Calcium-activated Cl(-) current contributes to delayed afterdepolarizations in single Purkinje and ventricular myocytes.

BACKGROUND: The ionic mechanism underlying the transient inward current (I(ti)), the current responsible for delayed afterdepolarizations (DADs), appears to be different in ventricular myocytes and Purkinje fibers. In ventricular myocytes, I(ti) was ascribed to a Na(+)-Ca(2+) exchange current, whereas in Purkinje fibers, it was additionally ascribed to a Cl(-) current and a nonselective cation current. If Cl(-) current contributes to I(ti) and thus to DADs, Cl(-) current blockade may be potentially antiarrhythmogenic. In this study, we investigated the ionic nature of I(ti) in single sheep Purkinje and ventricular myocytes and the effects of Cl(-) current blockade on DADs. METHODS AND RESULTS: In whole-cell patch-clamp experiments, I(ti) was induced by repetitive depolarizations from -93 to +37 mV in the presence of 1 micromol/L norepinephrine. In both Purkinje and ventricular myocytes, I(ti) was inward at negative potentials and outward at positive potentials. The anion blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) blocked outward I(ti) completely but inward I(ti) only slightly. The DIDS-sensitive component of I(ti) was outwardly rectifying, with a reversal close to the reversal potential of Cl(-) currents. Blockade of Na(+)-Ca(2+) exchange by substitution of extracellular Na(+) by equimolar Li(+) abolished the DIDS-insensitive component of I(ti). DIDS reduced both DAD amplitude and triggered activity based on DADs. Conclusions-In both Purkinje and ventricular myocytes, I(ti) consists of 2 ionic mechanisms: a Cl(-) current and a Na(+)-Ca(2+) exchange current. Blockade of the Cl(-) current may be potentially antiarrhythmogenic by lowering DAD amplitude and triggered activity based on DADs.

Ionic mechanism of delayed afterdepolarizations in ventricular cells isolated from human end-stage failing hearts.

BACKGROUND: Animal studies have shown that the Ca(2+)-activated Cl(-) current (I(Cl(Ca))) and the Na(+)/Ca(2+) exchange current (I(Na/Ca)) contribute to the transient inward current (I(ti)). I(ti) is responsible for the proarrhythmic delayed afterdepolarizations (DADs). We investigated the ionic mechanism of I(ti) and DADs in human cardiac cells. METHODS AND RESULTS: Human ventricular cells were enzymatically isolated from explanted hearts of patients with end-stage heart failure and studied with patch-clamp methodology. I(ti)s were elicited in the presence of 1 micromol/L norepinephrine by trains of repetitive depolarizations from -80 to +50 mV. DADs were induced in the presence of 1 micromol/L norepinephrine at a stimulus frequency of 1 Hz. I(ti) currents were inwardly directed over the voltage range between -110 and + 50 mV. Neither the Cl(-) channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid nor changes in [Cl(-)](i) affected I(ti) or DAD amplitude. This excludes an important role for I(Cl(Ca)). Blockade of Na(+)/Ca(2+) exchange by substitution of all extracellular Na(+) by Li(+), conversely, completely inhibited I(ti). In rabbit, I(Cl(Ca)) density in ventricular cells isolated from control hearts did not differ significantly from that in ventricular cells isolated from failing hearts. CONCLUSIONS: In contrast to many animal species, I(ti) and DADs in human ventricular cells from failing hearts consist only of I(Na/Ca). In rabbits, heart failure per se does not alter I(Cl(Ca)) density, suggesting that I(Cl(Ca)) may also be absent during DADs in nonfailing human ventricular cells.

Pacemaker synchronization of electrically coupled rabbit sinoatrial node cells.

