Delayed rectification in the cardiac Purkinje fiber is not activated by intracellular calcium

This study was designed to test the hypothesis that an outward current (Ix) responsible for action potential repolarization in the cardiac Purkinje fiber is activated by intracellular calcium (Cai). Pharmacological probes were combined with the measurement of membrane current and contractile activity under voltage clamp conditions. Experiments were designed to examine properties of Ix that have previously linked activation of this current to changes in Cai. The independence of Ix from Cai was demonstrated for each case tested. Thus, the results of these experiments support the view that Ix is not a calcium-activated current.     

Inactivation of calcium channel current in the calf cardiac Purkinje fiber. Evidence for voltage- and calcium-mediated mechanisms

We have studied the influence of divalent cations on Ca channel current in the calf cardiac Purkinje fiber to determine whether this current inactivates by voltage- or Ca-mediated mechanisms, or by a combination of the two. We measured the reversal (or zero current) potential of the current when Ba, Sr, or Ca were the permeant divalent cations and determined that depletion of charge carrier does not account for time-dependent relaxation of Ca channel current in these preparations. Inactivation of Ca channel current persists when Ba or Sr replaces Ca as the permeant divalent cation, but the voltage dependence of the rate of inactivation is markedly changed. This effect cannot be explained by changes in external surface charge. Instead, we interpret the results as evidence that inactivation is both voltage and Ca dependent. Inactivation of Sr or Ba currents reflects a voltage-dependent process. When Ca is the divalent charge carrier, an additional effect is observed: the rate of inactivation is increased as Ca enters during depolarizing pulses, perhaps because of an additional Ca-dependent mechanism.     

Photoalteration of calcium channel blockade in the cardiac Purkinje fiber.

Organic compounds that block calcium channel current (calcium antagonists) are important tools for the characterization of this channel. However, the practically irreversible nature of this block restricts the usefulness of this group of drugs. In this paper, we investigate the influence of light on calcium channel blockade by several organic compounds. Our results show that inhibition of calcium channel current by two dihydropyridine derivatives that contain an o-nitro moiety (nisoldipine and nifedipine) can be rapidly reversed by illumination. The energy range important to this reaction is for light wavelengths between 320 and 450 nm. Calcium channel inhibition by two other dihydropyridine derivatives (nicardipine and nitrendipine) as well as by D600, is not modulated by illumination. These results indicate that the photosensitivity of certain dihydropyridine calcium channel blockers make these compounds useful as reversible blockers of this channel.     

Phase resetting in a model of cardiac Purkinje fiber.

The phase-resetting response of a model of spontaneously active cardiac Purkinje fiber is investigated. The effect on the interbeat interval of injecting a 20-ms duration depolarizing current pulse is studied as a function of the phase in the cycle at which the pulse is delivered. At low current amplitudes, a triphasic response is recorded as the pulse is advanced through the cycle. At intermediate current amplitudes, the response becomes quinquephasic, due to the presence of supernormal excitability. At high current amplitudes, a triphasic response is seen once more. At low stimulus amplitudes, type 1 phase resetting occurs; at medium amplitudes, a type could not be ascribed to the phase resetting because of the presence of effectively all-or-none depolarization; at high amplitudes, type 0 phase resetting occurs. The modeling results closely correspond with published experimental data; in particular type 1 and type 0 phase resetting are seen. Implications for the induction of ventricular arrhythmias are considered.     

Adrenergic modulation of the delayed rectifier potassium channel in calf cardiac Purkinje fibers.

We have investigated the modulation of the delayed rectifier potassium channel in calf cardiac Purkinje fibers by the neurohormone norepinephrine. We find that 0.5 microM norepinephrine increases this K channel current by a factor of 2.7. A maximal increase of about four was found for concentrations of 1 microM and above. Norepinephrine produced a small (less than 5 mV) and variable shift of the K channel reversal potential toward more negative values. The kinetics of the potassium channel are well described by a two-exponential process, both in the absence and presence of norepinephrine. However, norepinephrine substantially decreases the slower time constant with no significant effect on the fast time constant. Potassium channel activation curves in the presence of norepinephrine are very similar to control curves except at large positive potentials. A simple sequential three-state model for this channel can reproduce these data both with and without norepinephrine. The logarithms of the rate constants derived from this model are quadratic functions of voltage, suggesting the involvement of electric field-induced dipoles in the gating of this channel. Most of the kinetic effects of norepinephrine appear to be on a single rate constant.     

