Akihiko Sunami

The E1784K mutation in SCN5A is associated with mixed clinical phenotype of type 3 long QT syndrome

Phenotypic overlap of type 3 long QT syndrome (LQT3) with Brugada syndrome (BrS) is observed in some carriers of mutations in the Na channel SCN5A. While this overlap is important for patient management, the clinical features, prevalence, and mechanisms underlying such overlap have not been fully elucidated. To investigate the basis for this overlap, we genotyped a cohort of 44 LQT3 families of multiple ethnicities from 7 referral centers and found a high prevalence of the E1784K mutation in SCN5A. Of 41 E1784K carriers, 93% had LQT3, 22% had BrS, and 39% had sinus node dysfunction. Heterologously expressed E1784K channels showed a 15.0-mV negative shift in the voltage dependence of Na channel inactivation and a 7.5-fold increase in flecainide affinity for resting-state channels, properties also seen with other LQT3 mutations associated with a mixed clinical phenotype. Furthermore, these properties were absent in Na channels harboring the T1304M mutation, which is associated with LQT3 without a mixed clinical phenotype. These results suggest that a negative shift of steady-state Na channel inactivation and enhanced tonic block by class IC drugs represent common biophysical mechanisms underlying the phenotypic overlap of LQT3 and BrS and further indicate that class IC drugs should be avoided in patients with Na channels displaying these behaviors.

Electrophysiological effects of ginseng and ginsenoside Re in guinea pig ventricular myocytes.

Panax ginseng is a folk medicine with various cardiovascular actions; however, its underlying mechanisms of action are not well known. In the present study, we examined the effects of ginseng and its main component, ginsenoside Re, on action potentials and membrane currents recorded from isolated guinea pig ventricular myocytes with the whole-cell patch clamp technique. Ginseng (1 mg/ml) shortened the action potential duration in a rate-dependent manner. Ginseng depressed the L-type Ca2+ current (I(Ca-L)) in a mode of both tonic block and use-dependent block, and enhanced the slowly activating component of the delayed rectifier K+ current (I(Ks)). Ginsenoside Re 3 microM exhibited similar electrophysiological effects to those of 1 mg/ml ginseng, but of slightly smaller magnitude. Inhibition of I(Ca,L) and enhancement of I(Ks) by ginsenoside Re appear to be one of the main electrophysiological actions of ginseng in the heart, although contributions from other ingredients should be considered.

Accessibility of mid-segment domain IV S6 residues of the voltage-gated Na+ channel to methanethiosulfonate reagents

The inner pore of the voltage‐gated Na+ channel is predicted by the structure of bacterial potassium channels to be lined with the four S6 α‐helical segments. Our previously published model of the closed pore based on the KcsA structure, and our new model of the open pore based on the MthK structure predict which residues in the mid‐portion of S6 face the pore. We produced cysteine mutants of the mid‐portion of domain IV‐S6 (Ile‐1575–Leu‐1591) in NaV1.4 and tested their accessibility to intracellularly and extracellularly placed positively charged methanethiosulfonate (MTS) reagents. We found that only two mutants, F1579C and V1583C, were accessible to both outside and inside 2‐(aminoethyl)‐methanethiosulfonate hydrobromide (MTSEA) Further study of those mutants showed that efficient closure of the fast inactivation gate prevented block by inside [2‐(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) at slow stimulation rates. When fast inactivation was inhibited by exposure to anthropleurin B (ApB), increasing channel open time, both mutants were blocked by inside MTSET at a rate that depended on the amount of time the channel was open. Consistent with the fast inactivation gate limiting access to the pore, in the absence of ApB, inside MTSET produced block when the cells were stimulated at 5 or 20 Hz. We therefore suggest that the middle of IV‐S6 is an α‐helix, and we propose a model of the open channel, based on MthK, in which Phe‐1579 and Val‐1583 face the pore.

Two components of use-dependent block of Na+ current by disopyramide and lidocaine in guinea pig ventricular myocytes.

