Introduction

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In human atrial myocytes the main repolarizing current that activates during the plateau-shaped action potential is a large voltage-dependent outward K⁺ current (I_o). This current is composed of an initial fast-inactivating component, followed by a large slowly inactivating component; the two components are usually designated I_t and I_sus, respectively (Escande et al., 1987). The ratio between I_t and I_sus depends on age (Escande et al., 1985) and on clinical conditions such as atrial dilation (Le Grand et al., 1994) and chronic arrhythmia (Van Wagoner et al., 1997) and is controlled by intracellular regulatory factors (Tessier et al., 1999). This current is sensitive to a number of pharmacological agents and is probably an important target of antiarrhythmic drugs used to treat atrial tachyarrhythmia (Wang et al., 1995). The molecular basis of this current and the mechanisms involved in its regulation are thus a focus of attention.

A large part of this current is probably the functional expression of hKv1.5 channels, which are abundant in human atrial myocardium (Tamkun et al., 1991; Fedida et al., 1993; Wang et al., 1993). These channels, when expressed in cell lines, carry a rapidly activating outward current that, in control conditions, inactivates slowly and has pharmacological characteristics similar to those of I_sus (Snyders et al., 1993). Indeed, cloned hKv1.5 channels are remarkably sensitive to a number of compounds, including antiarrhythmic agents (Snyders and Yeola, 1995; Delpon et al., 1999). By binding to a hydrophobic site in the internal mouth of the pore of Kv channels, some of these compounds block the channels in the open position, with therapeutically valuable use-dependent inhibition of the repolarizing current (Snyders and Yeola, 1995; Yeola et al., 1996; Longobardo et al., 1998). In addition, the frequency-dependent inhibition of the outward K⁺ current by drugs that block the channels in the open state can also arise from modulation of the intrinsic gating mechanisms, which govern the rate of channel inactivation (Baukrowitz and Yellen, 1996a; Rasmusson et al., 1998). This has been described with tetraethylammonium (TEA) compounds, which, by blocking the channel pore, reduce the outward K⁺ flow into the pore and, in turn, decrease the occupancy of K⁺ binding sites involved in the regulation of pore closure, thus leading to fast inactivation of shaker channels (Baukrowitz and Yellen, 1996b).

View larger version (14K): [in this window] [in a new window]   Structure

Bertosamil is a structural analog of tedisamil, a new bradycardic class III antiarrhythmic agent with potential therapeutic efficacy in both atrial and ventricular tachyarrhythmia (Wallace et al., 1995; Fischbach et al., 1999; Singh, 1999). We have previously reported that bertosamil accelerates the apparent rate of inactivation of the outward current in human atrial myocytes (Tessier et al., 1997), acting in the same way as tedisamil on the outward current in rat ventricular myocytes (Dukes and Morad, 1989). The effect of bertosamil on the outward K⁺ current of human atrial myocytes is also characterized by its irreversibility, enhanced current inactivation with membrane depolarization, a shift in its voltage-dependent activation, and an acceleration of the time to peak current, raising the possibility that the drug causes profound changes in the gating properties of channels carrying the outward current. Because bertosamil mainly affects I_sus, the underlying mechanism may involve modulation of hKv1.5 channels.

This study was undertaken to investigate 1) the mechanism of action of bertosamil on cloned hKv1.5 channels expressed in the Chinese hamster ovary (CHO) cell line, and 2) whether the same mechanism affects endogenous channels in human atrial myocytes, and, if so, how it contributes to the modulation of the outward current by bertosamil.

	Materials and Methods

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Cell Preparation and Transfection. CHO cells were cultured in Ham's F-12 medium (nutrient mixture; Invitrogen, Cergy Pontoise, France), 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Invitrogen). hKv1.5 cDNA was generated by polymerase chain reaction from human heart RNA, checked by sequencing, and inserted into the pIRES2-EGFP expression vector (CLONTECH, Palo Alto, CA), the internal ribosome entry sequence generating a dual cistronic mRNA. Transfection was done using the FuGENE 6 kit (Roche Molecular Biochemicals, Mannheim, Germany), with 6 µl of transfection reagent and 2 µg of plasmid DNA per 35-mm dish; 36 h after the outset of transient transfection, cells were trypsinized and plated. Voltage-clamp recording revealed typical hKv1.5 currents in 90 to 100% of cells expressing green fluorescent protein.

