Katsushige Ono

Mechanism of cAMP-dependent modulation of cardiac sodium channel current kinetics.

beta-Adrenergic modulation is one of the most important regulatory mechanisms of ion channel function. Only recently, however, have beta-adrenergic effects on cardiac Na+ channel activity been recognized, and some diversity of effects has been reported in different preparations. We report studies of protein kinase A-dependent phosphorylation effects on cardiac Na+ current using the macropatch on-cell mode voltage-clamp technique to maintain cytoplasmic composition intact. During the first 5 minutes after addition of 8-(4-chlorophenylthio)cAMP to the bath, the midpoints of both voltage-dependent availability and conductance shifted in the hyperpolarizing direction an average of -7.5 +/- 2.8 mV (n = 31). Moreover, these effects were not species specific; similar results were obtained in canine, rabbit, and guinea pig myocytes, and a similar shift occurred after exposure to 5 microM isoproterenol. Maximum conductance did not change, nor did single-channel conductance. The shifts of conductance and voltage-dependent availability that were induced by protein phosphorylation were distinct from and independent of the slow background shift in kinetics. We measured the background shift to be less than 0.3 mV/min and to be restricted to the channels within the patch. Pretreatment of cells with a blocker of protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89), prevented the effect of 8-(4-chlorophenylthio)cAMP while not affecting the background shift in kinetics. Although clearly not the result of addition of a negatively charged phosphate to the inside face of the channel, cAMP-dependent phosphorylation affects the voltage-dependent kinetics, as expected, by an electrostatic interaction with the voltage sensor.

Voltage-Dependent and Frequency-Independent Inhibition of Recombinant Cav3.2 T-Type Ca2+ Channel by Bepridil

Effects of bepridil on the low voltage-activated T-type Ca2+ channel (CaV3.2) current stably expressed in human embryonic kidney (HEK)-293 cells were examined using patch-clamp techniques. Bepridil potently inhibited ICa,T with a markedly voltage-dependent manner; the IC50 of bepridil was 0.4 µmol/l at the holding potential of –70 mV, which was 26 times as potent as that at –100 mV (10.6 µmol/l). Steady-state inactivation curve (8.4 ± 1.7 mV) and conductance curve (5.9 ± 1.9 mV) were shifted to the hyperpolarized potential by 10 µmol/l bepridil. Bepridil exerted the tonic blocking action but not the use-dependent block. Bepridil had no effect on the recovery from inactivation of T-type Ca2+ channels. Thus, high efficacy of bepridil for terminating atrial fibrillation and atrial flutter may be considered to be attributed, at least in a part, to the T-type Ca2+ channel-blocking actions.

A direct effect of forskolin on sodium channel bursting

A long-lasting component of current through voltage-dependent Na channels is believed to contribute to the plateau phase of the cardiac action potential. Here we report that in cardiac ventricular myocytes forskolin increases the contribution of a very slow component of decay (τ=36±16 ms,n=13) in ensemble currents in response to step depolarizations to 0 mV. Long-lasting bursts of openings (mean duration of 27±14 ms,n=10) accounted for this behavior. The slow time constant of decay was not altered by forskolin (5–50 μM). Rather, an increase in the probability of bursting behavior produced a forskolin concentration-dependent increase in the amplitude of this very slow component. This action of forskolin was not the result of stimulation of adenylyl cyclase because it was not affected when cAMP-dependent phosphorylation was inhibited by the protein kinase inhibitor H-89, and it could not be mimicked by addition of isoproterenol, membrane-permeant cAMP [8-(4-chloro-phenylthio)-cAMP], or the phosphatase inhibitor okadaic acid. In addition, bursting was not augmented by guanosine 5′-O-(3-thiotriphosphate) (GTP [γS]) either applied to the bath or directly to the intracellular face of the channel in inside-out macropatches. Further-more, 1,9-dideoxy-forskolin, which does not stimulate adenylyl cyclase and 6-(3-dimethylaminopropionyl)-forskolin, a hydrophilic derivative of forskolin, also augmented late channel activity. Comparison of the characteristics of bursts in the presence of forskolin with those occurring in its absence suggested that the increase in the frequency of long-lasting bursts produced by forskolin represents a direct interaction of forskolin with the channel that augments, by up to tenfold, the probability that channels will have delayed inactivation.

Short- and long-term amiodarone treatments regulate Cav3.2 low-voltage-activated T-type Ca2+ channel through distinct mechanisms.

