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(Circulation Research. 1997;81:202-210.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of L-Type Ca²⁺ Channel Current in Rat Ventricular Myocytes by Terfenadine

Shi Liu, Russell B. Melchert, , Richard H. Kennedy

From the Department of Medicine, Division of Cardiology (S.L.), the Department of Biopharmaceutical Sciences (R.B.M.), and the Department of Pharmacology and Toxicology (S.L., R.H.K.), University of Arkansas for Medical Sciences, Little Rock.

	Abstract

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Abstract To elucidate possible mechanisms underlying the cardiotoxicity of terfenadine, the effect of this antihistamine on L-type Ca²⁺ channel current (I_Ca,L) was studied in adult rat ventricular myocytes using the whole-cell patch-clamp technique. Myocytes were held at -70 mV and internally dialyzed and externally perfused with Na⁺- and K⁺-free solutions; exposure to terfenadine (10^-9 to 5x10^-6 mol/L) resulted in a concentration-dependent inhibition of peak I_Ca,L, with a half-maximum inhibition concentration (IC₅₀) of 142 nmol/L. The terfenadine-induced inhibition of I_Ca,L was not mediated via effects on histamine H₁ receptors, because 1 µmol/L triprolidine, a more selective and potent H₁ antagonist, had no effect on I_Ca,L. In this study, we found that terfenadine (1) increased both the fast and slow time constants of I_Ca,L inactivation, (2) shifted the steady state inactivation of I_Ca,L to more negative potentials, and (3) elicited a tonic block and a use-dependent block of I_Ca,L. The terfenadine-induced tonic and use-dependent block and the steady state inhibition of I_Ca,L were voltage dependent. Both tonic and use-dependent blocks of I_Ca,L by terfenadine at -40 mV were greater than that at -70 mV, and blocks were partially released by applying a long hyperpolarizing prepulse to -90 mV. These results suggest that terfenadine binds to L-type Ca²⁺ channels in inactivated and rested states and inhibits I_Ca,L predominantly by interacting with the inactivated state with an apparent dissociation constant of 60 nmol/L. Open-state block could be observed only at high concentrations of terfenadine. The high-affinity interaction of terfenadine with the inactivated state of L-type Ca²⁺ channels may play an important role in its cardiotoxicity under pathophysiological conditions, such as ischemia.

Key Words: terfenadine • Ca²⁺ channel • cardiac myocyte • rat • use-dependent block

	Introduction

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Terfenadine, a commonly prescribed nonsedating histamine-H₁ receptor antagonist, has been associated with cardiotoxicity, including fatal ventricular arrhythmias and sudden death.¹ Studies involving the mechanisms underlying these cardiotoxicities have shown that terfenadine blocks delayed rectifier K⁺ channels in feline and guinea pig ventricular myocytes^{2 3} as well as K⁺ channels cloned from human heart.⁴ Until recently, little was known about the effects of terfenadine on other ion channels that may also contribute to its cardiotoxic effects. A recent study in guinea pig ventricular myocytes showed that terfenadine, at concentrations >3 µmol/L, also blocks Ca²⁺ and Na⁺ channels.³ However, the mechanism(s) underlying the inhibition of these channels has not been established.

Receptor binding studies on rat cerebral cortex have shown that terfenadine antagonizes [³H]nitrendipine binding to Ca²⁺ channels in a manner comparable to diphenylalkylamines.⁵ Since terfenadine is structurally similar to this class of Ca²⁺ channel blockers, it is reasonable to hypothesize that terfenadine blocks L-type Ca²⁺ channels by a mechanism similar to that of phenyl-alkylamines, such as verapamil and D-600. In the present study, we characterized the inhibitory effect of terfenadine on I_Ca,L in cultured adult rat ventricular myocytes.

	Materials and Methods

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Myocyte Preparation
Single ventricular myocytes were isolated from the hearts of adult male Sprague-Dawley rats (250 to 300 g) as previously described.⁶ Briefly, hearts were rapidly excised and retrogradely perfused on a Langendorff perfusion apparatus, at 37°C, via the aorta with an oxygenated buffer solution followed by a Ca²⁺-free buffer solution with the following composition (mmol/L): NaCl 110, KCl 3.8, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25, (CaCl₂ 0.2), and glucose 11 (pH 7.4 in 95% O₂/5% CO₂). Hearts were then perfused with a collagenase-containing solution (0.5 mg/mL) and rinsed with Ca²⁺-free buffer. Isolated ventricular myocytes were incubated in culture dishes containing antibiotic-free bicarbonate-buffered culture medium 199 (60%, GIBCO) with 36% Earle's balanced salt solution composed of (mmol/L) NaCl 116, KCl 4.7, NaH₂PO₄ 0.9, MgSO₄ 0.8, NaHCO₃ 26, and glucose 5.6, along with 4% fetal bovine serum (GIBCO) (pH 7.4 in 5% CO₂/95% air at 37°C). Rod-shaped myocytes were used after 24 to 48 hours in culture.