Atrial Myocyte Isolation. With approval from the ethics committee of our institution, myocytes were enzymatically isolated from samples of right atrial appendage obtained from 10 patients aged 44 to 78 years undergoing heart surgery for coronary insufficiency, mitral valve disease, or aortic valve disease. Most patients were on pharmacological treatments, which were stopped at least 10 h before surgery (Ca²⁺ channel blockers, -adrenergic antagonists, diuretics, angiotensin-converting enzyme inhibitors, or nitric oxide donors). The isolation procedure has been described in detail elsewhere (Tessier et al., 1999). Briefly, small fragments of atrial appendage were cut up and washed in Krebs-Ringer solution containing 35 mM NaCl, 4.75 mM KCl, 1.19 mM KH₂PO₄, 16 mM Na₂HPO₄, 10 mM HEPES, 10 mM glucose, 25 mM NaHCO₃, 134 mM saccharose, and 30 mM 2,3-butane-dione oxime, pH 7.4, gassed with 95% O₂, 5% CO₂ and gently agitated at 37°C. Cells were dissociated by several enzymatic steps by using the same Krebs-Ringer solution without 2,3-butane-dione oxime and containing collagenase (type IV; Sigma, St. Quentin Fallavier, France) and protease (type XXIV; Sigma). Isolated myocytes were resuspended in a solution containing 2 mM Ca²⁺ and incubated at 37°C with continuous gassing with 21% O₂, 5% CO₂ for at least 1 h before use.

Current Measurements. Currents were recorded by using the patch-clamp technique in the whole cell configuration with borosilicate glass pipettes (tip resistance 1.5-2 M) connected to the input stage of the patch-clamp amplifier (Axoclamp 200A; Axon Instruments, Union City, CA). Resistance in series was compensated to obtain the fastest capacity transient current (percentage of resistance compensation between 0 and 5%); the capacitive and leakage currents were not compensated. Currents were filtered at 5 kHz, digitized with a Labmaster (Lab Rac, Scientific Solution, Mentor, OH), and stored on the hard disk of a personal computer. Data were acquired and analyzed with Acquis 1 software (G. Sadoc, Centre National de la Recherche Scientifique Unité Propre de Recherche 2212, Gif/Yvette, France).

Solutions and Drugs. Cells were continuously bathed in an external solution containing 137 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose, adjusted to pH 7.3 with NaOH. For potassium current measurements in control myocytes, Na⁺ was replaced by an equimolar concentration of choline chloride, Ca²⁺ channels were blocked with 5 × 10⁴ M Cd²⁺, and 10⁵ M atropine was added to the external solution to prevent muscarinic receptor activation. In experiments testing the effects of TEA and increasing external K⁺ concentration ([K⁺]_o), NaCl in the external solution was replaced with an equimolar concentration of TEA or KCl. Patch pipettes were filled with an internal solution containing 115 mM K-aspartate, 5 mM KCl, 5 mM MgATP, 5 mM Na-pyruvate, 3 mM MgCl₂, 5 mM EGTA, and 10 mM HEPES, adjusted to pH 7.2 with KOH. In experiments testing the effects of internal TEA, K-aspartate in the internal solution was replaced with an equimolar concentration of TEA. In some experiments, KCl was replaced by 140 mM cesium chloride (Cs⁺). Bertosamil [3-isobutyl-7-isopropyl-9,9-pentamethylene-3,7-diazabicyclo-(3.3.1) nonane sesquihydrogenfumarate; KC 10784] was a gift from Dr. Ziegler (Solvay Pharma Deutschland, Hannover, Germany). Because bertosamil is a water-soluble drug, it was dissolved in the external or internal solution. 4-Aminopyridine was obtained from Sigma and was dissolved in the extracellular solution. All experiments were carried out at room temperature (22-24°C).

