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Short- and Long-Term Amiodarone Treatments Regulate Ca_v3.2 Low-Voltage-Activated T-type Ca²⁺ Channel through Distinct Mechanisms

Department of Cardiovascular Science (N.Y., T.K., T.U., S.I., K.O.) and Internal Medicine (N.Y., H.Y.), Oita University School of Medicine, Oita, Japan

Received November 28, 2005; accepted January 26, 2006

	Abstract

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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 (Ca_v 3.2) Ca²⁺ channel. The Ca_v3.2 _1H subunit derived from human heart was stably transfected into cells [human embryonic kidney (HEK)-Ca_v3.2] 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 (I_Ca.T). Amiodarone blockade of I_Ca.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-Ca_v3.2 cells were incubated with 5 µM amiodarone for 72 h, I_Ca.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 (): 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 Ca_v3.2 Ca²⁺ channel created by long-term amiodarone treatment were functionally maintained. We conclude that amiodarone exerts its short- and long-term inhibitory actions on I_Ca.T via distinct blocking mechanisms.

I_Ca.T is characterized by its activation at low voltage, rapid inactivation, and slow deactivation (Matteson and Armstrong, 1986) and may function in cardiac pacemaker activity under physiological conditions (Kawano and Dehaan, 1990). I_Ca.T seems to play a particularly important role in pathophysiological or remodeled cardiovascular tissue (Ertel et al., 1997): atrial I_Ca.L was down-regulated by sustained rapid atrial pacing in dogs, whereas I_Ca.T was not reduced (Yue et al., 1997). Furthermore, there is evidence that T-type Ca²⁺ channel blockade may attenuate the functional remodeling caused by atrial fibrillation (Fereh et al., 1999). I_Ca.T may provide a continuing leak of Ca²⁺ into the cell, and I_Ca.T inhibition may therefore be beneficial for preventing electrical remodeling caused by atrial tachycardia.

Cohen et al. (1992) demonstrated that amiodarone inhibits I_Ca.T and shifts the steady-state inactivation curve in the hyperpolarization direction in guinea pig atrial myocytes. However, because the activation voltage range and the inactivation voltage range of I_Ca.L and I_Ca.T overlap, distinct isolation of one current from another contains possible errors. For example, other Ca²⁺ channel currents in the heterogeneous Ca²⁺ channel expression system, such as in cardiac myocytes, may interfere with pharmacological evaluations of I_Ca.T using a wide variety of experimental protocols. The aim of this study was to examine the detailed short- and longterm effects of amiodarone on I_Ca.T by the use of the conventional whole-cell patch-clamp technique and the heterologous expression system of the human _1H T-type (Ca_v3.2) Ca²⁺ channel.

	Materials and Methods

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Expression of Ca²⁺ Channel Proteins and Cell Culture. The 1 subunit of Ca_v3.2 T-type Ca²⁺ channel (_1H) subunit derived from human heart, which forms cardiac I_Ca.T, was stably expressed in human embryonic kidney (HEK) 293 cells with no auxiliary subunits (HEK-Ca_v3.2 cells). A profile and a procedure for channel expression were described in detail in a previous report (Cribbs et al., 1998). In brief, cDNA encoding the human cardiac _1H subunit (CACNA1H) was inserted into the transfection vector pcDNA3, and HEK 293 cells were transfected with 2 µg of this vector by using calcium phosphate precipitation. HEK 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/l streptomycin in an atmosphere of 95% O₂ and 5% CO₂ at 37°C. This medium was supplemented with 300 mg/l G418 (neomycin analog) for the selection of recombinant HEK-Ca_v3.2 cells.