Electrophysiological Measurements
Ventricular myocytes were placed on the heated stage of an inverted microscope (Nikon Diaphot) and perfused with a control Tyrode's solution. Cells were patch-clamped in the whole-cell configuration by conventional techniques⁷ using a patch-clamp amplifier (Axopatch 200A, Axon Instruments) as previously described.⁶ Briefly, patch electrodes were fabricated from borosilicate glass (7052, Garner Glass Co) and filled with a pipette solution consisting of (mmol/L) CsOH 100, aspartate 70, CsCl 11, TEA 15, MgCl₂ 2, Mg-ATP 5, EGTA 10, CaCl₂ 0.1, pyruvic acid 5, glucose 5.6, Tris₂-phosphocreatine 5, Li₄-GTP 0.4, HEPES 5, and Tris base 5 (pH adjusted to 7.2 with CsOH). Filled pipette electrodes had a tip resistance of 2 to 5 M. After the whole-cell configuration was achieved, cells were voltage-clamped at -70 mV. A period of 10 to 15 minutes was allowed before the experiment; thereafter, rundown of I_Ca,L was small. Series resistance was <10 M and electronically compensated (90%) to reduce associated artifacts. The recorded currents were filtered at 1 to 2 kHz through a four-pole low-pass Bessel filter and sampled at 5 kHz with a PC/AT computer using PClamp 6.0 software (Axon Instruments) through an Axon TL-1 Labmaster DMA acquisition system. To normalize measured membrane currents to C_m, the capacity current transient recorded in response to a 5-mV hyperpolarizing pulse was integrated and divided by the given voltage to give total C_m for each cell.

To measure whole-cell I_Ca,L, myocytes were perfused with an external solution consisting of (mmol/L) NMDG chloride 145, MgCl₂ 0.8, CaCl₂ 2, 4-AP 2, HEPES 5, and Tris base 5 (pH adjusted to 7.4 with CsOH). Under these conditions, K⁺ currents were suppressed by internal Cs⁺ and TEA, as well as by external K⁺-free solutions containing 4-AP. The Na⁺ current was suppressed by the use of Na⁺-free NMDG solutions. The Na⁺-K⁺ pump current was inactivated in K⁺-free bath solutions and Na⁺-free pipette solutions. Membrane current associated with Na⁺-Ca²⁺ exchange was eliminated by the Na⁺-free and low-Ca²⁺ (10 mmol/L EGTA) pipette solutions. Cd²⁺ (0.2 mmol/L) was used to verify the efficiency of the isolation of I_Ca,L.

I_Ca,L was elicited by a single 250-ms voltage pulse to +10 mV from the holding potential of -70 mV once every 15 s. The amplitude of I_Ca,L was measured as the peak inward current with reference to the current at the end of the test pulse. The I-V relationship of I_Ca,L was obtained by plotting the peak current amplitude in response to voltage pulses to potentials between -60 and +70 mV from the holding potential in 10-mV increments at 0.2 Hz. f and d relationships were determined using a gapped double-pulse protocol; a 1-s prepulse to potentials between -90 and +60 mV was followed by a 10-ms return to the holding potential and then a fixed 250-ms test pulse to +10 mV. Data obtained for f and d were fit by the Boltzmann distribution by using a Marquardt-Levenberg nonlinear least-squares curve-fitting algorithm. Some recorded currents were corrected by subtracting residual currents in the presence of Cd²⁺ (eg, those in Fig 1). All experiments were conducted at 37°C.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of terfenadine on peak ICa,L in cultured adult rat ventricular myocytes. Myocytes were voltage-clamped at -70 mV, and ICa,L was repetitively elicited by a single voltage pulse to +10 mV once every 15 s in a Na+- and K+-free solution containing 2 mmol/L 4-AP ( and trace 1). A, Exposure to 0.3 µmol/L terfenadine (TFD) caused a 40% decrease in peak ICa,L ( and trace 2). Subsequent exposure to 1.0 µmol/L TFD resulted in further suppression of peak ICa,L by 93% ( and trace 3). Addition of 0.2 mmol/L Cd2+ completely blocked peak ICa,L (). Upon washout of TFD and Cd2+, ICa,L partially recovered (trace 4). Inset, Current traces in the absence and presence of TFD were corrected by subtracting the residual current in the presence of Cd2+ from those where indicated and superimposed. Calibration bars are as follows: horizontal, 20 ms; vertical, 0.5 nA. Cm was 126 pF. B, Effect of terfenadine on peak ICa,L cannot be mimicked by triprolidine. Exposure of a myocyte to 1 µmol/L triprolidine, a potent and selective H1 antagonist, had little effect on peak ICa,L (). After removal of triprolidine, subsequent exposure to 1 µmol/L TFD resulted in 75% inhibition of ICa,L (). When the cell was exposed to 5 µmol/L TFD, ICa,L was almost completely blocked (). Upon removal of TFD, ICa,L partially recovered. Complete inhibition of ICa,L was observed in the presence of 0.2 mmol/L Cd2+ (). Cm was 122 pF.

