Faculty Bibliography

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ

Direct measurements of free Ca2+ in heart cells are needed for an understanding of the regulation of contractility. We developed and used Ca2+ -sensitive microelectrodes with fine tips, stable properties and ample sensitivity to free Ca2+ in the sub-micromolar range. In quiescent ventricular muscle, measurements which passed tests for electrode sealing and cell viability gave a mean free Ca2+ concentration of 0.26 microM. During contractures, we recorded Ca2+ transients rising as high as 10 microM. In studying the effects of catecholamines on free Ca2+ and force, we found evidence that adrenaline can reduce myofibrillar Ca2+ sensitivity in intact heart muscle

1. The possibility that the transient outward current of calf cardiac Purkinje fibres depends on intracellular calcium was investigated using a two micro-electrode voltage clamp. 2. Upon removal of Cao and replacement with Sr or Ba, the transient outward current was strongly suppressed. At the same time a large slow inward current was revealed. 3. Partial removal of Cao with replacement by Mg also diminished the transient outward current. The inhibition was not due to voltage shifts in the inactivation curve. 4. The kinetics of the peak transient outward current were compared with the kinetics of peak twitch force, an approximate measure of the level of Cai. The two signals were related in a linear manner during beat-dependent changes with trains of voltage clamp depolarizations. 5. Tension and transient outward current were also found to inactivate with a similar dependence on pre-potential and recover from inactivation along a similar time course. Both processes activated with membrane depolarization in a similar manner. 6. Intracellular injection of EGTA reduced the magnitude of the transient outward current and the twitch contraction. 7. The inhibition of outward current following EGTA injection was more pronounced for strong depolarizations. With pulses negative to - 10 mV, there was often little apparent change in the peak net outward current. 8. All lines of evidence support the hypothesis that the transient outward current is activated by intracellular Ca. 9. The functional significance of the transient outward current is discussed. Since a Ca-activated outward current would automatically offset slow inward Ca current, such a system may help prevent arrhythmogenic slow responses in the His-Purkinje network

Rhythmic oscillations in the membrane potential of heart cells are important in normal cardiac pacemaker activity as well as cardiac arrhythmias. Two fundamentally different mechanisms of oscillatory activity can be distinguished at the cellular and subcellular level. The first mechanism, referred to as a surface membrane oscillator, can be represented by a control loop in which membrane potential changes evoke delayed conductance changes and vice versa. Since the surface membrane potential is a key variable within the control loop, the oscillation can be interrupted at any time by holding the membrane potential constant with a voltage clamp. This mode of oscillation seems to describe spontaneous pacemaker activity in the primary cardiac pacemaker (sinoatrial node) as well as other regions (Purkinje fibre, atrial or ventricular muscle). In all tissues studied so far, the pacemaker depolarization is dominated by the slow shutting-off of an outward current, largely carried by potassium ions. The second mechanism can be called an internal oscillator since it depends upon a subcellular rhythm generator which is largely independent from the surface membrane. Under voltage clamp, the existence of the internal oscillation is revealed by the presence of oscillations in membrane conductance or contractile force which occur even though the membrane potential is held fixed. The two oscillatory mechanisms are not mutually exclusive; the subcellular mechanism can be preferentially enhanced in any given cardiac cell by conditions which elevate intracellular calcium. Such conditions include digitalis intoxication, high Cao, low Nao, low or high Ko, cooling, or rapid stimulation. Several lines of evidence suggest that the subcellular mechanism involves oscillatory variations in myoplasmic calcium, probably due to cycles of Ca uptake and release by the sarcoplasmic reticulum. The detailed nature of the Cai oscillator and its interaction with the surface membrane await further investigation

1. The three-micro-electrode voltage clamp method (Adrian, Chandler & Hodgkin, 1970) was adapted for the study of regenerative inward currents in cardiac muscle. The adequacy of measurements of slow inward current in cardiac Purkinje fibres was assessed. 2. Membrane current density is reported simultaneously by total applied current (IT), along with a longitudinal voltage difference signal (delta V), recorded between two intracellular micro-electrodes. 3. Non-linear cable calculations show that delta V is a more faithful measure of membrane current density than IT as peak inward current increases. Quantitative agreement between delta V and IT only occurs when both signals report the membrane characteristics that would be obtained with an ideal longitudinal space clamp. 4. Agreement between delta V and IT is thus a useful criterion for satisfactory experimental measurements which we applied to the slow inward current. This component was elicited by depolarizing steps from a holding potential near =45 mV in the presence of tetrodotoxin. The IT signal was compared directly with delta V/R, where R is an effective longitudinal resistance that was experimentally determined. 5. delta V/R and IT showed very good agreement in both peak amplitude and time course at all potentials studied. 6. Radial non-uniformity during the measured peak slow inward current was estimated by calculations assuming clefts 200 A wide with a uniform distribution of ionic channels. The calculated voltage span from surface to centre was always less than 5 mV, and the measured I-V characteristics showed little distortion. 7. In another check, I-V characteristics and slow response membrane action potentials were compared. The measured peak current showed good agreement with the product (total preparation capacitance) x (rate of rise). 8. The experimental and theoretical analysis suggest that the measurements of slow inward current are a good approximation to genuine membrane properties

1. Rabbit Purkinje fibres were studied using micro-electrode recordings of electrical activity or a two-micro-electrode voltage clamp. Previous morphological work had suggested that these preparations offer structural advantages for the analysis of ionic permeability mechanisms. 2. Viable preparations could be obtained consistently by exposure to a K glutamate Tyrode solution during excision and recovery. In NaCl Tyrode solution, the action potential showed a large overshoot and fully developed plateau, but no pacemaker depolarization at negative potentials. 3. The passive electrical properties were consistent with morphological evidence for the accessibility of cleft membranes within the cell bundle. Electrotonic responses to intracellular current steps showed the behaviour expected for a simple leaky capacitative cable. Capacitative current transients under voltage clamp were changed very little by an eightfold reduction in the external solution conductivity. 4. Slow current changes attributable to K depletion were small compared to those found in other cardiac preparations. The amount of depletion was close to that predicted by a cleft model which assumed free K diffusion in 1 micron clefts. 5. Step depolarizations over the plateau range of potentials evoked a slow inward current which was resistant to tetrodotoxin but blocked by D600. 6. Strong depolarizations to potentials near 0 mV elicited a transient outward current and a slowly activating late outward current. Both components resembled currents found in sheep or calf Purkinje fibres. 7. These experiments support previous interpretations of slow plateau currents in terms of genuine permeability changes. The rabbit Purkinje fibre may allow various ionic channels to be studied with relatively little interference from radial non-uniformities in membrane potential or ion concentration