Whole-Cell Current Recordings. HEK-Ca_v3.2 cells were seeded onto glass-bottomed dishes and were incubated in culture medium for 12 to 24 h without amiodarone before electrophysiological measurements. Macroscopic I_Ca.T was recorded in the conventional whole-cell patch-clamp technique using an EPC-9 amplifier (HEKA Electronik, Lambrecht, Germany), at room temperature (22-25°C). Patch pipettes were pulled from 75-mm plain capillary tubes (Drummond Scientific Company, Broomall, PA) with a micropipette puller (model P-97; Sutter Instrument Company, Novato, CA) and fire-polished subsequently. The electrode had a resistance of 1.0 to 5.0 M when the pipette was filled with the pipette solution (see below). Capacitance was minimized, and series resistance was compensated electrically as much as possible without oscillation (60-75%). Current signals were eight-pole Bessel-filtered at 3.3 kHz, digitally sampled at 10 kHz, and stored on a 400-MHz Intel Pentium II-based computer using Microsoft Windows 98 operating system under control of a data acquisition program, Pulse/PulseFit (version 8.11; HEKA Electronik). To investigate the channel availability (steady-state inactivation), conventional double-pulse protocol was applied every 5 s: test pulses of 400 ms at -10 mV after prepulses of 1000 ms from -100 to 0 mV (increment = 5 mV) were applied. Electrophysiological data were collected 3 min or later after application of amiodarone for the evaluation of its short-term effects. To examine the long-term effects of amiodarone, we cultured HEK-Ca_v3.2 cells with or without 5 µM amiodarone using 0.05% dimethyl sulfoxide (DMSO) as vehicle for 72 h on glass-bottomed dishes, followed by amiodarone-free culture medium (Dulbecco's modified Eagle's medium) for 12 to 24 h. Voltage-dependent effects of amiodarone on I_Ca.T were appreciated in the conductance transforms. Relative conductance (activation) values were calculated as follows: Fractional conductance = I_Ca.T/G_max (V_t-V_rev) (A), where I_Ca.T represents the current amplitude at the test potential (V_t), and G_max is the maximal conductance value obtained from linear regression line of each current-voltage (I-V) relation extrapolated through the estimated reversal potential (V_rev). Voltage-dependent activation and availability (steady-state inactivation) were evaluated by a Boltzmann equation fit to the normalized data to peak currents measured in protocols as follows: Fraction = 1/{1 + exp[(V-V_1/2)/k]} (B), where the normalized data (Fraction) were expressed as a function of voltage (V), the test potential in the case of conductance, and the conditioning potential in the case of voltage-dependent availability. Parameters estimated by the fit were the half-point of the relationship (V_1/2) expressed in millivolts, and k is the slope factor. Recovery from inactivation was evaluated by depolarizing pulse pairs; the prepulses at 0 mV for the duration of 500 ms followed by a test pulse to 0 mV for 200 ms at intervals varying from 5 ms to 3 s. The time course of recovery was fitted by a single exponential equation. Doseresponse curves were obtained by the following Hill equation as follows: Fraction = 1/{1 + (IC₅₀/[C]^h)} (C), where [C] is the concentration of amiodarone, IC₅₀ is the half-maximal blocking concentration of I_Ca.T by amiodarone, and h is the Hill coefficient.

Solutions. The recording chamber was filled with bath solution of the following composition: 120 mM tetraethylammonium chloride, 6 mM CsCl, 5 mM 4-aminopyridine, 0.5 mM MgCl₂, 0.5 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate, 10 mM HEPES, 1.8 mM CaCl₂, and 10 mM glucose (pH was adjusted to 7.4 with 1 N tetraethylammonium hydroxide solution). The patchclamp electrode was filled with pipette solution of 130 mM CsCl, 2 mM MgCl₂, 2 mM ATP, 0.5 mM GTP, 5 mM EGTA, and 5 mM HEPES (pH was adjusted to 7.3 with 1 N CsOH). Amiodarone hydrochloride was dissolved in DMSO to prepare a stock solution. On the day of the experiment, aliquots of the stock solution were diluted with the bath solution. Data were acquired by using computer software (Pulse/PulseFit, version 8.11; HEKA Electronik), and all curve fittings and figures were made on SigmaPlot (version 6.1; SPSS, Inc., Chicago, IL).