Low-voltage-activated T-type Ca2+ channels have been recognized recently in the mechanisms underlying atrial arrhythmias. However, the pharmacological effects of amiodarone on the T-type Ca2+ channel remain unclear. We investigated short- and long-term effects of amiodarone on the T-type (Cav 3.2) Ca2+ channel. The Cav3.2 α1H subunit derived from human heart was stably transfected into cells [human embryonic kidney (HEK)-Cav3.2] cultured with or without 5 μM amiodarone. Patch-clamp recordings in the conventional whole-cell configuration were used to evaluate the actions of amiodarone on the T-type Ca2+ channel current (ICa.T). Amiodarone blockade of ICa.T occurred in a dose- and holding potential-dependent manner, shifting the activation and the steady-state inactivation curves in the hyperpolarization direction, when amiodarone was applied immediately to the bath solution. However, when the HEK-Cav3.2 cells were incubated with 5 μM amiodarone for 72 h, ICa.T density was significantly decreased by 31.7 ± 2.3% for control,-93.1 ± 4.3 pA/pF (n = 8), versus amiodarone,-56.5 ± 3.2 pA/pF (n = 13), P < 0.001. After the prolonged administration of amiodarone, the activation and the steadystate inactivation curves were shifted in the depolarization direction by -7.1 (n = 41) and -5.5 mV (n = 37), respectively, and current inactivation was significantly delayed [time constant (τ): control, 13.3 ± 1.1 ms (n = 6) versus amiodarone, 39.6 ± 5.5 ms (n = 6) at -30 mV, P < 0.001)]. Nevertheless, short-term inhibitory effects of amiodarone on the modified T-type Cav3.2 Ca2+ channel created by long-term amiodarone treatment were functionally maintained. We conclude that amiodarone exerts its short- and long-term inhibitory actions on ICa.T via distinct blocking mechanisms.

Denervation and reinnervation of the heart after aortic surgery, estimated by 123I-metaiodobenzylguanidine scintigraphy.

PurposeTo investigate whether sympathetic nerve injury occurs during aortic surgery and how reinnervation takes place afterward.MethodsImaging with 123I-metaiodobenzylguanidine (MIBG) was performed in 12 patients (aortic group) who underwent aortic surgery (ascending replacement 3, ascending-arch replacement 9) before and 3 weeks after surgery. In 8 of 12 patients, MIBG scintigraphy was performed 1 and 2 years after surgery. Twelve patients (control group) who underwent open-heart surgery (mitral valve repair: 11; tricuspid valve replacement: 1) were studied using MIBG scintigraphy. The heart-to-mediastinum (H/M) activity ratio was obtained from planar images. The myocardial single-photon-emission computed tomography image was divided into five segments and the regional tracer uptake was scored from 0 = absent to 3 = normal uptake.ResultsNo significant difference in the H/M ratio in either early and delayed planar scans was observed between both groups before surgery. The H/M ratios significantly decreased 3 weeks after surgery in the aortic group, whereas there was no significant change in the control group. The H/M ratio did not recover to the preoperative level within 2 years. In these 8 patients, the regional uptake of MIBG improved in the anterior and septal regions 1 year after surgery.ConclusionDuring ascending or ascending-arch replacement, the sympathetic nerve was globally denervated and slight reinnervation was observed within 2 years. The anterior and septal regions showed a rapid reinnervation, whereas other regions did not.

CHAPTER 12 – Sodium Channels

This chapter describes the function of the voltage-gated Na + channel and its metabolic regulation, at concentrated on modulation and defects in molecular function. Activation of Na + channels is believed to result from a voltage-driven conformational change, which opens a transmembrane pore through the protein. Channel opening is triggered by membrane depolarization, exerting an electrical force on the voltage sensor located within the transmembrane electrical field of the S4 segment in each domain. The movement of the gating charges through the membrane is directly measured as an outward gating current. Studies of the cardiac Na + channel at the molecular level utilizing the patch-clamp technique and a recombinant expression system shed light on the process of how the Na + channel behaves in terms of molecular function in physiological and pathophysiological situations. The discovery of the Na + channel gene defects that cause LQT and Brugada syndromes represents a major advancement in the field. The powerful combination of molecular genetics with detailed studies of the structure and function of recombinant Na + channels can provide insight into the mechanistic factors that cause the clinical phenotypes of many inherited diseases related to the Na + channel. It also offers the possibility of developing specific therapeutic approaches to disease management that are based on the specific functional properties of the Na + channel in physiological and pathophysiological conditions.

β-adrenergic regulation of cardiac Na+ channel

Calcium (Ca2+) channels in the heart are controlled by adrenergic neurotransmitters through adenosine 3′5′-cyclic monophosphate (cAMP)-mediated phosphorylation of the channel [1]. In biochemical studies, cardiac Na+ channels were also seen to undergo phosphorylation by cAMP, thereby suggesting the regulation of these channels by adrenergic neurotransmitters [2]. Although cardiac excitation and impulse transmission critically depends on the availability of voltage-gated Na+ channels, there is a paucity of information on effects of catecholamines or cAMP on the fast Na+ system [3,4] and available results are inconsistent.