Chemicals
Most reagents were purchased from Sigma Chemical Co. ATP (Sigma), GTP (Sigma), TEA (Sigma), and 4-AP (Sigma) were directly added when needed. A stock solution of terfenadine was prepared in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in extracellular solutions was <0.01% and had no effect on I_Ca,L (data not shown). After the addition of these chemicals, the pH of the solutions was readjusted as necessary.

Statistics
Values are presented as mean±SEM. Data obtained from the same myocyte were used to express the results in terms of percentage. Statistical significance was evaluated by the two-tailed paired Student's t test. When more than one test concentration was compared, data were evaluated by one-way ANOVA. Differences at a value of P<.05 were considered significant.

	Results

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Concentration-Dependent Steady State Block of Peak I_Ca,L Induced by Terfenadine
The acute effect of terfenadine on I_Ca,L was examined by monitoring peak I_Ca,L elicited by repeatedly applying a single pulse to +10 mV from a holding potential of -70 mV before, during, and after exposure to terfenadine. Fig 1 shows results from experiments demonstrating that terfenadine elicits a concentration-dependent inhibition of peak I_Ca,L. Exposure of a ventricular myocyte to 0.3 µmol/L terfenadine resulted in an 40% decrease in peak I_Ca,L in 15 minutes (open circles in Fig 1A). Upon increase of the concentration of terfenadine to 1 µmol/L, peak I_Ca,L was further reduced to 7% of the control level within 10 minutes (open triangles). Subsequent addition of 0.2 mmol/L Cd²⁺ completely blocked peak I_Ca,L in 15 s (open diamonds), confirming that the current was I_Ca,L. Upon removal of terfenadine, I_Ca,L partially recovered to a pseudo–steady state level in 10 minutes. The inset of Fig 1A, which shows superimposed current traces in the absence and presence of different concentrations of terfenadine, suggests that terfenadine not only reduces peak I_Ca,L but also alters its kinetics.

To determine whether the inhibitory effect of terfenadine on peak I_Ca,L is mediated via histamine H₁ receptors, similar experiments were performed using triprolidine, one of the most selective and potent H₁ antagonists. Fig 1B demonstrates that exposure for 10 minutes to 1 µmol/L triprolidine had little effect on I_Ca,L (solid squares). Similar observations were found in three other experiments, and the averaged I_Ca,L in the presence of 1 µmol/L triprolidine was 94.4±4.2% of control levels (n=4). After removal of triprolidine for 5 minutes, exposure of this cell to 1 µmol/L terfenadine caused a dramatic decrease in peak I_Ca,L in 10 minutes (open triangles), similar to that shown in Fig 1A. Subsequent exposure to 5 µmol/L terfenadine almost completely blocked peak I_Ca,L (open inverted triangles). After washout of terfenadine, application of 0.2 mmol/L Cd²⁺ caused a complete inhibition of I_Ca,L (open diamonds). These results suggest that terfenadine inhibits cardiac I_Ca,L directly via direct action on the channel rather than indirectly via the histamine H₁ receptor. Therefore, all subsequent experiments were performed in the absence of H₁ antagonists.