The effects of intercellular coupling conductance on the activity of two electrically coupled isolated rabbit sinoatrial nodal cells were investigated. A computer-controlled version of the "coupling clamp" technique was used in which isolated sinoatrial nodal cells, not physically in contact with each other, were electrically coupled at various values of ohmic coupling conductance, mimicking the effects of mutual interaction by electrical coupling through gap junctional channels. We demonstrate the existence of four types of electrical behavior of coupled spontaneously active cells. As the coupling conductance is progressively increased, the cells exhibit: (a) independent pacemaking at low coupling conductances, (b) complex dynamics of activity with mutual interactions, (c) entrainment of action potential frequency at a 1:1 ratio with different action potential waveforms, and (d) entrainment of action potentials at the same frequency of activation and virtually identical action potential waveforms. The critical value of coupling conductance required for 1:1 frequency entrainment was <0.5 nS in each of the five cell pairs studied. The common interbeat interval at a relatively high coupling conductance (10 nS), which is sufficient to produce entrainment of frequency and also identical action potential waveforms, is determined most by the intrinsically faster pacemaker cell and it can be predicted from the diastolic depolarization times of both cells. Evidence is provided that, at low coupling conductances, mutual pacemaker synchronization results mainly from the phase-resetting effects of the action potential of one cell on the depolarization phase of the other. At high coupling conductances, the tonic, diastolic interactions become more important.

Decreased inward rectifier current in adult rabbit ventricular myocytes maintained in primary culture: a single-channel study.

OBJECTIVE: Regulation of ion channel function in heart has been shown to be affected by changes in the cellular environment. Recently it was shown that rabbit ventricular myocytes kept in primary culture, show a strong reduction in inward rectifier current (IK1). The aim of the present study was to elucidate the mechanism underlying this decrease in IK1, using single-channel measurements. In addition, we studied the effects of primary culture on the ATP-regulated K+ (K.ATP) channel, also a member of the inwardly rectifying K+ channel family. METHODS: Adult rabbit ventricular myocytes were cultured for up to 3 days in Ham's F-10 medium complemented with 1% rabbit serum and 5% glutamine. IK1 and K.ATP channel activity was studied in the inside-out patch configuration of the patch-clamp technique with equimolar K+ concentrations (140 mM K+) on the intra- and extracellular side. Single channel characteristics were determined at various times during culture and compared to those present in freshly isolated myocytes. RESULTS: IK1 channels in freshly isolated myocytes (day 0) had a single-channel conductance of 56.1 +/- 2.5 pS (mean +/- SEM) and an open probability of 0.64 +/- 0.05 (mean +/- SEM). Neither the single-channel conductance nor the open probability (Po) underwent significant changes during culture. The mean number of channels per patch, however, was drastically reduced from 1.2 +/- 0.3 (mean +/- SEM) at day 0 to 0.17 +/- 0.06 at day three. K.ATP channel density and open probability, on the other hand, were both increased with an optimum at day two. Po increased from 0.27 +/- 0.06 at day 0 to 0.63 +/- 0.06 at day three. The mean number of channels per patch was 2.29 +/- 0.57 and 3.25 +/- 0.48 at days 0 and 3 respectively. The unitary current amplitude at -50 mV remained unchanged, suggesting no change in the K.ATP single-channel conductance. CONCLUSIONS: The decrease in IK1 in rabbit ventricular myocytes as has been observed during primary culture is the result of a reduction in the number of active channels and not of altered kinetic or conductive channel properties. The increase in K.ATP channel activity under the same conditions suggests that gene expression of both channel types is differently regulated.

Phentolamine blocks ATP sensitive potassium channels in cardiac ventricular cells.