Drugs. Amiodarone hydrochloride was a gift of Taisyo Pharmaceutical Co. (Tokyo, Japan). Tri-iodothyronine (T₃) was purchased from Wako Pure Chemical Ind. (Osaka, Japan), and all other chemicals were from Sigma Chemical Co. (St. Louis, MO).

Statistics. Group data are shown as mean ± S.E. Analysis of variance and Tukey-Kramer procedure were used for multiple comparisons, and Student's t test was used for comparison between two means. Differences were considered significant when p values were <0.05.

	Results

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In conventional whole-cell patch-clamp experiments using HEK-Ca_v3.2 cells, T-type Ca²⁺ currents (I_Ca.T) were recorded. Figure 1A shows representative examples of I_Ca.T change with () or without () acute amiodarone application. Peak current was rapidly decreased by the application of 2 µM amiodarone and was partially reversed upon washout. Although these currents were obtained with the conventional, whole-cell configuration, that is, with the intracellular milieu dialyzed in pipette solution, currents did not exhibit significant "run-down". For example, current peak amplitude was -1.12 nA at 1 min and -1.10 nA at 11 min after obtaining the whole-cell mode in this patch (Fig. 1A, d and e), and the percentage amplitude of I_Ca.T at 11 min was 98.2 ± 1.0% when I_Ca.T at 1 min after obtaining the whole-cell mode was assigned as 100% (n = 5, P > 0.01). Figure 1, B and C, illustrates the short-term effects of 1 µM amiodarone on representative whole-cell current traces elicited by a depolarization pulse of 300-ms duration, ranging from -80 to +50 mV in 5-mV steps, applied from the holding potential of -100 mV, and an I-V relationship constructed using group data, indicating a reduction of the maximum current by 25.4 ± 8.0% (n = 23). To analyze the dose-dependent inhibitory effect of amiodarone, the activation and the steady-state inactivation (availability) curves were compared (Fig. 1D). Both the activation and the steady-state inactivation curves were shifted by amiodarone in the hyperpolarization direction in a dose-dependent manner. The average values for potentials of half-activation (V_1/2, activation) and half-inactivation (V_1/2, inactivation) under control condition and the presence of amiodarone are summarized in Table 1. Dosedependent significant shifts of activation and steady-state inactivation curves by amiodarone were observed; that is, 2 and 5 µM amiodarone shifted the activation and the steadystate inactivation curves in the hyperpolarization direction by 4.1 ± 1.4 mV (n = 19) and 9.5 ± 1.9 mV (n = 6), respectively (paired test, P < 0.001). DMSO as a vehicle (control) at the concentration of 0.1% had no significant effect on I_Ca.T (data not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 1. Short-term amiodarone effects on ICa.T. A, typical time course of the peak ICa.T recorded from HEK-Cav3.2 cells with () or without 2 µM amiodarone (). Amiodarone (2 µM) was immediately applied to the bath solution for 2 min. Five different current traces of ICa.T at -10 mV obtained µ from a pair of different patches (, ) are shown before (a), during application of 2 M amiodarone (b), during washing-out period (c), and at 1 (d) and 11 min (e) after obtaining the whole-cell mode in the absence of amiodarone. ICa.T was elicited from the holding potential of -100 mV at the test potential of -10 mV with the stimulation frequency of 0.2 Hz. B, representative whole-cell current families before (top traces) and after (bottom traces) application of 1 µM amiodarone (Ami) obtained from an HEK-Cav3.2 cell. Currents were elicited from a holding potential of -100 mV at the stimulation frequency of 0.2 Hz. C, I-V relationship with (, n = 23) or without 1 µM amiodarone (, n = 78). Amiodarone (1 µM) decreased the maximum peak ICa.T by 25.4 ± 8.0% (paired comparison with n = 23). D, dose-dependent shifts of activation and steady-state inactivation curves caused by amiodarone. Curves were fitted by the Boltzmann equation in each data group. Control (, n = 78 for activation and n = 55 for inactivation), 2 µM amiodarone (, n = 19 for activation and n = 14 for inactivation), and 5 µM amiodarone (, n = 6 for activation and n = 9 for inactivation). E, time course for development of use-dependent block of ICa.T by 2.5 µM amiodarone. ICa.T was recorded by a train of 50 pulses consisting of 100-ms duration at a test potential -10 mV from the holding potential of -100 mV with the stimulation frequency of 1 Hz. Shown are data for controls (, n = 6) and for short-term 2.5 µM amiodarone application (, n = 6). Each point in the presence of amiodarone indicates fractional peak currents normalized by the value for the first pulse of the train (, pulse 0), which was obtained before drug application. A train of 50 pulses, beginning with pulse 1, was applied 3 min after the application of amiodarone, during which no electrical stimulation was applied. The fitting curve for control data () was reconstructed (boldface line), in which the point for the control current number 1 (, 1.0) was reassigned to the current number 1 during short-term amiodarone application (, 0.643), yielding a drug-independent use-dependent block component during short-term amiodarone application. Ratios for tonic block (TB) and drug-delimited use-dependent block (UDB) during short-term amiodarone application are identified.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Comparison of short- and long-term effects of amiodarone. A, representative whole-cell current traces obtained from an HEK-Cav 3.2 cell cultured for 72 h without amiodarone (control, DMSO), with 5 µM amiodarone [Ami(C)], and with 5 µM amiodarone plus 0.12 nM T3 [Ami(C) + T3(C)]. B, I-V relationships for ICa.T with or without long-term amiodarone treatment corrected by cell capacitance. Control (DMSO) (, n = 66), long-term treatment of 5 µM amiodarone [Ami(C)] (, n = 41), long-term treatment of 5 µM amiodarone with 0.12 nM T3 [Ami(C)+ T3(C)] (broken line without symbols, n = 6). C, shifts of activation and steady-state inactivation curves by long-term amiodarone treatment. Curves were fitted by the Boltzmann equation in each data group. Control (DMSO) (, n = 66), Ami(C) (, n = 41), and Ami(C) + T3 (C) (broken line without symbols, n = 6).