Fig 2A shows the I-V relationships of I_Ca,L in the absence and presence of terfenadine. These results show that terfenadine induced a concentration-dependent suppression of peak I_Ca,L without altering the reversal potential. However, it is noticeable that the voltage dependence of peak I_Ca,L was slightly shifted toward more negative potentials in the presence of terfenadine. The concentration-response relationship for the terfenadine-induced inhibition of I_Ca,L at +10 mV from the holding potential of -70 mV is summarized in Fig 2B. The normalized peak amplitudes of I_Ca,L in the presence of different concentrations of terfenadine with respect to the control level were fit by the Hill equation to yield a half-maximum inhibition concentration (IC₅₀) of 142±27 nmol/L and a Hill coefficient of 0.8.

View larger version (25K): [in this window] [in a new window]   Figure 2. A, Effect of terfenadine (TFD) on the I-V relationship of ICa,L. TFD suppressed the peak I-V curve of ICa,L (0.3 µmol/L TFD, , n=5; 1.0 µmol/L TFD, , n=7) compared with control (, n=12). B, Concentration-dependent inhibition of TFD on peak ICa,L. The dose-response curve for TFD-induced inhibition of ICa,L was constructed from mean relative peak ICa,L measured in response to a voltage pulse to +10 mV in each concentration of TFD. Numbers in parentheses represent the number of cells. Data are mean±SEM and fit by a Hill equation: (1-Imin)/{1+[IC50/(TFD)]n}+Imin (solid line), where Imin represents the relative current in the maximally effective concentration. The concentration that produces a half-maximum inhibition (IC50) was 1.42±0.27x10-7 mol/L, and the Hill coefficient (n) was 0.8. C, Voltage dependence of the TFD-induced steady state inhibition of ICa,L. The TFD-induced steady state inhibition of ICa,L at the holding potential (Eh) of 40 mV (solid bars) was greater than that at -70 mV (open bars) at three different concentrations. Inset, Protocol and superimposed current traces in the absence and presence of TFD at -70 and -40 mV, respectively. Bars=10 ms (horizontal) and 1 nA (vertical).

Many Ca²⁺ channel antagonists, including verapamil⁸ and dihydropyridines,^{8 9} elicit a voltage-dependent block of Ca²⁺ channels. We then determined whether the inhibition of peak I_Ca,L induced by terfenadine was voltage dependent. Cells were given a double 50-ms test pulse to +10 mV from two different holding potentials (-70 and -40 mV) once every 15 s. The two test pulses were separated by a 390-ms rest at -70 mV and a 10-ms depolarization to -40 mV (see the inset of Fig 2C). Fig 2C shows the relative amplitudes of peak I_Ca,L elicited from 70 mV (open bars) and -40 mV (solid bars) in the presence of three different concentrations of terfenadine after a 12- to 15-minute exposure to each concentration. The inset in Fig 2C illustrates superimposed current traces recorded in the absence and presence of 1.0 and 5.0 µmol/L terfenadine at -70 and -40 mV, respectively. The steady state inhibition of I_Ca,L was significantly greater at more depolarized potentials (eg, at -40 mV) than at the rested state (eg, at -70 mV) at all concentrations of terfenadine. The ratio of the steady state block at -70 mV to that at -40 mV also displayed a concentration dependence (0.79±0.03, n=4; 0.91±0.01, n=12; and 0.97±0.01, n=6; for 0.3, 1, and 5 µmol/L terfenadine, respectively). This terfenadine-induced voltage-dependent block of I_Ca,L is consistent with closed-state (ie, rested) and inactivated-state block. The results showing that low concentrations of terfenadine displayed greater voltage-dependent block also suggest that terfenadine interacts with the inactivated state of Ca²⁺ channels with a higher affinity.

Effect of Terfenadine on the Inactivation and Activation Kinetics of I_Ca,L
As shown in the inset of Fig 1, inactivation of I_Ca,L in the presence of terfenadine displayed a crossover phenomenon when superimposed with that in its absence, indicating an alteration of the inactivation kinetics. The time course of inactivation of I_Ca,L can be best described by a double-exponential function, except for the small currents observed in the presence of high concentrations of terfenadine, which display a single-exponential process of inactivation. Fig 3 shows _f and _s in the absence and presence of terfenadine. Exposure of myocytes to 0.3 µmol/L terfenadine resulted in a significant increase in _f at potentials between -10 and +50 mV (Fig 3A) and in _s at potentials between +10 and +50 mV (Fig 3B). The large variation in the effects of terfenadine on _f at -10 mV was due to less accurate curve fitting because of the smaller amplitude of I_Ca,L. However, in spite of this variation, statistical analysis did show a significant increase in _f at -10 mV. A higher concentration of terfenadine (1 µmol/L) did not further increase _f or _s measured at +10 mV (10.6±1.3 ms [n=7] [solid triangle in Fig 3A] and 41.1±7.5 ms [n=5], respectively) compared with _f and _s measured at 0.3 µmol/L (9.1±1.3 ms [n=6] and 41.8±6.8 ms [n=6], respectively). The increased time constants of I_Ca,L inactivation by terfenadine resulted in a crossover of the decay phase of I_Ca,L when compared before and during exposure to terfenadine.