OBJECTIVE: The alpha adrenoceptor antagonist phentolamine prevents ischaemia related arrhythmias in rat, guinea pig, and cat heart. This effect has been related to the attenuation of ischaemia induced shortening of the action potential and has been ascribed to its alpha adrenoceptor antagonist properties. The aim of this study was to examine the effect of phentolamine on the ATP sensitive potassium channel (KATP), because this channel seems to be involved in action potential shortening during ischaemia. METHODS: Single channel experiments were performed on inside-out and outside-out patches of isolated rabbit ventricular cells at room temperature. Cells were isolated with conventional isolation techniques. Pipette and bath solution contained (in mmol.litre-1): K-gluconate 140, KCl 10, and HEPES-KOH 10 (pH 7.4). RESULTS: Excision of the patch always resulted in KATP channel activity [single channel conductance 60(SD 2.8) pS n = 4], which could be completely blocked by 5 mM ATP. In 22 of 26 patches the addition of 5 microM phentolamine to the intracellular side of the membrane reduced KATP channel activity. In 17 of these patches the effect was reversible. In four patches no effect was observed. Open probability decreased by 94% (n = 12). Addition of 50 microM phentolamine resulted in the disappearance of channel activity in six of eight patches which was reversible in four patches. In outside-out patches 5 microM phentolamine was only effective in 50% of the patches, reducing open probability by 98 to 100%. CONCLUSIONS: Phentolamine blocks ATP sensitive potassium channels in rabbit ventricular cells independently of the alpha adrenoceptor. This blocking effect probably occurs at the intracellular side of the membrane. The antiarrhythmic effect of phentolamine may at least partially be explained by blockade of KATP channels and may thus partly be independent of its effects on the alpha adrenoceptor.

Effects of cell-to-cell uncoupling and catecholamines on Purkinje and ventricular action potentials: implications for phase-1b arrhythmias.

OBJECTIVE: The delayed phase of ventricular arrhythmias during acute ischemia (phase-1b arrhythmia) is associated with depletion of catecholamines and cell-to-cell uncoupling between depressed depolarized intramural ischemic region and surviving cells in subepicardium and subendocardium. In the present study we determined the effects of uncoupling and catecholamines on development of proarrhythmic afterdepolarizations. METHODS: Depressed depolarized ischemic region was simulated by a passive electronic circuit with a potential of -73, -53, -33 or -13 mV. Using patch-clamp methodology, single sheep Purkinje and ventricular cells were coupled to the simulated ischemic region via a variable conductance. By varying coupling conductance, we were able to selectively study the effects of various degrees of uncoupling. RESULTS: At strong coupling, cells were inexcitable and depolarized to potentials near those of the simulated ischemic region. Excitability, action potential duration and resting potential increased with progressive uncoupling. In a critical range of uncoupling, ventricular and "high-plateau" Purkinje cells developed early afterdepolarizations when the potential of the simulated ischemic region was -13 mV. Norepinephrine (1 microM) frequently induced early and delayed afterdepolarizations in both ventricular and Purkinje cells, but these afterdepolarizations were only present during uncoupling when the potential of the simulated ischemic region was -33 mV or more positive. CONCLUSIONS: In a critical range of uncoupling, afterdepolarizations were present when the potential of the simulated ischemic region was -33 or -13 mV, suggesting that triggered activity plays a role in phase-1b arrhythmias when surviving layers uncouple from a highly depolarized intramural ischemic region.

Injury current modulates afterdepolarizations in single human ventricular cells.

OBJECTIVE: Injury current (I(injury)) and afterdepolarizations are thought to play an important role in arrhythmias that occur during acute ischemia. However, little is known about the effects of I(injury) on afterdepolarizations. The present study was designed to study the effect of I(injury) on afterdepolarizations and action potentials in single human ventricular cells. METHODS: The patch-clamp technique was used to record action potentials and to apply I(injury) to human ventricular cells. In these cells, early and delayed afterdepolarizations (EADs and DADs) were induced by 1 microM norepinephrine. I(injury) was simulated by coupling cells via a variable coupling resistance to a passive resistance circuit with a potential of 0, -20, or -40 mV, mimicking a depolarized ischemic region. RESULTS: At all potentials, I(injury) induced depolarization of the resting membrane potential and action potential shortening. Flowing from 0 mV, I(injury) induced EADs by itself and aggravated the EADs and DADs that were induced by norepinephrine. Flowing from -40 mV, I(injury) abolished the noradrenaline-induced EADs and DADs. CONCLUSIONS: Our results demonstrate that I(injury) may either prevent or promote the occurrence of afterdepolarizations in human ventricle. The latter holds if conduction is slowed to such an extent that it permits flow of current from depolarized ischemic cells at plateau level to cells in phase 3 or phase 4.