We studied the kinetics of the use-dependent block of the Na+ current (INa) by disopyramide and lidocaine. INa was recorded from isolated guinea pig ventricular myocytes by using the whole-cell patch-clamp technique. The use-dependent block of INa by disopyramide with 20- and 200-msec depolarizing pulses developed in two exponential functions. The degree of the use-dependent block and the amplitude of the fast (Af) and slow (As) components with the short (20-msec) pulse protocol were comparable to those with the long (200-msec) pulse protocol. When pH was raised from 7.3 to 8.0, disopyramide increased Af without a change in As. At pH 6.5, INa block developed with a single exponential function revealing only the slow component. The fast and slow components of INa block by disopyramide could be explained by binding of the uncharged and charged forms, respectively, to the activated state of the channel. Development of INa block by lidocaine also was expressed by two exponentials at all pulse durations (5-200 msec). As pulse durations were prolonged or holding potentials were depolarized, the degree of the use-dependent block and Af increased. When pH was lowered to 6.5, the short pulse produced only the slow component, whereas the long pulse caused two exponentials with decreased Af and increased As. Internal application of QX-314, a permanently charged lidocaine analogue, produced a single exponential block of INa with a very slow onset rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Use-dependent block of Na+ currents by mexiletine at the single channel level in guinea-pig ventricular myocytes.

1 The mechanism of use‐dependent block of Na+ current by mexiletine was studied at the single channel level in guinea‐pig ventricular myocytes by the patch‐clamp techniques. All experiments were performed using stimulation protocols to enable us to analyze the strict dependence of changes in channel properties on channel use. 2 In cell‐attached patches, bath or pipette application of mexiletine (40 μm) produced a use‐dependent reduction of the peak average current without changes in single channel conductance. Null sweeps were increased and the number of openings per sweep decreased with successive pulses, whereas no significant change in the mean open time was detected during the train. 3 Block by mexiletine became greater when pulse duration was extended beyond the period in which channels were open, suggesting that block progressed without channel opening. 4 At near threshold potentials, mexiletine decreased the later occurrence of first openings. Additionally, late openings were reduced in a use‐dependent way. 5 We conclude that mexiltine binds to the inactivated closed states of the Na+ channel and then causes a failure of late openings as well as early, which results in null sweeps on subsequent depolarization.

The catalytic subunit of cyclic AMP-dependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes

We investigated the effects of the purified catalytic subunit (C subunit) of the cAMP-dependent protein kinase (A-kinase) on the cardiac Na+ channel currents. Single Na+ channel currents in guinea-pig ventricular myocytes were recorded using the patch clamp technique of the inside-out configuration. Application of C subunit decreased the peak average current and slowed the current decay, effects which were caused by decrease in the open probability of Na+ channels and increase in the first latency, whereas the unitary current amplitude and mean open times were not affected. We conclude that the cardiac Na+ channel is directly modulated by phosphorylation process through A-kinase.

States and sites of actions of flecainide on guinea-pig cardiac sodium channels

The states and sites of actions of flecainide on sodium channels were investigated in guinea-pig single cardiac cells, using the whole-cell voltage-clamp technique at 22 degrees C. External application of flecainide caused tonic and use-dependent block of the sodium current (INa). The tonic block and the steady state use-dependent block increased with increasing drug concentrations. The dose-response curve for the use-dependent block was fitted by the equation for 1:1 drug-receptor binding and yielded a KD of 7.0 microM flecainide. At 5 microM flecainide, the use-dependent block of INa with 10 and 200 ms depolarizing pulses at an interpulse interval of 400 ms was 31.1 +/- 2.7 (mean +/- S.E.) and 36.8 +/- 2.7%, respectively. The two values were not significantly different. The block developed as a single exponential function with onset rate of 0.041 +/- 0.005/pulse. Recovery from flecainide block consisted of two components as reported previously. The mean time constant of the initial fast component was 48 +/- 17 ms, which was comparable but significantly longer than that in the absence of the drug. The late slow component was only seen after drug application and the time constant was 26 +/- 7 s at -100 mV. Internal application of 5 and 50 microM flecainide for 30 min after rupture of the cell membrane produced a non-significant block and values of 1.7 +/- 0.8 and 6.9 +/- 2.4%, respectively, for the use-dependent block of INa.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple ionic mechanisms of early afterdepolarizations in isolated ventricular myocytes from guinea-pig hearts.