Cadmium-induced blockade of the cardiac fast Na channels in calf Purkinje fibres

The fast transient inward current elicited by depolarizations above about -60 mV in calf Purkinje fibres was found to be depressed by Cd in concentrations less than 1 mM. The Cd-sensitive current, which strongly depended on external Na, was recorded in the presence of 2 mM MnCl2 and was blocked by TTX, indicating that a contamination from slow Ca-dependent currents could be discounted. The current reduction caused by Cd was also observed in nominally Ca-free solutions. The Cd-induced depression of the fast Na current was not accompanied by changes in the current kinetic parameters, as revealed by comparing inactivation curves and peak current voltage relations at different Cd concentrations, and could be attributed to a voltage-independent channel blocking action. Half-blockade occurred at 0.182 +/- 0.06 mM (n = 4). Plots of peak current amplitude as a function of the Cd concentration showed that the cooperation of two Cd ions was required to block a single channel.     

A cleft model for cardiac Purkinje strands.

Conduction of the action potential in cardiac muscle is complicated by its multicellular structure, with narrow intercellular clefts and cell-to-cell coupling. A model is developed from anatomical data to describe cardiac Purkinje strands of variable diameter and different internal arrangements of cells. The admittance of the model is solved analytically and fit to results of cable analysis. Using the extracted specific membrane and cell electrical parameters (Rm = 13 K omega cm2, Cm = 1.5 mu F/cm2, Ri = 100 mu cm, and Re = 50 omega cm), the model correctly predicted conduction velocity and filling of capacitance at the onset of a voltage step. The analysis permits more complete studies of the factors controlling conduction velocity; for instance, the effect on conduction velocity of a capacity in the longitudinal current circuit is discussed. Predictions of the impedance and phase angle were also made. Measurements of the frequency dependence of phase angle may provide a basis for separating cleft membrane properties from those of the surface membrane and may aid the measurement of nonlinear membrane properties in muscle.     

The mechanism of rectification of iK1 in canine Purkinje myocytes.

We have characterized the inward rectifying background potassium current, iK1, of canine cardiac Purkinje myocytes in terms of its reversal potential, voltage activation curve, and "steady-state" current-voltage relation. The latter parameter was defined from the difference current between holding currents in the presence and absence of 20 mM cesium. Our data suggest that iK1 rectification does not arise exclusively from voltage-dependent gating or exclusively from voltage-dependent blockade by internal magnesium ions. The voltage activation curve constructed from tail currents fit to a Boltzmann two-state model predicts less outward current than is actually observed. The magnesium-dependent rectification due to channel blockade is too fast to account for the time-dependent gating of iK1 that gives rise to the tail currents. We propose a new model of rectification that assumes that magnesium blockade of the channel occurs simultaneously with voltage-dependent gating. The new model incorporates the kinetic schema elaborated by Matsuda, H. (1988. J. Physiol. 397:237-258) to explain the appearance of subconducting states of the iK1 channel in the presence of blocking ions. That schema suggested that iK1 channels were composed of three parallel pores, each of which could be blocked independently. In our model we considered the consequences of partial blockade of the channel. If the channels are partially blocked at potentials where normally they are mostly gated closed, and if the partially blocked channels cannot close, then blockade will have the paradoxical result of enhancing the current carried by iK1.     

Impulse responses of automaticity in the Purkinje fiber

We examined the effects of brief current pulses on the pacemaker oscillations of the Purkinje fiber using the model of McAllister , Noble, and Tsien (1975. J. Physiol. [Lond.]. 251:1-57). This model was used to construct phase-response curves for brief electric stimuli to find "black holes," where rhythmic activity of the Purkinje fiber ceases. In our computer simulation, a brief current stimulus of the right magnitude and timing annihilated oscillations in membrane potential. The model also revealed a sequence of alternating periodic and chaotic regimes as the strength of a steady bias current is varied. We compared the results of our computer simulations with experimental work on Purkinje fibers and pointed out the importance of modeling results of this kind for understanding cardiac arrhythmias.     