Data Analysis. I_peak was measured as the difference between the amplitude of the peak current and the zero current; the amplitude of the maintained current I_ss was measured as the difference between the current measured at the end of the 750-ms test pulse and the zero current; the amplitude of the initial fast-inactivating component of the outward current I_t was calculated as the difference between I_peak and I_ss. The extent of I_o or hKv1.5 current inactivation was quantified by measuring the fraction of outward K⁺ current inactivation, defined as the extent of inactivation = (I_total  I_{steady state})/I_total. The tail current decay was quantified as (tail current beginning  tail current end)/tail current beginning. The time course of I_o or hKv1.5 current inactivation elicited by a 750-ms test pulse was best fitted by the sum of two exponential functions,

(1)

whereas the current elicited by a 5-s test pulse was best fitted by three exponential functions: y(t) = A exp(t/₁) + B exp(t/₂) + C exp(t/₃) + E, where A, B, and C are amplitude terms; t is time; ₁, ₂, and ₃ are time constants of the fast and slow inactivation phases; and D and E are the amplitude of the steady-state component (eq. 1). The procedure used to normalize the current consisted of dividing raw values of the digitized current by its maximum, by using Fig.P version 6.0 software (Biosoft, Cambridge, UK).

(2)

(3)

(4)

Statistical Analysis. Data are presented as means ± S.E.M. Student's paired or unpaired t test was used to determine the significance of differences, as appropriate. P values of <0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Bertosamil Accelerates hKv1.5 Current Inactivation. In CHO cells expressing hKv1.5 channels, 750-ms test pulses from 60 to +50 mV delivered at 0.1 Hz elicited a rapidly activating outward current with a very slow rate of inactivation and a small extent of inactivation (0.09 ± 0.03, n = 12; Fig. 1A). External application of 10 µM bertosamil progressively inhibited the current (at +50 mV, I_ss: 862 ± 248 versus 434 ± 132 pA in control and bertosamil conditions, respectively, n = 12, P < 0.01; Fig. 1A), with a more marked effect on the current recorded at the end of the 750-ms test pulse (I_ss) than on the peak (I_peak). At steady state (reached after around 20 pulses at 0.1 Hz), this resulted in an increase in the extent of current inactivation compared with control conditions (0.41 ± 0.05, n = 12, P < 0.001) and a slow tail current (extent of decay: 0.67 ± 0.05 versus 0.24 ± 0.06, n = 7, P < 0.001; Fig. 1B). The effects of bertosamil on the currents were observed when the drug was applied at rest as illustrated in Fig. 1C, which shows the current traces recorded in control conditions and after bertosamil application during a 2-min rest period. The current recorded at the resumption of stimulation in the absence of drug (to avoid additional drug binding to the channel in its open state, trace 2) was characterized by a marked acceleration of time-dependent inactivation, resulting in steady-state suppression of I_ss and a large transient component (I_t) (extent of inactivation: 0.11 ± 0.04 versus 0.56 ± 0.06, n = 5, P < 0.05). Further application of bertosamil (traces 3 and 4) inhibited both I_peak and I_ss (I_peak: 21.08 ± 5.10%; extent of inactivation: 0.46 ± 0.12, n = 5, N.S.) without changing the current kinetics. Analysis of the concentration-dependent effects of external bertosamil on the I_peak yielded IC₅₀ values of 39 µM (n_H of 1.0 ± 0.2) and 11 µM (n_H of 0.9 ± 0.2) for I_ss (P < 0.05) (eq. 2 under Materials and Methods; Fig. 2, A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effects of extracellular bertosamil on the hKv1.5 current in CHO cells. A and B, cell was pulsed at +50 mV from a holding potential of 60 mV and then repolarized to 40 mV for 150 ms (at 0.1 Hz) to record the deactivation current (tail current). A, traces of current in the absence () and presence of 10 µM external bertosamil (BTe, ); B, tail current in control () and bertosamil () conditions. Cell capacitance, 46 pF. C, current traces recorded in control conditions (1), after superfusion of the cell with external 10 µM bertosamil during a 2-min rest period, at the resumption of stimulation (2), at the beginning (3) and steady-state (4) effects of 10 µM bertosamil applied during a train of test pulses (0.1 Hz). Cell capacitance, 32 pF.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent effects of external bertosamil on the hKv1.5 current. Concentration dependence of effect of bertosamil on Ipeak (A) and Iss (B), expressed as the ratio of current amplitude before and after application of various concentrations. Each point is the average of six to eight measurements; vertical lines show the S.E.M. and are visible when larger than the symbols.