Ionic mechanisms of early afterdepolarization (EAD) induced by the K(+)-free solution or veratridine were studied with guinea-pig ventricular myocytes using the patch-clamp technique of whole-cell and cell-attached patch configurations. In the K(+)-free solution, myocytes exhibited prolonged action potential duration with humps on the final repolarization phase, which eventually turned into EAD starting around -70 mV and induced triggered activity. Application of 0.5 mM Cd2+ inhibited the development of EAD and caused depolarization of maximum diastolic potentials around -30 mV, although Cd2+ did not prevent prolongation of the action potential. Application of 50-100 microM Ni2+ or 30 microM tetrodotoxin had little effects on EAD and diastolic potentials. The background current-voltage relation examined by a ramp voltage clamp showed inhibition of the inward rectifier K+ current, induction of steady inward current between -40 and -10 mV, and increase in the outward tail current upon repolarization in the K(+)-free solution. Cd2+ completely blocked the steady inward current at the plateau level and partially depressed the delayed outward K+ current, while Ni2+ had no effects on the background I-V relation. Tetrodotoxin showed a mild inhibitory effect on the inward component of the background current negative to -50 mV, but left the steady inward current at the plateau level. Therefore, EAD in the K(+)-free condition is mainly formed by decreased inward rectifier K+ current, activation of the L-type Ca2+ current, and time-dependent decay of the delayed outward K+ current upon repolarization. Application of 25-100 microM veratridine caused marked prolongation of action potential with appearance of regenerative EADs. Action potential prolongation and EADs were partially abolished by Cd2+ and completely eliminated by tetrodotoxin. The single channel current recordings showed a decreased current amplitude, and prolonged and delayed openings of the Na+ channel currents by veratridine. Thus, an ensemble average current showed markedly prolonged decay time constant of 609 msec in veratridine from 3.6 msec in the control. These results indicate that veratridine-induced EAD is mainly formed by altered properties of the Na+ channel current and partly by the L-type Ca2+ current due to slowed repolarization. Thus, EAD can be induced by different ionic mechanisms depending on the basal conditions.

Involvement of local anesthetic binding sites on IVS6 of sodium channels in fast and slow inactivation

Local anesthetics (LAs) block Na(+) channels with a higher affinity for the fast or slow inactivated state of the channel. Their binding to the channel may stabilize fast inactivation or induce slow inactivation. We examined the role of the LA binding sites on domain IV, S6 (IVS6) of Na(+) channels in fast and slow inactivation by studying the gating properties of the mutants on IVS6 affecting LA binding. Mutation of the putative LA binding site, F1579C, inhibited fast and slow inactivation. Mutations of another putative LA binding site, Y1586C, and IVS6 residue involved in LA access and binding, I1575C, both enhanced fast and slow inactivation. None of the mutations affected channel activation. These results suggest that the LA binding site on IVS6 is involved in slow inactivation as well as fast inactivation, and these two gatings are coupled at the binding site.

New Observations on the Mechanisms of Antiarrhythmic Actions of Disopyramide on Cardiac Membranes

Electrophysiologic effects of disopyramide on cardiac membranes were studied using the microelectrode technique applied to papillary muscles and the suction pipette whole-cell clamp method applied to isolated ventricular myocytes from guinea-pig hearts. In contrast to previous reports, the development and recovery from the Vmax blocks of action potentials by disopyramide and lidocaine were best expressed by 2 exponential functions, not 1, suggesting that there might be 2 different processes for the sodium current block. A near-therapeutic concentration (11 microM) of disopyramide depressed Vmax at frequencies of 0.1 to 2.0 Hz decreased action potential amplitude and shortened plateau phase in ventricular myocytes. Action potential duration was prolonged by the drug in most of the preparations but shortened in some. Disopyramide prolonged the refractory period and increased threshold current for excitation. The drug was also shown to depress both the calcium current and the delayed outward potassium current. These multiple actions of disopyramide may explain its variety of antiarrhythmic properties.