Fast Sodium Current Research Articles

Inhibitory effects of the antiestrogen agent clomiphene on cardiac sarcolemmal anionic and cationic currents.

The aim of this study was to determine the effects of the antiestrogen agent clomiphene on cardiac anionic and cationic sarcolemmal ion channels. Whole-cell recordings were made from rat and guinea pig ventricular myocytes. Clomiphene inhibited the volume-regulated chloride current [I(Cl,vol), activated by cell swelling after hypotonic shock (approximately 145 mOsM)] with an IC(50) value of approximately 9.4 microM. In contrast, at concentrations up to 100 microM, clomiphene failed to inhibit both the chloride current activated by cyclic AMP (I(Cl,cAMP)) and the anionic background current (I(AB)). At 10 microM, clomiphene blocked the voltage-gated fast sodium current and the L-type calcium current (I(Ca,L)) in both species. The voltage-independent fractional block of I(Ca,L) induced by clomiphene (10 microM) was approximately 82%, this concentration also inhibited the inwardly rectifying K(+) current with a fractional current block of approximately 26% at -90 mV. Fractional block of outward current at +70 mV in rat was approximately 25%, implying that delayed rectifying K(+) channels were also affected by clomiphene. We conclude that clomiphene shows selectivity for I(Cl,vol) over I(Cl,cAMP) and I(AB) and therefore represents a useful tool for studying chloride conductances in isolated ventricular myocytes with interfering currents blocked. However, due to its effects on cation conductances it would be of little value in this regard for other types of in vitro or in vivo experiments.

Dissipation of the excitation wave fronts.

An excitation wave in cardiac tissue will fail to propagate if the transmembrane voltage at its front rises too slowly and does not excite the tissue ahead of it. Then the sharp voltage profile of the front will dissipate, and the subsequent spread of voltage will be purely diffusive. This mechanism is impossible in FitzHugh-Nagumo type systems. Here a simplified mathematical model for this mechanism is suggested. The model has exact traveling front solutions, and gives conditions for the front dissipation. In particular, a front will dissipate if it is not allowed to propagate faster than a certain nonzero speed. This critical speed depends only on the properties of the fast sodium current.

Functional modulation of human brain Na v1.3 sodium channels, expressed in mammalian cells, by auxiliary β1, β2 and β3 subunits

Voltage-gated sodium channels consist of a pore-forming α subunit and two auxiliary β subunits. Excitable cells express multiple α subtypes, designated Na v1.1–Na v1.9, and three β subunits, designated β1, β2 and β3. Understanding how the different α subtypes, in combination with the various β subunits, determine sodium channel behavior is important for elucidating the molecular basis of sodium channel functional diversity. In this study, we used whole-cell electrophysiological recording to examine the properties of the human Na v1.3 α subtype, stably expressed in Chinese hamster ovary cells, and to investigate modulation of Na v1.3 function by β1, β2 and β3 subunits. In the absence of β subunits, human Na v1.3 formed channels that inactivated rapidly ( τ inactivation≈0.5 ms at 0 mV) and almost completely by the end of 190-ms-long depolarizations. Using an intracellular solution with aspartate as the main anion, the midpoint for channel activation was ∼−12 mV. The midpoint for inactivation, determined using 100-ms conditioning pulses, was ∼−47 mV. The time constant for repriming of inactivated channels at −80 mV was ∼6 ms. Coexpression of β1 or β3 did not affect inactivation time course or the voltage dependence of activation, but shifted the inactivation curve ∼10 mV negative, and slowed the repriming rate ca. three-fold. β2 did not affect channel properties, either by itself or in combination with β1 or β3. Na v1.3 expression is increased in damaged nociceptive peripheral afferents. This change in channel expression levels is correlated with the emergence of a rapidly inactivating and rapidly repriming sodium current, which has been proposed to contribute to the pathophysiology of neuropathic pain. The results of this study support the hypothesis that Na v1.3 may mediate this fast sodium current.