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of terfenadine on time constants of ICa,L inactivation. Terfenadine (0.3 µmol/L) significantly increased both f and s ( [A] and [B], respectively, n=4 to 6; control, and , n=7 to 12). Terfenadine at 1.0 µmol/L did not further increase f ( [A], n=7).

At concentrations of <1 µmol/L, terfenadine did not significantly alter the rate of I_Ca,L activation. However, 1 µmol/L terfenadine slightly increased the 10% to 90% rise time of peak I_Ca,L from 0.86±0.07 ms in control to 1.14±0.06 ms (n=7, P<.01). Note that 1 to 3 µmol/L nifedipine reduced I_Ca,L to a level similar to that induced by 1 µmol/L terfenadine without altering the kinetics of activation.

Effects of Terfenadine on f and d
The voltage-dependent steady state block of I_Ca,L may result from a decrease in the steady state availability of Ca²⁺ channels. Therefore, we examined the effect of terfenadine on f in Ca²⁺ channels using a gapped double-pulse protocol before and after a 12- to 15-minute exposure to terfenadine. Myocytes were given a 1-s prepulse to potentials between -90 and +40 mV, followed by a 10-ms return to the holding potential of -70 mV and then a fixed 250-ms test pulse to +10 mV. The pulse protocol was initiated once every 12 or 15 s to minimize the use-dependent block by terfenadine. Fig 4A shows that terfenadine elicited a concentration-dependent hyperpolarizing shift of f. Interestingly, the effect of terfenadine on the apparent f was biphasic and could be best described by the sum of two Boltzmann distributions. In the absence of terfenadine, f was described by a V_h of -21.5±0.2 mV and a slope factor (k) of 4.8±0.2 mV/e-fold change (n=8). Exposure to 0.3 µmol/L terfenadine resulted in biphasic shift in the apparent f with V_h values of -80.0±0.8 and -29.2±0.2 mV (n=6, P<.001) and corresponding k values of 5.1±0.8 and 5.2±0.2 mV (P>.05). Similarly, in the presence of 1 µmol/L terfenadine, the two V_h values were -80.4±1.3 and -32.9±0.4 mV (n=4, P<.001) with corresponding k values of 5.1±1.2 and 5.4±0.4 mV (P>.05). The ratio of fractional unblocked channels at -40 mV to that at -70 mV (ie, 0.83 and 0.64 at 0.3 and 1 µmol/L terfenadine, respectively) is compatible with the unblocked I_Ca,L described in Fig 2C. These results show that the terfenadine-induced inhibition of Ca²⁺ channels is consistent with the inactivated block. In addition, the change in apparent f at subthreshold potentials (or more negative potentials) suggests that terfenadine also binds to the closed state of Ca²⁺ channels (see "Discussion").

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of terfenadine (TFD) on f and d. A, f was measured using a double-pulse protocol, which contains a 1-s prepulse between -90 and +40 mV, followed by a 10-ms return to -70 mV, and then a 250-ms test pulse to +10 mV at a 12- or 15-s interval. In control conditions, f was well described by a Boltzmann equation: I/Imax=1/[1+exp(V-Vh)/k], where I/Imax represents a ratio of currents to the maximum current (measured at -90 mV); Vh (a half-maximum inactivation potential) was -21.5±0.2 mV, and k was 4.8±0.2 mV (dashed line, n=8). In the presence of TFD, the apparent f was fit by two Boltzmann distributions; Vh values were -80±0.8 and -29.2±0.2 mV, respectively, at 0.3 µmol/L ( and solid line, n=6) and -80.4±1.3 and -32.9±0.4 mV at 1.0 µmol/L ( and solid line, n=4). Values for k were 5.1±0.8 and 5.2±0.2 mV at 0.3 µmol/L and 5.1±1.2 and 5.4±0.4 mV at 1.0 µmol/L. B, d was determined by a ratio of conductances [G=I/(V-Vrev), where Vrev was the apparent reversal potential from each I-V curve] to maximum conductance (Gmax). Data were also fit by a Boltzmann equation: G/Gmax=1/{1+exp[(Vh-V)/k]}, which yields Vh (a half-maximum activation potential) of -4.0±0.3 mV and k of 5.9±0.3 mV in control ( and dashed line, n=5). TFD (0.3 µmol/L) shifted Vh to -5.9±0.4 mV without altering k (5.8±0.0.3 mV) ( and solid line, n=5).