Effects of Intracellular Application of Bertosamil on hKv1.5 Current. To determine whether the rest-dependent effect of bertosamil arose from drug binding to an internal site, cells were dialyzed with an internal solution containing 50 µM bertosamil to obtain a complete drug effect. Figure 3A shows the superimposition of currents elicited by a test pulse from 60 to +50 mV at 0.1 Hz, just after breaking the patch, and at various times of cell dialysis with a solution containing 50 µM bertosamil. Internal bertosamil application caused gradual and marked inhibition of I_ss, whereas I_peak was not significantly modified; at steady state, this resulted in a current with a large extent of inactivation (0.60 ± 0.03, n = 12, P < 0.001) and a slow tail current (extent of tail current decay: 0.06 ± 0.03, n = 12, P < 0.001; Fig. 2B). To compare the rate of inactivation of hKv1.5 current between control and internal bertosamil condition, membrane was depolarized during 5 s to allow the slow inactivation of the control current (eq. 1). This gave a ₁ of 680.4 ± 140.3 versus 29.4 ± 4.1 ms (P < 0.001) and ₂ of 3700.1 ± 772.5 versus 295.1 ± 38.3 ms (P < 0.001) in control (n = 7) and bertosamil (n = 8), respectively (₃ was around to 4 s). After 5 min of internal bertosamil dialysis at rest (time required to obtained steady-state dialysis of the cell) the current recorded at the first pulse was already characterized by a large I_t and a suppressed I_ss, with no changes during subsequent pulses (Fig. 3C). This suggested that the gradual decrease in I_ss observed upon intracellular application of bertosamil during a train of pulses (Fig. 3A) resulted mainly from slow dialysis of the drug. External application of 10 µM bertosamil in the presence of intracellular drug caused a additional inhibition of both I_peak and I_ss (I_peak: 29.26 ± 4.43%, n = 4, n = 5, P < 0.05; I_ss: 24.70 ± 4.10%, n = 4, P < 0.05; Fig. 3D). Figure 4 shows representative traces of currents elicited by 10-mV incremental 750-ms test pulses delivered at a frequency of 0.1 Hz from a holding potential of 60 mV in CHO cells dialyzed with control (Fig. 4A) or bertosamil-containing (Fig. 4B) internal solution. The peak outward current-voltage relationship, normalized to control, was identical in the two conditions, whereas I_ss was reduced in intracellular bertosamil conditions; the effect was significant at potentials above 0 mV (normalized currents at +50 mV, 0.76 ± 0.02 versus 0.32 ± 0.04 in control and bertosamil conditions, respectively, n = 11, P < 0.0001; Fig. 4, C and D). The activation curve of the current was not modified by internal bertosamil (V_1/2: 4.1 ± 1.3 in C versus 2.5 ± 1.2 in intracellular bertosamil (BT_i), n = 8, N.S.; k: 23.0 ±1.5 in C versus 26.4 ± 1.8, n = 8, N.S.) (eq. 4 under Materials and Methods; Fig. 4E). These results suggested that bertosamil modulated I_hKv1.5 by binding to distinct extra- and intracellular sites on the channel, the latter mediating the acceleration of the apparent rate of current inactivation.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   A, effects of intracellular bertosamil (BTi): superimposition of currents elicited by test pulses from 60 to +50 mV and repolarized to 40 mV (at 0.1 Hz), just after breaking the patch () and at steady-state dialysis (5 min) () with a 50 µM bertosamil-containing internal solution; B, tail currents in control () and bertosamil () conditions. Cell capacitance, 18 pF. C, trace of current elicited by the same protocol as in A after 5 min of intracellular dialysis with an internal solution containing 50 µM bertosamil at rest. Cell capacitance, 21 pF. D, superimposition of current traces elicited by test pulses from 60 to +50 mV and then to 40 mV (at 0.1 Hz) at the steady-state effect of internal application of 50 µM bertosamil () and upon external application of 10 µM bertosamil (). Cell capacitance, 15 pF.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effects of internal bertosamil on the voltage dependence of the hKv1.5 current. Traces of currents elicited by 10-mV incremental test pulses from 60 to +50 mV (at 0.1 Hz) in control conditions () (A) and at the steady-state effect of 50 µM bertosamil () (B). Cell capacitance, 17 pF. C, peak and steady-state current-voltage (I-V) relationships of hKv1.5 currents; Iss, steady-state current (); Ipeak, peak current (). Values are means ± S.E.M. (n = 8) of measurements with different CHO cells. D, peak () and steady-state () I-V relationships of hKv1.5 currents in the presence of 50 µM intracellular bertosamil. E, activation curve of Ipeak in control conditions () and on 50 µM bertosamil exposure (). Error bars in C, D, and E (S.E.M.) are visible when larger than the symbols.