View larger version (29K): [in this window] [in a new window]   Fig. 5. Effects of short-term amiodarone application on ICa.T with long-term amiodarone treatment for 72 h. A, representative ICa.T families obtained from an HEK-Cav3.2 cell with long-term amiodarone treatment [Ami(C)] and short-term effects of 1 µM amiodarone [Ami(C) + 1 µM Ami]. B, I-V relationship of ICa.T treated with 5 µM amiodarone [Ami(C)] (, n = 9) and short-term 1 µM amiodarone application [Ami(C) + 1 µM Ami] (, n = 9). C, voltage dependence of activation and steady-state inactivation curves of long-term amiodarone treatment (, n = 9), and short-term amiodarone treatment (1 µM) and effects on continually treated ICa.T (, n = 9). Note that the activation and steady-state inactivation curves were significantly shifted, with short-term application of 1 µM amiodarone, in the hyperpolarization direction by 8.6 ± 1.1 mV (n = 9) and 8.0 ± 1.5 mV (n = 11), respectively (P < 0.001). D, time course for the development of use-dependent block of ICa.T recorded from HEK-Ca 3.2 cells treated with long-term 5 µM amiodarone with or without short-term 2.5 µM amiodarone application. The same pulse protocol consisting of va train of 50 pulses described in Fig. 1E was applied. Shown are data for long-term 5 µM amiodarone [Ami(C)] (, n = 6) and short-term 2.5 µM amiodarone application on ICa.T recorded from HEK-Cav3.2 cells treated with long-term 5 µM amiodarone [Ami(C) + 2.5 µM Ami] (, n = 6). The fitting curve for long-term amiodarone data () was reconstructed (boldface line), in which the point for the current number 1 (, 1.0) was reassigned to the current number 1 during short-term amiodarone application (, 0.605), yielding a drug-independent use-dependent component during short-term amiodarone application. Ratios for tonic block (TB) and drug-delimited use-dependent block (UDB) during short-term amiodarone application are identified.