Anticonvulsant profile and mechanism of action of propranolol and its two enantiomers

The anticonvulsant properties of the ß-adrenoceptor antagonist propranolol and its two enantiomers were examined in various screening tests in order to characterize the anticonvulsant profile as well as the possible molecular mechanism of action. These compounds dose-dependently raised the threshold for tonic electroshock seizures in mice and were effective in the traditional maximal electroshock test (ED 50s 15– 20 mg kg −1i.p.). In combination with clinically used antiepileptics, the anticonvulsant effectiveness of the latter was significantly increased. In the pentylenetetrazol (85 mg kg −1s.c.) seizure threshold test, ( ±)- and ( +)-propranolol were not effective in preventing clonic seizures. In unrestrained rats with chronically implanted electrodes in the dorsal hippocampus, propranolol and its ( +)-enantiomer equieffectively reduced the duration of electrically-evoked hippocampal afterdischarges (10 and 20 mg kg −1i.p.) and raised the focal stimulation threshold (20 mg kg −1i.p.). In amygdala-kindled rats, both drugs ( ≥ 10 mg kg−1 i.p.) reduced the seizure severity from stage 5 (generalized clonic–tonic) to stage 3 (unilateral forelimb) seizures. Furthermore, whole-cell patch-clamp experiments showed that ( +)- as well as ( −)-propranolol ( 10−6to 10−4M) depressed the fast inward sodium current in a concentration- and use-dependent manner in cultured rat cardiomyocytes and inhibited picrotoxin-induced burst firing activity of mouse spinal cord neurones in culture. In conclusion, propranolol and its two enantiomers have anticonvulsant effects in models for generalized tonic–clonic and complex partial seizures which may be accounted for by the sodium channel blocking and not by the ß-adrenoceptor blocking activity.

Ionic Current Basis of Electrocardiographic Waveforms

Body surface electrocardiograms and electrograms recorded from the surfaces of the heart are the basis for diagnosis and treatment of cardiac electrophysiological disorders and arrhythmias. Given recent advances in understanding the molecular mechanisms of arrhythmia, it is important to relate these electrocardiographic waveforms to cellular electrophysiological processes. This modeling study establishes the following principles: (1) voltage gradients created by heterogeneities of the slow-delayed rectifier (I(Ks)) and transient outward (I(to)) potassium current inscribe the T wave and J wave, respectively; T-wave polarity and width are strongly influenced by the degree of intercellular coupling through gap-junctions. (2) Changes in [K+]o modulate the T wave through their effect on the rapid-delayed rectifier, I(Kr). (3) Alterations of I(Ks), I(Kr), and I(Na) (fast sodium current) in long-QT syndrome (LQT1, LQT2, and LQT3, respectively) are reflected in characteristic QT-interval and T-wave changes; LQT1 prolongs QT without widening the T wave. (4) Accelerated inactivation of I(Na) on the background of large epicardial I(to) results in ST elevation (Brugada phenotype) that reflects the degree of severity. (5) Activation of the ATP-sensitive potassium current, I(K(ATP)), is sufficient to cause ST elevation during acute ischemia. These principles provide a mechanistic cellular basis for interpretation of electrocardiographic waveforms.

Sodium Currents in Striatal Neurons from Dystonic dtsz Hamsters: Altered Response to Lamotrigine

Dystonic mutant dtsz hamsters are a model for paroxysmal dystonia. Handling/stress provoke the dystonic attacks. This phenomenon subsedes with maturation, but can be reinvoked when these animals receive sodium channel blockers such as lamotrigine, suggesting a dysfunction of striatal sodium channels. Voltage-gated fast sodium currents (INa+) were studied in acutely isolated striatal neurons from healthy and dtsz hamsters in whole-cell voltage clamp recordings. The action of lamotrigine was tested on (a) current/voltage relationship, (b) kinetics, and (c) steady-state inactivation and activation. Under control conditions, properties of INa+ were not different between healthy and dtsz neurons. With lamotrigine, however, (a) peak currents were significantly less depressed by the drug in neurons from dtsz hamsters as compared to healthy cells, and (b) the steady-state inactivation curve shift of INa+ was less pronounced in dtsz neurons. The results suggest that in dtsz hamsters, fast sodium currents in striatal neurons are more resistant to blockade. This sodium channel alteration might be causal for a functional imbalance between input and output structures of the basal ganglia under conditions of compromised I+Na.