Ionic Sensitivity of Bertosamil Effect on Current. In these experiments we examined the mechanism responsible for the apparent acceleration of I_hKv1.5 inactivation by bertosamil. First, we examined whether the nature of the monovalent cation flowing through the channels modulated the effect of bertosamil. Because the Cs⁺ current carried by hKv1.5 channels inactivates much less markedly than the K⁺ current (Fedida et al., 1999), we tested the effect of replacing internal K⁺ by 140 mM Cs⁺, which caused about a 5-fold reduction in current amplitude without changing the activation threshold (around 40 mV). As illustrated in Fig. 5A, the Cs⁺ current elicited by 750-ms pulses at +50 mV was inhibited by 10 µM external bertosamil applied during a train of pulses (11.45 ± 3.92%, n = 5, P < 0.05), but its extent of inactivation was unaffected (0.04 ± 0.01 versus 0.05 ± 0.02, n = 5, N.S.). The effects of bertosamil on the steady-state amplitude of the Cs⁺ current was significant at potentials above 0 mV (Fig. 5B). As controls, and to check that the drug effectively bound to the channels, we used an external solution containing 80 mM potassium to shift the reversal potential for K⁺ (around +15 mV versus 88 mV in control conditions), such that an inward potassium current could be recorded in the potential range of the voltage dependence activation of hKv1.5 channels (Fig. 5, C and D). Figure 5C shows a superimposition of inward K⁺ currents recorded during a test pulse at 20 mV, before and after bertosamil application, in the same CHO cell as in Fig. 5A. In contrast to its effect on the outward Cs⁺ current, bertosamil not only suppressed the inward K⁺ current but also increased its extent inactivation (0.12 ± 0.02 versus 0.34 ± 0.01, n = 4, P < 0.001; Fig. 5C). Figure 5D shows the effects of bertosamil on the 80 mM K⁺ currents at various potentials (n = 4). After bertosamil application in the presence of 80 mM external potassium and drug binding to the channels, the time-dependent inactivation of the Cs⁺ outward current (test pulse to +50 mV), recorded again in the absence of external potassium, was still not altered.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effects of bertosamil on the Cs+ current. A, superimposition of current traces elicited by test pulses from 60 to +50 mV and then to 40 mV (at 0.1 Hz) in CHO cells dialyzed with a pipette solution containing 140 mM Cs+, 0 mM K+ in control conditions () and upon external application of 10 µM bertosamil (). Cell capacitance, 25 pF. B, current-voltage relationships of the Cs+ current in control conditions () and after bertosamil exposure (). C, superimposition of current traces recorded in the same cell as in A (i.e., the same Cs+ internal solution) but bathed with an external solution containing 80 mM K+ and elicited by depolarizing pulses to 20 mV from a holding potential of 60 mV in the absence () and presence () of 10 µM external bertosamil. Cell capacitance, 40 pF. D, current-voltage relationships of the current recorded with 80 mM external K+ in control conditions () and after bertosamil exposure (). Each point is the average of six to eight measurements; vertical lines show the S.E.M. and are visible when larger than the symbols.