Because amiodarone reportedly exerts short- and longterm effects on various cardiac ion channels, we examined the long-term effects of amiodarone on I_Ca.T. I_Ca.T recorded from HEK-Ca_v3.2 cells treated with 2 µM amiodarone for 72 h revealed a significant delay in recovery from inactivation in the absence of amiodarone in the bath solution [: 2 µM long-term effects of amiodarone (n = 6), 618 ± 71 ms]. Figure 2A shows the representative I_Ca.T from HEK-Ca_v3.2 cells with and without amiodarone treatment for 72 h. HEK-Ca_v3.2 cells undergoing long-term treatment with amiodarone expressed significantly smaller I_Ca.T than those cells not treated with amiodarone (DMSO control cells), indicating a reduction of the maximum peak I_Ca.T by 31.7 ± 2.3%, although amiodarone was excluded from both the culture medium and the bath solution for more than 12 h (Fig. 2, A and B). The activation and steady-state inactivation curves obtained from HEK-Ca_v3.2 cells cultured with 5 µM amiodarone were shifted in the depolarization direction by -7.1 and -5.5 mV, respectively (Fig. 2C and Table 1). Because there are a number of studies on the interaction of amiodarone and thyroid hormones in clinical and experimental studies (Guo et al., 1997; Bosch et al., 1999; Shahrara and Drvota, 1999), we conducted experiments for the evaluation of long-term amiodarone effects on I_Ca.T with or without thyroid hormone T₃. As shown in Fig. 2, exposure of HEK-Ca_v3.2 cells to T₃ for 72 h did not modify the long-term effect of amiodarone on I_Ca.T, in terms of current amplitude and voltage dependence of activation and steady-state inactivation curves.

The short-term application of amiodarone shifted the activation and steady-state inactivation curves in the hyperpolarization direction, and long-term treatment of HEK-Ca_v3.2 cells by amiodarone shifted the same curves in the depolarization direction. Therefore, we found that I_Ca.T in HEK-Ca_v3.2 cells subjected to long-term amiodarone treatment was diminished because of a mechanism that is different compared with those in short-term treatment.

To study the affinity of amiodarone for the distinct channel states (i.e., activation, inactivation, and resting states), we examined whether the time course for recovery from inactivation of I_Ca.T was modulated by short- and long-term amiodarone treatment. Thus, we examined the recovery time course from inactivation by using the pulse protocol illustrated in Fig. 3. Recovery from inactivation was significantly delayed by immediately applied amiodarone at a concentration of 2 µM [time constant : control (n = 6), 490 ± 35 ms; 2 µM amiodarone (n = 6), 1081 ± 95 ms] (p < 0.001), which is consistent with the findings shown in Fig. 1E. Recovery from inactivation recorded in HEK-Ca_v3.2 cells subjected to longterm amiodarone treatment for 72 h was significantly delayed in the absence of amiodarone in the bath solution, which indicates that gating properties of I_Ca.T might be modified by long-term amiodarone treatment.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Time course of recovery from inactivation of ICa.T. Curves were fitted to the data using a single exponential equation yielding time constant (). control (, n = 6), 490 ± 35 ms; short-term 2 µM amiodarone (, n = 5), 1081 ± 95 ms; long-term 2 µM amiodarone (, n = 5), 618 ± 71 ms. Inset, the pulse protocol.