Recent findings on the dromotropic actions of the class III antiarrhythmic agents

Class III antiarrhythmic agents have been considered to lengthen the myocardial effective refractory period (ERP) without any significant effects on the conduction velocity. However, recent investigations have clarified the positive or negative dromotropic effects of these agents. Amiodarone, a representative class III agent, exerts negative dromotropism by suppressing the fast sodium current responsible for conduction in acute administration (class I effects). Chronic amiodarone causes prolongation of ERP (class III effects), which is sometimes associated with negative dromotropism based on the alteration of passive or active membrane properties. Sotalol shows neither significant positive nor negative dromotropism under the normoxic condition, whereas this agent is reported to exert positive dromotropism mediated by the cAMP-dependent facilitation of gap junctional electrical coupling under the hypoxic condition. Some pure class III agents such as nifekalant are suspected to elicit 'apparent' positive dromotropism in the premature impulse propagation. This is explained by the right and upward shift of the strength-interval curve, which theoretically transforms the graded premature response to the all-or-none response. Although the clinical relevancy of these phenomena remains to be investigated, such variable dromotropism of the individual class III agent may contribute to the better understanding and development of antiarrhythmic agents.

The antidotal effect of α 1-acid glycoprotein on amitriptyline toxicity in cardiac myocytes

Tricyclic antidepressants in overdose cause toxicity marked by prolongation of the QRS interval of the electrocardiogram. These drugs are bound to α 1-acid glycoprotein (AAG) with high affinity in plasma. Animal studies have shown that the administration of AAG shortens the QRS prolongation induced by tricyclic antidepressants. In order to clarify the pharmacological mechanism involved and to obtain clinically relevant evidence at the cellular level, whole-cell patch clamp techniques were performed in single guinea-pig ventricular myocytes to elicit the time and voltage-dependent fast sodium currents using both normal and modified physiological solutions. Cells stayed viable for much longer when they were placed in normal physiological solutions, providing sufficient recording time for consistently reproducible, clinically relevant toxicological results to be obtained. Amitriptyline (AMI) produced a concentration-dependent blockade of sodium currents with an approximate IC 50 of 0.69 μM. AAG reversed this blockade in a concentration-dependent fashion at concentrations ranging from 3.2 to 12.8 μM. Using the same experimental conditions, AAG also reversed the blockade of sodium current by quinidine, a class I antiarrythmic drug. Albumin did not reverse the blockade of sodium channels by AMI. The results indicate that AAG is a potential antidote for tricyclic antidepressant overdose.

Voltage-gated calcium and sodium currents of starburst amacrine cells in the rabbit retina.

The voltage-gated calcium and sodium currents of starburst amacrine cells were examined in slices of the adult rabbit retina. ON-center starburst amacrine cells were targeted for whole-cell recording by prelabeling the retina with the nuclear dye 4'-6-diamidino-2-phenylindole hydrochloride (DAPI). Calcium currents were isolated using an external Ringer that contained tetrodotoxin to block sodium currents and barium to block potassium channels. When starburst amacrine cells were stepped to holding potentials positive to -50 mV, a series of voltage-dependent calcium currents were activated. The calcium current peaked at -10 mV. The calcium currents kinetics were mainly sustained in nature, showing only a small amount of slow inactivation. Nickel (100 microM), a T-type channel blocker, had no effect on the calcium current. Application of the L-type channel agonist BAY K8644 (1-2.5 microM) had small variable effects on the calcium current while the L-type channel antagonist nifedipine (10 microM) had no effect. However, addition of a reported N-type calcium channel antagonist, omega-conotoxin G6A (1 microM), blocked a large portion of the calcium current, as did a more nonselective antagonist, omega-conotoxin M7C (200 nM). Agatoxin 4A (500 nM) reduced a smaller sustained calcium current component, implying a P/Q-type calcium channel was present on these neurons. In addition to the calcium currents, a fast voltage-gated sodium current was observed in many starburst cells. This current could be blocked by tetrodotoxin (200-500 nM). The differing kinetics and durations of the sodium and calcium currents could play important roles in the regulation of synaptic release and in the coordination of spiking by starburst amacrine cell dendrites during retinal development and in the encoding of motion across the retinal surface.