Immunological studies on the Purkinje cells from rat and mouse cerebella. I. Evidence for antibodies characteristic of the Purkinje cells.

Antibodies have been raised against an enriched preparation of isolated rat cerebellar Purkinje cells. The immunoglobulins were labeled with ¹²⁵I and the strength and specificity of the serum determined by a direct binding assay on cerebellar membranes. About 2% of the ¹²⁵I-labeled IgG bound to an excess of cerebellar membranes. Absorption with heart and liver membranes removed 80.5% of the ¹²⁵I-labeled IgG binding to cerebellar membranes; absorption with cerebrum membranes removed 13% more; the remaining 6.5% were directed specifically against cerebellar membranes. An enriched ¹²⁵I-labeled anti-Purkinje antibody population was prepared by absorption and subsequent elution from cerebellar membranes. The absorption pattern with heart, liver, and cerebrum membranes resembled that found with the total population of IgG except that the nonspecific binding was significantly diminished. The Purkinje cell degeneration (pcd) mouse mutant was used to assess the specificity of the serum toward the Purkinje cells. After absorption of the enriched anti-Purkinje antibodies with heart, liver, and cerebrum membranes, the binding of labeled IgG to membranes prepared from pcd/pcd cerebella was about one-fourth that found with control cerebella. The direct immunoperoxidase technique performed on smears of purified Purkinje and granule cells shows that the unabsorbed serum stains both classes of cells, but that after absorption with liver, heart, and cerebrum membranes, only the Purkinje cells react positively.     

Hydrogen ion modulation of Ca channel current in cardiac ventricular cells. Evidence for multiple mechanisms

We have investigated the effects of H ions on (L-type) Ca channel current in isolated ventricular cells. We find that the current amplitude is enhanced in solutions that are alkaline relative to pH 7.4 and reduced in solutions acidic to this pH. We measured pH0-induced shifts in channel gating and analyzed our results in terms of surface potential theory. The shifts are well described by changes in surface potential caused by the binding of H ions to negative charges on the cell surface. The theory predicts a pK of 5.8 for this binding. Gating shifts alone cannot explain all of our observations on modulation of current amplitude. Our results suggest that an additional mechanism contributes to modification of the current amplitude.     

The relation of Vmax to INa, GNa, and h infinity in a model of the cardiac Purkinje fiber.

The inward sodium current in cardiac muscle is difficult to study by voltage clamp methods, so various indirect experimental measures have been used to obtain insight into its characteristics. These methods depend on the relationship between maximal upstroke velocity of the action potential (Vmax) and the sodium current (INa), usually defined in terms of the Hodgkin-Huxley model. These relationships were explored using an adaptation of this model to cardiac Purkinje fibers. In general Vmax corresponded to INa, and it could be used to determine the relationship of membrane potential to GNa, and h infinity. The results, however, depended on the method of stimulation of the action potential, and an optimal stimulation method was determined. A commonly used experimental technique called "membrane responsiveness" was shown to distort seriously the properties of steady-state gating inactivation that is supposed to measure. Estimation of the changes in maximal sodium conductance, such as those produced by tetrodotoxin (TTX), would be accurately measured. Some experimental results have indicated a voltage-dependent effect of TTX. Characteristics of the measures of TTX effect under those conditions were illustrated. In summary, calculations with a model of the cardiac Purkinje fiber action potential provide insight into the accuracy of certain experimental methods using maximal upstroke velocity as a measure of INa, and cast doubt on other experimental methods, such as membrane responsiveness.     

Ionic mechanisms of pacemaker activity in cardiac Purkinje fibers.