View larger version (21K): [in this window] [in a new window]   Fig. 4. ICa.T relaxation (inactivation) with or without long-term amiodarone treatment. The decay of the current was fitted by a single exponential equation. Inactivation time constant () was plotted against the test potential. Each plot was constructed for three groups of data: the control (DMSO) condition (, n = 6), short-term treatment with 5 µM amiodarone (, n = 6), and long-term treatment with amiodarone [Ami(C)] (, n = 6). a, representative current traces at -30 mV (indicated by arrows) with or without long-term amiodarone treatment that were normalized to the peak current. b, representative current traces with or without longterm amiodarone treatment at the maximum current [control at -30 mV, Ami(C) at -25 mV, indicated by arrows) normalized to the peak current. *, P < 0.05 versus control (nonpaired): **, P < 0.01 versus control (nonpaired); ***, P < 0.001 versus control (nonpaired).

Finally, we obtained IC₅₀ values for amiodarone in shortterm application on I_Ca.T, long-term application on I_Ca.T, and short-term application on I_Ca.T from the HEK-Ca_v3.2 cells that received long-term treatment with amiodarone. As a tonic-blocking action, short-term amiodarone application inhibited I_Ca.T with an IC₅₀ value of 2.4 µM, whereas application of amiodarone for 72 h lessened I_Ca.T with an IC₅₀ value of 9.4 µM (Fig. 6). The short-term effect of amiodarone on I_Ca.T in HEK-Ca_v3.2 cells that received long-term treatment with amiodarone exhibited an IC₅₀ value of 2.8 µM (Fig. 6).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Dose-response relationship of short- and long-term actions of amiodarone on ICa.T. Short- and long-term effects of amiodarone were evaluated by a pulse protocol composed of a holding potential of -100 mV to a test potential for the maximum current (-25 to -35 mV) at the stimulation frequency of 0.2 Hz. Short-term effect (, n = 5-23), longterm effect (, n = 5-13), and short-term effect on cells treated with 5 µM long-term amiodarone (, n = 4-11) on ICa.T. Data were fitted by the Hill equation yielding IC50 values of 2.4, 9.4, and 2.8 µM, respectively. Therapeutic plasma concentration (1-3 µM) is indicated by shading (Singh et al., 1989).

	Discussion

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Short-Term Effects of Amiodarone on Cardiac Ca²⁺ Channels. Although short-term application of amiodarone promptly decreased I_Ca.T, we could not reverse I_Ca.T completely upon washout of the drug (Fig. 1A). As already pointed out, amiodarone is a highly lipophilic drug (oil/water partition coefficient, log P = 5.95, and pK_a = 8.7 at 37°C) (Chatelain et al., 1986). Short-term effects of amiodarone cannot be consistently and completely reversed during shortterm experiments in the literature (Raatikainen et al., 1996; Kodama et al., 1997).

In 1992, using guinea pig atrial cells, Cohen et al. demonstrated that amiodarone at a concentration of 10 µM decreased I_Ca.T, shifting the steady-state inactivation curve in the hyperpolarization direction. As far as we know, this is the only published report showing an inhibitory effect of amiodarone on I_Ca.T. However, in their report, no blocking mechanism or the inhibitory potency of amiodarone on I_Ca.T was observed. In our study, we used a heterologous expression system of _1H (Ca_v3.2) in HEK 293 cells to examine the detailed pharmacological effects of amiodarone on homogenous I_Ca.T. It has been shown that some calcium antagonists demonstrate a general affinity for blocking both I_Ca.L and I_Ca.T. For example, bepridil (Yatani et al., 1986; Uchino et al., 2005), mibefradil (Mehrke et al., 1994), and efonidipine (Masumiya et al., 1998, 2000) block I_Ca.L and I_Ca.T with different efficacies and affinities. Lubic et al. (1994) studied the interaction of amiodarone with receptors for the 1,4-dihydropyridine in rat and rabbit myocardial membranes and found that amiodarone completely inhibited the specific binding to myocardial membrane receptors by 1,4-dihydropyridine Ca²⁺ channel blockers. These results provide evidence that amiodarone modifies current kinetics by directly interacting with the Ca²⁺ channel and/or its immediate lipidic environment.