Sodium currents in isolated rat CA1 pyramidal and dentate granule neurones in the post-status epilepticus model of epilepsy

Status epilepticus (SE) was induced in the rat by long-lasting electrical stimulation of the hippocampus. After a latent period of 1 week, spontaneous seizures occurred which increased in frequency and severity in the following weeks, finally culminating after 3 months in a chronic epileptic state. In these animals we determined the properties of voltage-dependent sodium currents in acutely isolated CA1 pyramidal neurones and dentate granule cells using the whole-cell voltage-clamp technique. The conductance of the fast transient sodium current was larger in SE rats (84±7 nS versus 56±6 nS) but related to a difference in cell size so that the neurones had a similar specific sodium conductance (control: 7.8±0.8 nS/pF, SE: 6.7±0.8 nS/pF). Current activation and inactivation were characterised by a Boltzmann function. After SE the voltage dependence of activation was shifted to more negative potentials (control: −45.1±1.4 mV, SE: −51.5±2.9 mV, P<0.05). In combination with a small shift in the voltage dependence of inactivation to more depolarised potentials (control: −68.8±2.3 mV, SE: −66.3±2.3 mV), it resulted in a window current that was much increased in the SE neurones (median: 64 pA in control, 217 pA in SE, P<0.05). The peak of this window current shifted to more hyperpolarised potentials (control: −44 mV, SE: −50 mV, P<0.05). No differences were found in the sodium currents analysed in dentate granule cells of control and SE animals. The changes observed in CA1 neurones after SE contribute to enhanced excitability in particular when membrane potential is near firing threshold. They can, at least partly, explain the lower threshold for epileptic activity in SE animals. The comparison of CA1 with DG neurones in the same rats demonstrates a differential response in the two cell types that participated in very similar seizure activity.

Anticonvulsant and sodium channel blocking activity of higher doses of clenbuterol.

Clenbuterol, a lipophilic beta2-adrenoceptor agonist, was investigated in various seizure models of experimental epilepsy. In the maximal electroshock seizure threshold test, clenbuterol (> or =4 mg/kg i.p.) increased the electroconvulsive threshold for tonic seizures in mice. In the traditional maximal electroshock seizure (MES) test in mice, ED50 values of 11 mg/kg i.p. or s.c. were determined. In both models, the beta2-receptor antagonist ICI 118.551 did not antagonize the anticonvulsant activity of clenbuterol. Combinations of clenbuterol with standard antiepileptics revealed additive anticonvulsant effects. Repeated administration of clenbuterol (5 mg/kg s.c., twice daily for 14 days) to mice did not significantly influence its anticonvulsant potency or the effectiveness of phenobarbital in the MES test. In various chemically-induced seizure tests with tonic convulsions, clenbuterol inhibited or tended to suppress the tonic phase. However, this drug was not effective in preventing clonic seizures in the pentylenetetrazol (85 mg/kg s.c.) seizure threshold test. In the rotarod ataxia test (mice), a minimal "neurotoxic" dose (TD50) of 41 mg/kg i.p. was determined. In unrestrained rats with chronically implanted electrodes in the dorsal hippocampus, clenbuterol (2 mg/kg and 4 mg/kg i.p.) significantly reduced the duration of electrically evoked hippocampal afterdischarges. In amygdala-kindled rats, clenbuterol (5 mg/kg and 10 mg/kg i.p.) reduced the seizure severity to stage 3. Additional studies indicated that clenbuterol (6 mg/kg i.p.) increased the heart rate and decreased the blood pressure, but this drug did not alter the plasma level of the two tested antiepileptics phenobarbital and carbamazepine. Furthermore, in whole-cell voltage-clamp experiments on cultured neonatal rat cardiomyocytes, clenbuterol (1-100 microM) depressed the fast sodium current in a concentration- and frequency-dependent manner. In conclusion, the anticonvulsant effects of higher doses of clenbuterol against generalized tonic-clonic and complex partial seizures seem to be related to the inhibition of voltage-dependent sodium channels and not to the modulation of beta-adrenoceptors.