Human SCN5A gene mutations alter cardiac sodium channel kinetics and are associated with the Brugada syndrome.

BACKGROUND: Primary dysrhythmias other than those associated with the long QT syndrome, are increasingly recognized. One of these are represented by patients with a history of resuscitation from cardiac arrest but without any structural heart disease. These patients exhibit a distinct electrocardiographic (ECG) pattern consisting of a persistent ST-segment elevation in the right precordial leads often but not always accompanied by a right bundle branch block (Brugada syndrome). This syndrome is associated with a high mortality rate and has been shown to display familial occurrence. METHODS AND RESULTS: Pharmacological sodium channel blockade elicits or worsens the electrocardiographic features associated with this syndrome. Hence, a candidate gene approach directed towards SCN5A, the gene encoding the alpha-subunit of the cardiac sodium channel, was followed in six affected individuals. In two patients missense mutations were identified in the coding region of the gene: R1512W in the DIII-DIV cytoplasmic linker and A1924T in the C-terminal cytoplasmic domain. In two other patients mutations were detected near intron/exon junctions. To assess the functional consequences of the R1512W and A1924T mutations, wild-type and mutant sodium channel proteins were expressed in Xenopus oocytes. Both missense mutations affected channel function, most notably a 4-5 mV negative voltage shift of the steady-state activation and inactivation curves in R1512W and a 9 mV negative voltage shift of the steady-state activation curve in A1924T, measured at 22 degrees C. Recovery from inactivation was slightly prolonged for R1512W channels. The time dependent kinetics of activation and inactivation at -20 mV were not significantly affected by either mutation. CONCLUSIONS: Two SCN5A mutations associated with the Brugada syndrome, significantly affect cardiac sodium channel characteristics. The alterations seem to be associated with an increase in inward sodium current during the action potential upstroke.

Cl- current blockade reduces triggered activity based on delayed afterdepolarisations.

OBJECTIVES: Increasing evidence suggests that a Ca2+-activated Cl- current (ICl(Ca)) contributes to the transient inward current (Iti), the current responsible for proarrhythmic delayed after-depolarisations (DADs). Because the equilibrium potential for Cl- ions (ECl) in myocytes is around - 50 mV, activation of the ICl(Ca) results in an inward depolarising current at resting membrane potential and ICl(Ca) may thus be responsible for a part of the depolarisation during a DAD. In this study, we investigated the ionic nature of Iti and the effects of Cl- current blockade on DADs. METHODS AND RESULTS: The ionic mechanisms of Iti and underlying DADs were studied in sheep ventricular myocytes using the patch-clamp methodology. The DADs were induced in the myocytes by exposure to 1 µM noradrenaline and the Iti were elicited by repetitive depolarisations from -93 mV to +37 mV in the presence of the drug. The current-voltage relation of Iti reversed in sign around -20 mV. The outward Iti was completely blocked by the anion current blocker 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), whereas the inward Iti was only slightly affected. The DIDS-sensitive component of Iti was outwardly rectifying with a reversal potential close to ECl. The DIDS-insensitive component of Iti was abolished by blockade of the Na+-Ca2+ exchanger by substitution of extracellular Na+ by equimolar Li+. Interestingly, DIDS reduced the DAD amplitude and triggered activity based on DADs. CONCLUSION: In sheep ventricular myocytes, Iti consists of two ionic mechanisms: a Cl- current and a Na+-Ca2+ exchange current. Blockade of the Cl- current may be potentially antiarrhythmic by lowering DAD amplitude and triggered activity based on DADs.