Rhythmic activity in cardiac Purkinje fibers can be analyzed by using the voltage clamp technique to study pacemaker currents. In normally polarized preparations, pacemaker activity can be generated by two distinct ionic mechanisms. The standard pacemaker potential (phase 4 depolarization) involves a slow potassium current, IK2. Following action potential repolarization, the IK2 channels slowly deactivate and thus unmask a steady background inward current. The resulting net inward current causes the slow pacemaker depolarization. Epinephrine accelerates the diastolic depolarization by promoting more complete and more rapid deactivation of IK2 over the pacemaker range of potentials. The catecholamine acts rather selectively on the voltage dependence of the gating mechanism, without altering the basic character of the pacemaker process. The nature of the pacemaker depolarization is altered by intoxication with high concentrations of cardiac glycosides or aglycones. These compounds promote spontaneous impulses in Purkinje fibers by a mechanism that supersedes the ordinary IK2 pacemaker. The digitalis-induced depolarization is generated by a transient inward current that is either absent or very small in untreated preparations. The transient inward current is largely carried by sodium ions. Its unusual time course probably reflects an underlying subcellular event, the oscillatory release of calcium ions from intracellular stores.     

Membrane currents following activity in canine cardiac Purkinje fibers.

Recent experiments in canine Pukinje fibers (Gadsby and Cranefield, 1979) have shown that following a period of sodium loading in K+-free solution a slowly decaying outward current is observed. This current has been attributed to the activity of the electrogenic Na+-K+ exchange pump. In the present paper we show that similar slowly decaying outward currents are observed following prolonged periods of overdrive with action potentials or with brief depolarizing voltage clamp pulses. The dependent of the prolonged outward current on the duration and frequency of the preceding period of overdrive and on the potential following overdrive is reported. We also present results which indicate that a large portion of this current can be induced by phasic Na+ loading through the fast-inward channel.     

Low conductance sodium channels in canine cardiac Purkinje cells.

Low conductance sodium (Na) channels have been observed in nerve, skeletal muscle, and cardiac cells. In cardiac tissues the higher amplitude, more commonly observed Na channel was first investigated in detail by Cachelin et al. (Cachelin, A.B., J.E. de Peyer, S. Kokubun, and H. Reuter, 1983, J. Physiol. (Lond.), 340:389-402). They also reported low amplitude Na channel events. We have studied this low conductance Na channel in single canine cardiac Purkinje cells using cell-attached patches. Patch pipette solutions contained either 140 or 280 mM NaCl, and cells were bathed in a solution of 150 mM KCl to bring their resting potential close to zero. In 140 mM Na+, during steps to -50 mV, the lower and higher openings had amplitudes of 0.57 +/- 0.2 and 1.2 +/- 0.2 pA (means +/- SD of Gaussian fits). In 280 mM Na+ at -50 mV, amplitudes were 0.72 +/- 0.2 and 1.55 +/- 0.2 pA. Over a substantial voltage range, the lower events had amplitudes of about one-third that of the higher events. The frequency of the low conductance openings varied in different patches from zero to 22% of total openings. Histograms of open durations and latencies at several voltages suggested no difference in kinetics between the two channel events. The behavior of the low conductance channels was more consistent with a second population of channels rather than a second open state.     

Evidence for glycosylphosphatidylinositol anchoring of intralumenal alkaline phosphatase of the calf intestine.

1. Considerable amounts of intestinal alkaline phosphatase (AP) were found intralumenally in all animal species investigated, i.e. calf, pig, goat, rat, mouse, guinea pig, hen and carp. The ratios between the total activity of AP found intralumenally and the total intestinal activity vary considerably. Calves and pigs show the highest, i.e. 0.77 and 0.44, respectively, while rodents have much lower ratios. Only 20-34% of the intralumenal alkaline phosphatase (IAP) of the calf and pig is soluble and not within the sediment after centrifugation at 135,000 x g for 60 min. whereas the IAP of rodents is soluble in the range of 60-72% of the total IAP. 2. For the IAP of the mucosa and chyme of calf, all criteria were found which are generally used, indicating a glycosylphosphatidylinositol (GlcPtdIns) anchor as proved by strong hydrophobicity using Triton X-114 phase partitioning, phenyl-Sepharose binding and enzyme aggregation, and the susceptibility to phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and papain digestion. 3. More than 80% of the mucosa alkaline phosphatase (MAP) of the proximal part of the intestine and of the particulate fraction of IAP exhibit these criteria indicating the presence of the GlcPtdIns-anchor structure, whereas the anchor content of the soluble intralumenal enzyme decreases from the pylorus to the ileocecal junction. 4. MAP partially purified to a specific activity of 1747 IU/mg retains the anchor structure. 5. The results presented indicate that the release of large amounts of AP into the chyme is realized without splitting the GlcPtdIns anchor. The possible intralumenal function of this form of AP is discussed.     