Ectopic activity in ventricular cells induced by early afterdepolarizations developed in Purkinje cells.

The development of early afterdepolarizations (EADs) in Purkinje fibers and their propagation to ventricular muscle cells are studied by computer modeling. The Purkinje-ventricular system has been simulated by a two-dimensional model of a Purkinje fiber (PF) connected to a thin sheet of ventricular muscle tissue (VMT). EADs are induced in the PF by enhancing the fast second inward current, iCa,f, and blocking the delayed K+ current, iK, while the VMT is kept under physiological conditions. Different phenomena are observed depending on the EAD conditions applied. For 70% iK blockade and iCa,f enhancement greater than 60%, a single phase 3 EAD developed in the PF propagates to the VMT generating an ectopic beat. For 80% iK blockade and iCa,f enhancement in the range from 0% to 70%, multiple ectopic beats appear in the VMT. However, for iK blockades over 80%, action potentials in PF cells do not repolarize and the ectopic activity in the VMT disappears. In our simulations, the ionic mechanism underlying phase 3 EAD development is the reactivation of the fast sodium current in the PF. Our results demonstrate that there exists a critical range of EAD conditions that favor the development of EADs in the PF and their propagation to the VMT as ectopic activity. This phenomenon could underlie the genesis of some triggered arrhythmias.

Riluzole inhibits the persistent sodium current in mammalian CNS neurons.

The effects of 0.1-100 microM riluzole, a neuroprotective agent with anticonvulsant properties, were studied on neurons from rat brain cortex. Patch-clamp whole-cell recordings in voltage-clamp mode were performed on thin slices to examine the effects of the drug on a noninactivating (persistent) Na+ current (INa,p). INa,p was selected because it enhances neuronal excitability near firing threshold, which makes it a potential target for anticonvulsant drugs. When added to the external solution, riluzole dose-dependently inhibited INa,p up to a complete blocking of the current (EC50 2 microM), showing a significant effect at therapeutic drug concentrations. A comparative dose-effect study was carried out in the same cells for the other main known action of riluzole, the inhibitory effect on the fast transient sodium current. This effect was confirmed in our experiments, but we found that it was achieved at levels much higher than putative therapeutic concentrations. Only the effect on INa,p, and not that on fast sodium current, can account for the reduction in neuronal excitability observed in cortical neurons following riluzole treatment at therapeutic concentrations, and this might represent a novel mechanism accounting for the anticonvulsant and neuroprotective properties of riluzole.

Effects of voltage trajectory on action potential voltage threshold in simulations of cat spinal motoneurons

A single-cell neuronal model was used to investigate the effect of membrane trajectory on voltage threshold (Vth) for action potential generation. Previous results suggested that hyperpolarization of Vth could be produced by a rapid membrane depolarization, but this effect is limited to the first spike in a train. This study shows rapid current injections hyperpolarize Vth because they are more effective in activating the sodium current underlying spiking. The hyperpolarization of Vth induced by rapid membrane depolarization becomes less effective in altering Vth when other mechanisms of enhancing the fast sodium current underlying action potentials are activated.

The small sodium-channel blocking factor in the cerebrospinal fluid of multiple sclerosis patients is probably an oligopeptide.

An endogenous factor that is able to reduce the fast transient sodium current of excitable cells has been reported to exist in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. This was confirmed with nine clinically definite MS patients in the acute relapse. In order to purify and chemically identify the factor, microconcentration and gel filtration high-performance liquid chromatography (HPLC) were applied. After each purification step the activity-containing fraction was determined using a biological assay. With all CSFs the activity was contained in the fraction corresponding to 600-800 Da molecular weight, indicating that the factor is chemically homogeneous. The biological activity of the CSF specimens was not correlated to the laboratory CSF data; however, it was correlated to the area under the 210 nm UV light absorption peak in the corresponding chromatogram, i.e. the 600-800 Da MW fraction. As the factor was degradable by acid hydrolysis and a carboxypeptidase, it is suggested that it might be a small peptide.