Voltage clamp measurements of the hyperpolarization-activated inward current I(f) in single cells from rabbit sino-atrial node.

1. The kinetics and ion transfer characteristics of the hyperpolarization-activated inward current, I(f), have been studied in single cells obtained by enzymatic dispersion from the rabbit sino-atrial (S-A) node. These experiments were done to assess the role of I(f) in the generation of the pacemaker depolarization in the S-A node. 2. The activation and the deactivation of I(f) in these single cells are accompanied by significant conductance increases and decreases respectively, confirming earlier findings from multicellular man-made strips of rabbit S-A node, and from mammalian Purkinje fibres. 3. The steady-state activation of I(f) lies between -40 and -120 mV, and its voltage dependence can be described by a Boltzmann relation with the half-activation point at approximately -70 mV. 4. The delay or sigmoidicity in both the onset of I(f) and the deactivation of the tail currents can be accounted for semi-quantitatively by using a second-order Hodgkin-Huxley kinetic scheme. 5. The reversal potential for I(f) is -24 +/- 2 mV (mean +/- S.E.M., n = 6). It does not change significantly as a function of the amount of I(f) which is activated, indicating that ion accumulation or depletion phenomena are not important variables controlling the time course of I(f), or its selectivity. 6. The fully-activated current-voltage relationship for I(f) is approximately linear with a slope conductance of 12.0 +/- 0.88 nS per cell (mean +/- S.E.M., n = 6). 7. A simple mathematical model based on the measured values of maximum conductance, reversal potential, and kinetics of I(f) has been developed to simulate the size and time course of I(f) during typical spontaneous pacemaker activity in rabbit sino-atrial node cells. The calculations show that I(f) can change significantly during pacing and suggest that this current change is, at least in part, responsible for the pacemaker depolarization.

Pacemaker activity of the rabbit sinoatrial node. A comparison of mathematical models.

In the past decade, three mathematical models describing the pacemaker activity of the rabbit sinoatrial node have been developed: the Bristow-Clark model, the Irisawa-Noma model, and the Noble-Noble model. In a comparative study it is demonstrated that these models, as well as subsequent modifications, all have several drawbacks. A more accurate model, describing the pacemaker activity of a single pacemaker cell isolated from the rabbit sinoatrial node, was constructed. Model equations, including equations for the T-type calcium current, are based on experimental data from voltage clamp experiments on single cells that were published during the last few years. In contrast to the other models, only a small amount of background current contributes to the overall electrical charge flow. The action potential parameters of the model cell, its responses to voltage clamp steps and its current-voltage relationships have been computed. The model is used to discuss the relative contribution of membrane current components to the slow diastolic depolarization phase of the action potential.

A transient outward current in isolated cells from the crista terminalis of rabbit heart.

Voltage-clamp experiments were carried out with the objective of identifying and characterizing the time- and voltage-dependent properties of a transient outward current recorded in single myocytes from the crista terminalis region of the rabbit heart. A collagenase enzymic dispersion procedure similar to that described by Desilets & Horackova (1982) was used to obtain these viable individual myocytes. Transmembrane ionic currents were recorded using a single micro-electrode voltage-clamp technique. In experiments aimed at studying a tetrodotoxin-resistant transient inward current, (ICa); a transient outward current was consistently recorded following blockade of ICa with Cd2+ (5 X 10(-4) M). The time and voltage dependence of the activation and inactivation of this current were measured. Its steady-state inactivation curve spans the voltage range -70 to -10 mV, and it is activated between -20 and +10 mV. The reversal potential of this transient outward current is approximately -75 mV in [K+]O 5 mM, suggesting that it is carried mainly by K+. This transient outward current can be inhibited completely by external application of 4-aminopyridine (4-AP, 3 mM). The time- and voltage-dependent properties, the reversal potential, and the sensitivity to 4-AP of this transient outward current are all very similar to those of a transient outward current first identified in molluscan neurones. Hence, we have labelled it, IA. Selective inhibition of IA and knowledge of its voltage- and time-dependent properties yield specific predictions concerning its role in the action potential of isolated crista terminalis cells. Consistent with these predictions, a decrease in stimulus rate is found to decrease the duration of the action potential and vice versa; and application of effective doses of 4-AP results in a substantial lengthening of the action potential. These results are discussed in terms of the possible physiological role of IA in subsidiary or follower pace-maker tissue, and the anatomical and physiological heterogeneity of the sino-atrial node region of the rabbit heart.