Bertosamil Effects Are Suppressed by Internal TEA. External application of 30 mM TEA had a small but significant effect on the current recorded in the presence of intracellular bertosamil (I_peak: 16.75 ± 2.60%, P < 0.05; rate of inactivation: _fast: 33.2 ± 4.4 versus 44.4 ± 5.0 ms, n = 7, P < 0.01; extent of inactivation: 0.64 ± 0.05 versus 0.56 ± 0.06, n = 7, P < 0.05; Fig. 6A). An effect on the current obtained during bertosamil treatment was also observed when the [K⁺]_o was increased (40 mM [K⁺]_o versus 5 mM). High [K⁺]_o reduced the amplitude and rate (I_peak: 22.21 ± 2.69%, P < 0.01; _fast: 47.34 ± 6.10 versus 68.98 ± 8.30 ms, n = 7, P < 0.01) and the extent of current inactivation (0.57 ± 0.07 versus 0.49 ± 0.07, n = 7, P < 0.05; Fig. 6B).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   A, in CHO cells expressing hKv1.5 channels, superimposed currents evoked by 750-ms step depolarization from a holding potential of 60 to +50 mV (at 0.1 Hz) before () and after exposure to 30 mM TEA (), recorded at the steady-state effect of internal 50 µM bertosamil. Cell capacitance, 33 pF. B, superimposed current tracings with 750-ms depolarizing pulses from a holding potential of 60 to +50 mV (at 0.1 Hz) at the steady-state effect of internal 50 µM bertosamil, recorded with 5 mM () and 40 mM () external K+. Cell capacitance, 14 pF. The left-hand panels show raw data, and the right-hand panels show data after normalization to peak current values.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effects of internal TEA on the outward hKv1.5 current. A, CHO cell was pulsed, at 0.1 Hz, to +50 mV from a holding potential of 60 mV and then repolarized to 40 mV for 150 ms to record the tail current; current traces were obtained just after breaking the patch () and at the steady-state effect of 30 mM internal TEA (). B, tail currents elicited on return to 40 mV after 750-ms depolarizing pulses at +50 mV, at the beginning () and steady state of internal TEA application (). Cell capacitance, 15 pF. C, using the same depolarizing protocol as in A, superimposition of current traces obtained at the steady-state effect of 30 mM internal TEA () and upon 10 µM bertosamil exposure (). Cell capacitance, 18 pF. D, concentration-dependent effect of TEA on the bertosamil effect, expressed as changes in the extent of outward current inactivation. Results are expressed as means ± S.E.M., and stars indicate statistical differences relative to control; n = 8 at each concentration, except for 3 mM (n = 4).

Effects of Rate and Duration of Membrane Depolarization on Current Treated with Bertosamil. After bertosamil application, the current became highly sensitive to the frequency of membrane depolarization, as indicated by experiments with a protocol involving delivery of a pair of depolarizing pulses from 60 to +50 mV separated by an increasing time interval. In control conditions, the hKv1.5 current had almost entirely recovered after the shortest time interval tested (96.10 ± 0.75% with a 4-ms interval, n = 7, P < 0.05; Fig. 8A), whereas the amplitude of the current elicited by the first pulse slightly decreased (85.10 ± 0.20%, n = 7, P < 0.05; Fig. 8A). After intracellular application of bertosamil, I_t was suppressed at the shortest coupling interval and slowly recovered with increasing interval duration: 80% of I_t had recovered with an interval duration of 0.55 ± 0.21 s versus <5 ms in C (n = 7, P < 0.0001) and with rate constant ₁ = 142 ms and ₂ = 2687 ms, n = 7 (eq. 5 under Materials and Methods; Figs. 8B and 10). To discriminate between channel recovery from drug binding or from inactivation as the main mechanism responsible for the effect of the rate of stimulation on I_t, several protocols known to modulate channel inactivation were tested (Aldrich, 1981). As shown in Fig. 9, prolonged membrane depolarization with 5-s test pulses delivered at 0.1 Hz had a slight transient effect on control current, whereas in the presence of intracellular bertosamil it caused a marked suppression of I_peak (23.7 ± 3.6%, n = 8, P < 0.001) but not I_ss (3.9.1.7, n = 8, N.S.; Fig. 9). Likewise, after intracellular bertosamil application, an increase in the rate of membrane depolarization from 0.1 to 1 Hz caused a marked and persistent decrease in the peak current, with little effect on I_ss (n = 5) (Fig. 9, C and D). When the cell was again paced at the low frequency of 0.1 Hz, I_t showed slow partial recovery (Fig. 9). In human atrial myocytes, too, bertosamil accelerates the rate of I_o inactivation (Tessier et al., 1997) and slows its recovery from inactivation (₁ = 83 versus ₁ = 170 ms, n = 7, P < 0.001; time necessary for 80% current recovery: 0.25 ± 0.02 versus 1.32 ± 0.30 s, n = 7, P < 0.0001; Fig. 10); in addition, the current underwent a cumulative inactivation phenomenon at high rates of membrane depolarization as illustrated in Fig. 11. Taken together, these results indicate that bertosamil increases the sensitivity of both the hKv1.5 current and I_o in human atrial myocytes to the rate of stimulation and to cumulative inactivation phenomena.