Some mechanisms of antiarrhythmic effect of phosphoenolpyruvate

Depolarization automaticity was modeled on the papillary muscle from the guinea pig heart. Phosphoenolpyruvate (0.1 mM) 2-fold decreased the high-frequency calcium-dependent automaticity, but only weakly affected frequency of action potentials, whose upstroke was formed by fast sodium current or had a mixed nature. Phosphoenolpyruvate shifted the diastolic potential toward negative values, which depended on the amplitude of depolarization step. These effects developed 10–15 min after application of the preparation.

Ionic mechanisms of electrical remodeling in human atrial fibrillation

Atrial fibrillation (AF) is associated with a decrease in atrial ERP and ERP adaptation to rate as well as changes in atrial conduction velocity. The cellular changes in repolarization and the underlying ionic mechanisms in human AF are only poorly understood. Action potentials (AP) and ionic currents were studied with the patch clamp technique in single atrial myocytes from patients in chronic AF and compared to those from patients in stable sinus rhythm (SR). The presence of AF was associated with a marked shortening of the AP duration and a decreased rate response of atrial repolarization. L-type calcium current (ICa,L) and the transient outward current (Ito) were both reduced about 70% in AF, whereas an increased steady-state outward current was detectable at test potentials between -30 and 0 mV. The inward rectifier potassium current (IKI) and the acetylcholine-activated potassium current (IKACh) were increased in AF at hyperpolarizing potentials. Voltage-dependent inactivation of the fast sodium current (INa) was shifted to more positive voltages in AF. AF in humans leads to important changes in atrial potassium and calcium currents that likely contribute to the decrease in APD and APD rate adaptation. These changes contribute to electrical remodeling in AF and are therefore important factors for the perpetuation of the arrhythmia.

Action Potentials That Mimic Fibrillation Activate Sodium Current

Ventricular fibrillation (VF) has brief action potentials (50–70 ms) with short diastolic intervals (10–30 ms). Under these conditions ion channel activity may be grossly different to normal sinus rhythm (NSR). In particular, sodium channel activation may not contribute to the generation and propagation of action potentials during VF. This study determined if sodium channels can be activated when action potentials mimic VF. Isolated chick ventricular myocytes (n=7) were voltage-clamped to quantitate fast inward sodium current. The voltage clamp protocol simulated VF with a 10 pulse train at 10 Hz (100 ms cycle length (CL)) and depolarization interval (action potential duration) ranging from 90 to 20 ms. After each train a test pulse was delivered from holding (−80 mV) in 10-ms steps. The train preceded each step pulse. Peak sodium current for control and each VF protocol occurred at a membrane potential (Vm) of −10 mV. Sodium current was evident during brief resting intervals as short as 20 ms, albeit 10–20% of baseline. Resting intervals less than 60 ms shifted the sodium conductance activation curve from Vm0.5−30 mV to −22 mV membrane potential. Similar findings occurred when resting potential was at −65 mV, although there was less sodium current with all tested protocols. There was significantly less inactivation of sodium current when the prepulse was shorter (100 v 1000 ms). There was approximately 20% greater sodium current when the test pulse followed a short v long depolarized (>−80 mV) prepulse. Although the longer depolarization pulses produce approximately 20% greater sodium current at membrane potentials more negative than −80 mV. Lastly the time for half recovery of sodium current from activation was significantly less when the inactivating prepulse was short v long (45.9±9 v 118±20 ms,P <0.05). In conclusion, sodium current is evident when the diastolic rest interval is as brief as 10–20 ms. Rest interval, length of membrane depolarization and membrane potential interact to affect sodium channel activation, inactivation and recovery from inactivation. These data demonstrate that the brief action potentials at more depolarized membrane potentials seen during VF allow for inward sodium current upon depolarization, less sodium channel inactivation, and a faster recovery from inactivation, thereby compensating for a short diastolic rest interval. Therefore, it is likely that the inward sodium channel contributes to wave front propagation during ventricular fibrillation.

Dynamic selectivity filters in ion channels.

This work was supported by a Wellcome Trust (UK) International Prize Travelling Fellowship and the NIH (NS-11756).

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.