Slow inward current in aggregates of neonatal rat heart cells and its contribution to the steady state current-voltage relationship.

In voltage clamped neonatal rat heart cells a transient current is observed during depolarizing potential steps, which was identified as slow inward current (Isi) by its range of activation, by its reversal potential of approximately +50 mV and by its sensitivity to D600 or low external Ca2+. This Isi activates too fast to be detected by the present methods, which implies that activation is completed within milliseconds. The time constant of inactivation was weakly potential dependent and less than 30 ms between -40 mV and +20 mV. The f infinity curve of Isi had a sigmoidal shape with 90% and 10% values near -50 mV and -10 mV respectively, half maximum was at -25 mV. From double pulse experiments an estimate was obtained of the potential dependence and amplitude of steady state Isi. A maximum was expected around -30 mV. Steady state Isi appears to be present indeed in steady state current voltage relations, as the relative minimum at -30 mV in such relations is abolished by 5 X 10(-7) g/ml D600. Currents tails during hyperpolarizing steps from prepulse potentials near 0 mV are potential dependent in a way expected when Isi contributes to these current tails by a decrease in inactivation. Moreover, the current tails are diminished by D600 or Co2+. Consequences of steady state Isi are discussed.

Single delayed rectifier channels in the membrane of rabbit ventricular myocytes.

In rabbit ventricular cells, the delayed rectifier current (IK) has not been extensively studied, and properties of single IK channels still need to be determined. In this study, we present data on a voltage-dependent channel in rabbit ventricular cells; the properties indicate that it is an IK channel. Patch-clamp experiments were carried out on cell-attached and inside-out patches of rabbit ventricular cells. Single-channel currents were recorded at negative potentials as inward currents with 150 mM K+ in the pipette. Voltage-dependent channel activity was only present after the return from a depolarizing test pulse, indicating activation on depolarization. Single-channel conductance calculated from the current-voltage relation was 13.1 pS (pooled data, n = 8). The shift in reversal potential of the unitary currents, determined at 150 and 300 mM K+ at the intracellular side of the membrane, showed that the channels were highly permeable to potassium ions. Increase of the duration or the amplitude of the depolarizing test pulse increased channel activity. The time constant for activation at +30 mV was 187 msec (pooled data, n = 4). Half-activation potential was -4.9 +/- 3.8 mV (mean +/- SD), and the slope factor was 7.2 +/- 3.7 mV (mean +/- SD). Current tails, reconstructed from averaged single-channel currents, revealed that the time course of deactivation decreased from 694 +/- 73 msec at -80 mV to 136 +/- 39 msec at -110 mV. Additional evidence that the channel was indeed an IK channel was provided by the observation that the channel was blocked by 10(-7) M E-4031, a class III antiarrhythmic agent that has been shown to block a component of the macroscopic IK in guinea pig heart.

Effects of delayed rectifier current blockade by E-4031 on impulse generation in single sinoatrial nodal myocytes of the rabbit.

