the positive inotropic effect, and it could be observed in the absence of aftercontractions or other signs of toxicit.3. The response of myocardial I(si) and twitch force to ouabain depended strongly on a previous history of driven action potentials.4. Veratridine, a toxin that promotes Na entry through tetrodotoxin-sensitive channels, also increased I(si) and twitch force in driven ventricular muscle preparations.5. The effects of ouabain, action potential stimulation and veratridine are consistent with reported effects of K-poor solutions in indicating that elevation of intracellular Na can lead to enhancement of I(si). Additional experiments suggest that the link between Na(i) and I(si) involves intracellular Ca.6. When Cs-loaded Purkinje fibres were bathed in solutions containing Sr instead of Ca, enhancement of I(si) by strophanthidin was abolished even though a positive inotropic response persisted.7. After intracellular injection of Purkinje fibres with EGTA, I(si) no longer increased with strophanthidin, although it remained responsive to adrenaline.8. Clear-cut increases in I(si) were seen in Cs-loaded Purkinje fibres even at very low concentrations of strophanthidin (20-50 nM), where the occurence of Na pump inhibition has been questioned.9. Positive regulation of Ca entry by intracellular Ca may act as a facilitory mechanism that amplifies myocardial responsiveness to digitalis and other inotropic interventions. Through changes in I(si), small rises in diastolic free Ca might lead to large increases in the activator Ca transient during contraction