Short-term effects of amiodarone on blocking I_Ca.L have been reported as follows: 1) amiodarone decreased I_Ca.L in a concentration-dependent manner, with an IC₅₀ value of 0.36 to 5.8 µM; 2) amiodarone (5-16 µM) shifted the steady-state inactivation curve of I_Ca.L in the hyperpolarizing direction by 9 to 10 mV; and 3) amiodarone caused both tonic and usedependent reduction of I_Ca.L (Nishimura et al., 1989; Valenzuela and Bennett, 1991; Varró et al., 1996). Short-term inhibitory actions of amiodarone might be attributed to binding to a single receptor. The block depends on whether the membrane is held at rest or is hyperpolarized electrically for longer or shorter periods, and it depends on the frequency and type of stimulation (Hille, 1977). Short-term application of 2.5 µM amiodarone caused both tonic and use-dependent block of I_Ca.T (Fig. 1E), in which tonic block primarily reflects rested-state block. The fractional ratio of (tonic block)/(usedependent block) was approximately 2.1 for short-term 2.5 µM amiodarone application on I_Ca.T (Table 2). The lower the ratio, the stronger the gain of potency obtained during the high frequency of stimulation. Fractional tonic block by short-term 2.5 µM amiodarone on I_Ca.T (35.7%) and inhibition in cells that underwent long-term treatment with amiodarone on I_Ca.T (39.5%) were statistically identical (P = 0.45). Moreover, drug-delimited use-dependent block of short-term 2.5 µM amiodarone on I_Ca.T (17.2%) and inhibition in cells that underwent long-term treatment with amiodarone on I_Ca.T (15.0%) were also statistically identical (P = 0.66). These phenomena can be qualitatively explained by assuming that amiodarone preferentially binds to the resting or activated state rather than to the inactivated state of the _1H T-type Ca²⁺ channel, regardless whether the channel received long-term amiodarone treatment or not. Taken together, these data and ours indicate that the short-term blocking effects of amiodarone on I_Ca.L and I_Ca.T are alike, suggesting that amiodarone blocks these two types of Ca²⁺ channels via the same mechanism.

Long-Term Effects of Amiodarone on Cardiac Ion Channels. The most striking difference in the effects produced by short-versus long-term administration of amiodarone can be found on the shifts in activation and steady-state inactivation curves (Figs. 1D and 2C) and on current relaxation (Fig. 4). Desensitization of I_Ca.T by long-term amiodarone treatment is unlikely, because the potency of short-term amiodarone on I_Ca.T (IC₅₀ = 2.4 µM) and the potency of short-term amiodarone on I_Ca.T continually treated with amiodarone (IC₅₀ = 2.8 µM) are nearly identical (Fig. 6). It is widely recognized that the most prominent long-term effects of amiodarone include prolongation of the refractory period and action potential durations (Kodama et al., 1997, 1999), suggesting the inhibition of K⁺ currents (Kamiya et al., 2001). Their studies suggest that decreases in I_Kr, I_Ks, and I_to are due to down-regulation of channel proteins primarily caused by gene regulations for ion channel expression. A comprehensive study of cDNA microarrays of ion channels by long-term amiodarone treatment was reported by Le Bouter et al. (2004). They found that Na⁺ channel - and -subunits; L-type Ca²⁺ channel ₁, ₁, and ₂ subunits; T-type Ca²⁺ channel _1Gsubunit; Kv2.1, Kv1.5, and Kv4.2, ATP-sensitive K⁺ channels; Kir6.2; and SUR2 were down-regulated, and that other K⁺ channel - and -subunits, such as KNCA4, KCNK1, KCNAB1, and KCNE3, were up-regulated. However, possible remodeling of the T-type Ca²⁺ channel _1H subunit by long-term amiodarone treatment has not yet been studied. The cardiac T-type Ca²⁺ channel is composed of two subtypes of subunits, _1H and _1G. The _1H subunit differs from the _1G subunit in many respects (Perez-Reyes, 2003). Hagelüken et al. (1995) reported that amiodarone is a direct activator of G_i and G_o proteins, activating nonselective cation channels in HL-60 cells via these proteins. Lledo et al. (1992) and Lu et al. (1996) proposed that I_Ca.T could be modulated by hormones and neurotransmitters coupled to the G_q or G_i/G_o families. It is therefore speculated that amiodarone may exert its long-term effects on the Ca_v 3.2 T-type Ca²⁺ via G-protein(s). It is more interesting that _1H T-type Ca²⁺ channel kinetics were drastically modified by long-term amiodarone treatment, particularly on the current relaxation phase. Although no proteins or signals that modulate the kinetics of _1H have yet been identified, our results suggest the presence of functional auxiliary protein(s) regulated by amiodarone. Because the T-type Ca²⁺ channels are postulated to play important roles in pathological conditions of the heart, long-term amiodarone treatment may reverse the remodeling of I_Ca.T. Further study is needed to clarify the interaction of G_i/G_o, transcription factors for Ca_v3.2 channel expression, modulators for the promoter regions of the channel gene, and/or subsidiary proteins with the Ca_v3.2 proteins in the presence of amiodarone.