An upper limit for the electrogenic Na-K pump contribution to maximum diastolic potential in feline cardiac Purkinje fibers in steady state

We have estimated an upper limit for the electrogenic contribution of the Na-K pump to diastolic transmembrane potential. We simultaneously monitored the maximum diastolic potential and the extracellular space potassium activity during exposure to a very high concentration of ouabain. Exposure to ouabain caused a depolarization of approximately 3 mV (n = 33 experiments) over 34 +/- 3 s (mean +/- standard error) prior to any change in extracellular K activity. In four experiments, we monitored intracellular sodium activity and observed it to rise with approximately the same temporal lag (delay = 26 +/- 7 s). We also measured relative membrane conductance in one series of experiments and observed it to decrease to 91 +/- 2% of its control value by the time extracellular space K began to rise. Following the initial increase in extracellular space K activity the subsequent membrane depolarization is shown to be accurately predicted solely from the measured increase in extracellular space K activity as calculated from the Goldman equation. Limitations of the method and possible interpretations of the data are discussed. We interpret this ouabain-induced depolarization that occurs prior to the rise in external K to be an upper limit to the Na-K pump's electrogenic contribution to steady-state membrane potential.     

Divalent cation changes in cerebellar Purkinje cells after climbing fiber deafferentation.

A secondary ion mass spectrometry (SIMS) microscope was used to detect intracellular stores of calcium, magnesium, sodium and potassium. Measurements were made in semithin sections of fixed tissues of normal and climbing fiber deafferented cerebellar cortex. Quantitative data were collected from 150 microns diameter image fields in the molecular and granule layers. The results indicate smaller quantities of both calcium and magnesium in the deafferented cerebellar cortex compared to the normals, the molecular as well as the granule layer being affected. The results are discussed in terms of the usefulness and limitations of the SIMS microscope for histological preparations.     

Background K+ current in isolated canine cardiac Purkinje myocytes.

The current-voltage (I-V) relation of the background current, IK1, was studied in isolated canine cardiac Purkinje myocytes using the whole-cell, patch-clamp technique. Since Ba2+ and Cs+ block IK1, these cations were used to separate the I-V relation of IK1 from that of the whole cell. The I-V relation of IK1 was measured as the difference between the I-V relations of the cell in normal Tyrode (control solution) and in the presence of either Ba2+ (1 mM) or Cs+ (10 mM). Our results indicate that IK1 is an inwardly rectifying K+ current whose conductance depends on extracellular potassium concentration. In different [K+]0's the I-V relations of IK1 exhibit crossover. In addition the I-V relation of IK1 contains a region of negative slope (even when that of the whole cell does not). We also examined the relationship between the resting potential of the myocyte, Vm, and [K+]0 and found that it exhibits the characteristic anomalous behavior first reported in Purkinje strands (Weidmann, S., 1956, Elektrophysiologie der Herzmuskelfaser, Med. Verlag H. Huber), where lowering [K+]0 below 4 mM results in a depolarization.     

Transient state kinetics tutorial using the kinetics simulation program, KINSIM.

This article provides an introduction to a computer tutorial on transient state kinetics. The tutorial uses our Macintosh version of the computer program, KINSIM, that calculates the time course of reactions. KINSIM is also available for other popular computers. This program allows even those investigators not mathematically inclined to evaluate the rate constants for the transitions between the intermediates in any reaction mechanism. These rate constants are one of the insights that are essential for understanding how biochemical processes work at the molecular level. The approach is applicable not only to enzyme reactions but also to any other type of process of interest to biophysicists, cell biologists, and molecular biologists in which concentrations change with time. In principle, the same methods could be used to characterize time-dependent, large-scale processes in ecology and evolution. Completion of the tutorial takes students 6-10 h. This investment is rewarded by a deep understanding of the principles of chemical kinetics and familiarity with the tools of kinetics simulation as an approach to solve everyday problems in the laboratory.