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Effects of bertosamil on the kinetics of current reactivation. Representative traces of the recovery from inactivation of the hKv1.5 current in control conditions (A) and at the steady-state effect of 50 µM intracellular bertosamil (B) with a protocol consisting of two identical 750-ms pulses from 60 mV to +50 mV, with interpulse intervals from 4 to 5000 ms. Cell capacitance, 16 and 21 pF, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Effect of prolonged membrane depolarization on hkV1.5 current recorded in control condition (A) and after the steady-state internal application of 50 µM bertosamil (B). Cell capacitance, 26 and 22 pF. Effect of increasing the rate of membrane depolarization from 0.1 to 1 Hz on hkV1.5 current recorded in control condition (C) and after the steady-state internal application of 50 µM bertosamil in CHO (D). Cell capacitance, 27 and 25 pF, respectively. E, time course of inhibition of the peak current by high rate of depolarization and of its recovery at the resumption of the low rate of stimulation (0.1 Hz).

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Effects of bertosamil on the kinetics of current reactivation. A, time course of current recovery from inactivation, by using two 750-ms pulses from 60 to + 50 mV with interpulse intervals from 4 to 5000 ms, recorded in atrial myocytes in the absence () and presence of bertosamil () and in CHO cells treated with bertosamil ().

View larger version (14K): [in this window] [in a new window]   Fig. 11.   Effect of increasing the rate of membrane depolarization from 0.1 to 1 Hz after 10 pulses in atrial myocytes in the absence (A) and presence (B) of 50 µM internal bertosamil. Atrial myocyte capacitance, 112 and 138 pF, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The main finding of this study is that berstosamil, a structural analog of the antiarrhythmic agent tedisamil, has two effects on the current carried by hKv1.5 channels: 1) a use-dependent inhibition of the current observed during external drug application, and 2) an acceleration of time-dependent current inactivation when the drug is applied externally at rest, or internally, and that persists after drug washout. In human atrial myocytes, these effects of bertosamil on potassium channels may account for the changes in the time- and use-dependent properties of the outward potassium current.

Mechanisms of Effects of Bertosamil on hKv1.5 Channels. The effects of bertosamil on the current carried by hKv1.5 channels may partly be explained by blockade of the channel pore in its open state. Such a mechanism could account for the time-dependent decrease in the current that occurred when bertosamil was applied during a train of depolarization, together with the increased magnitude of the drug effect with depolarization and the slowing of the deactivation tail current. However, various lines of evidence indicate that, in addition to this classic open-channel blockade, the acceleration of the time-dependent decrease in the current could also be due to drug modulation of the rate of channel inactivation. First, the acceleration of hKv1.5 current decay has also been observed in the absence of bertosamil in the extracellular medium (applied at rest, Fig. 1C) and persisted for a long time after the drug was washed out. Second, the effects of bertosamil on hKv1.5 channels were modulated by interventions known to alter channel inactivation processes, such as the nature of the charge carrier, the extracellular concentration of K⁺, TEA, and the rate and duration of membrane depolarization. Third, in the presence of intracellular bertosamil, or after external application of the drug during a rest period, only the use-dependent inhibitory effect was observed when the cell was again exposed to bertosamil, suggesting the existence of distinct sites of action of this agent. Finally, the effect of prolonged or rapid depolarization on the current does not support the possibility that the acceleration of Kv1.5 current decay by bertosamil is due mainly to drug release from the channel at a rate sufficiently rapid to allow the current to recover by the next pulse and to be blocked again. Both protocols caused a persistent inhibition of the transient but not the steady-state component of the bertosamil-treated current, despite the use of a pulse interval long enough to allow drug release from the channel.

Physiological Consequences. Our findings support previous reports that bertosamil and its analog tedisamil modulate the outward potassium current in rat ventricular (Dukes and Morad, 1989) and human atrial myocytes (Tessier et al., 1997) by accelerating its time-dependent inactivation. In this study we found that, in bertosamil-treated myocytes, I_o recovers slowly from inactivation and undergoes marked cumulative inactivation when the rate of stimulation is increased, in a manner reminiscent of the behavior of the hKv1.5 current after drug application. The outward current in human atrial myocytes is the sum of distinct current components that may also be altered by bertosamil, thus preventing a direct extrapolation of the data obtained with cloned channels to the endogenous current. However, in a previous study we found that, in human atrial myocytes, bertosamil had little effect on the outward current that remained after application of 200 µM 4-aminopyridine, suggesting a prominent effect of the drug on hKv1.5 channels (Tessier et al., 1997).