The role of the delayed rectifier current (IK) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit. We used the class III antiarrhythmic drug E-4031, which blocks IK in rabbit ventricular myocytes. In single sinoatrial nodal cells, E-4031 (0.1 mumol/L) significantly prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. Half of the cells were arrested completely. At higher concentrations (1 and 10 mumol/L), spontaneous activity ceased in all cells. Three ionic currents fundamental for pacemaking, ie, IK, the long-lasting inward calcium current (ICa,L), and the hyperpolarization-activated current (I(f)), were studied by using the whole-cell and amphotericin-perforated patch technique. E-4031 blocked part of the outward current during depolarizing steps as well as the tail current upon subsequent repolarization (ITD) in a dose-dependent manner. E-4031 (10 mumol/L) depressed ITD (88 +/- 4%) (n = 6), reduced peak ICa,L at 0 mV (29 +/- 15%) (n = 4), but did not affect I(f). Lower concentrations did not affect ICa,L. Additional use of 5 mumol/L nifedipine demonstrated that ITD is carried in part by a calcium-sensitive current. Interestingly, complete blockade of IK and ICa,L unmasked the presence of a background current component with a reversal potential of -32 +/- 5.4 mV (n = 8) and a conductance of 39.5 +/- 5.6 pS/pF, which therefore can contribute both to the initial part of repolarization and to full diastolic depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed rectifier channels in human ventricular myocytes.

BACKGROUND: Previous studies have shown that in heart there are two kinetically distinct components of delayed rectifier current: a rapidly activating component (IKr) and a more slowly activating component (IKs). The presence of IKr and/or IKs appears to be species dependent. We studied the nature of the delayed rectifier current in human ventricle in whole-cell and single-channel experiments. METHODS AND RESULTS: Ventricular myocytes were obtained from hearts of patients with ischemic or dilated cardiomyopathy. Single-channel currents and whole-cell tail currents were recorded at negative potentials directly after return from a depolarizing step. Single-channel currents were measured in the cell-attached patch configuration with 140 mmol/L K+ in the pipette. In the present study, we identified a voltage-dependent channel with a single-channel conductance of 12.9 +/- 0.8 pS (mean +/- SEM, n = 5) and a reversal potential near to the K+ equilibrium potential, suggesting that the channel is selective to K+ ions. Channel activity was observed only after a depolarizing step and increased with the duration and amplitude of the depolarization, indicating time- and voltage-dependent activation. Activation at +30 mV was complete within 300 milliseconds, and the time constant of activation, determined in the whole-cell configuration, was 101 +/- 25 milliseconds (mean +/- SEM, n = 4). The voltage dependence of activation could be described by a Boltzmann equation with a half-activation potential of -29.9 mV and a slope factor of 9.5 mV. The addition of the class III antiarrhythmic drug E-4031 completely blocked channel activity in one patch. No indications for the presence of IKs were found in these experiments. CONCLUSIONS: The conformity between the properties of IKr and those of the K+ channel in the present study strongly suggests that IKr is present in human ventricle.

Contribution of L-type Ca2+ current to electrical activity in sinoatrial nodal myocytes of rabbits.

The role of L-type calcium current (ICa,L) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit, with the use of the amphotericin-perforated patch-clamp technique. Nifedipine, at a concentration of 5 microM, was used to block ICa,L. At this concentration, nifedipine selectively blocked ICa,L for 81% without affecting the T-type calcium current (ICa,T), the fast sodium current, the delayed rectifier current (IK), and the hyperpolarization-activated inward current. Furthermore, we did not observe the sustained inward current. The selective action of nifedipine on ICa,L enabled us to determine the activation threshold of ICa,L, which was around -60 mV. As nifedipine (5 microM) abolished spontaneous activity, we used a combined voltage- and current-clamp protocol to study the effects of ICa,L blockade on repolarization and diastolic depolarization. This protocol mimics the action potential such that the repolarization and subsequent diastolic depolarization are studied in current-clamp conditions. Nifedipine significantly decreased action potential duration at 50% repolarization and reduced diastolic depolarization rate over the entire diastole. Evidence was found that recovery from inactivation of ICa,L occurs during repolarization, which makes ICa,L available already early in diastole. We conclude that ICa,L contributes significantly to the net inward current during diastole and can modulate the entire diastolic depolarization.