Thyroid Hormone-Independent Effects of Amiodarone on I_Ca.T. It is known that amiodarone can cause either hypothyroidism (Martino et al., 1984) or hyperthyroidism (Martino et al., 1987). Cardiac electrophysiologic changes induced by long-term treatment with amiodarone closely resemble those induced by hypothyroidism (Kodama et al., 1997). However, hypothyroidism does not mimic all of the long-term effects of amiodarone (Lambert et al., 1987; Bosch et al., 1999). Guo et al. (1997) have demonstrated that 72-h exposure to 1 µM amiodarone significantly decreased the current density of I_to and I_K in the cardiac myocytes only when the cells were cocultured with T₃. In our study, downregulation of I_Ca.T in long-term treatment with amiodarone was mediated independently of thyroid hormone action. It is noteworthy that exposure of HEK-Ca_v3.2 cells to amiodarone in the presence of T₃ resulted in changes of channel kinetics and density of I_Ca.T that were identical with those observed in the absence of T₃ (Fig. 2). Based on these findings, we conclude that the inhibition and regulation of I_Ca.T as the result of long-term amiodarone treatment are exerted independently of its action through the modulation of thyroid hormone secretion.

In conclusion, as short-term effects, amiodarone binds directly to the _1H channel, decreases the current, and shifts activation and steady-state inactivation curves in the hyperpolarization direction. On the other hand, as long-term effects, amiodarone regulates the expression of the _1H, probably through unknown protein(s) and/or signaling molecules. The short- and long-term effects of amiodarone on I_Ca.T are completely different in their pharmacological actions, which indicate the complicated therapeutic activity of amiodarone on the regulation of T-type Ca²⁺ channel-related arrhythmias in clinical application.

	Acknowledgements

 
We thank Dr. E. Perez-Reyes (University of Virginia, Charlottesville, VA) for the generous gift of Ca_v3.2 vector constructions.

	Footnotes

 
ABBREVIATIONS: I_Ca.L, L-type Ca²⁺ channel current; I_Ca.T, T-type Ca²⁺ channel current; HEK, human embryonic kidney; Ami, amiodarone; DMSO, dimethyl sulfoxide; T₃, tri-iodothyronine; I-V, current-voltage.

Address correspondence to: Dr. Katsushige Ono, Department of Cardiovascular Science, Oita University School of Medicine, 1-1 Idaigaoka, Hasama, Yufu, Oita 879-5593, Japan. E-mail: ono{at}med.oita-u.ac.jp

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