The effect of terfenadine on d in I_Ca,L was examined by plotting G/G_max as a function of voltage pulses between -60 and +40 mV, as described in Fig 2A. Fig 4B shows that 0.3 µmol/L terfenadine caused a slight, but significant, shift of V_h from -4.0±0.3 mV (control, n=5) to -5.9±0.4 mV (n=5) without altering the slope factor (5.9±0.3 mV, control; 5.8±0.3 mV, terfenadine). These results may account for the small hyperpolarizing shift of the I-V curve in the presence of terfenadine shown in Fig 2A. It is also noticeable that nifedipine has no effect on the apparent d. However, the maximum conductance of I_Ca,L in the presence of terfenadine was significantly reduced to 0.19±0.02 nS/pF from the control level of 0.35±0.01 nS/pF (n=5).

Tonic and Use-Dependent Block
Verapamil and D-600 display varied degrees of use-dependent block with little tonic block of I_Ca,L.^{8 10 11} To determine whether terfenadine shows tonic and/or use-dependent block of I_Ca,L, we used a standard protocol illustrated in the inset of Fig 5C. Myocytes were given a train of 30 depolarizing pulses (200-ms duration) to +10 mV from a holding potential of -70 mV at 0.5 Hz in the control solution. After cessation of the test pulses, cells were exposed to different concentrations of terfenadine for 12 to 15 minutes, followed by stimulation with the same train of depolarizing pulses to +10 mV. The degree of difference in peak I_Ca,L elicited by the first pulse (I_Ca-P1) before and during exposure to terfenadine was defined as tonic block: 1-(I_{Ca-P1,with drug}/I_{Ca-P1,no drug}). Use-dependent block was defined as the degree of decrement in the amplitude of peak I_Ca,L elicited by the last pulse relative to that elicited by the first pulse: 1-(I_Ca-P30/I_Ca-P1). Since use-dependent suppression of I_Ca,L exists in the absence of drug, we defined the apparent terfenadine-associated use-dependent block as 1-[(I_Ca-P30/I_Ca-P1)_{with drug}/(I_Ca-P30/I_Ca-P1)_{no drug}].

View larger version (33K): [in this window] [in a new window]   Figure 5. Tonic and use-dependent block of ICa,L by terfenadine (TFD). A, Superimposed current traces of ICa,L in response to the pulse protocol shown in the inset of panel C in control conditions (holding potential was -70 mV). Myocytes were allowed a 2-minute rest before the train of thirty 200-ms depolarizing pulses to +10 mV was applied. The first postrest peak ICa,L (P1) was always smaller than the second peak with a more rapid inactivation. Little use-dependent block of ICa,L was observed during consecutive pulses (P2-30) in control conditions. B, In the presence of 0.3 µmol/L TFD for 15 minutes, there was small inhibition of the first ICa,L (P1, tonic block). During consecutive pulses, ICa,L was gradually suppressed (use-dependent block). Bars=10 ms (horizontal) and 1 nA (vertical). Cm was 170 pF. C, Kinetics of the onset of TFD-induced use-dependent block of ICa,L. IPX/IP1 indicates normalized current. The TFD-induced use-dependent block of ICa,L was best fit by a biexponential function in all three concentrations (0.1 µmol/L, , n=4; 0.3 µmol/L, , n=7; 1 µmol/L, , n=7; and solid lines, best-fit curves).

Fig 5 shows a representative example of the tonic and use-dependent block of I_Ca,L induced by three concentrations of terfenadine. Fig 5A illustrates that in control conditions rat ventricular myocytes display a small use-dependent suppression of peak I_Ca,L (open squares in Fig 5C). Note that after a 2-minute rest, the first postrest I_Ca,L (I_Ca-P1) was smaller than the subsequent I_Ca,L. This postrest depression for a series of voltage pulses from a holding potential of -70 mV has been well described by Hryshko and Bers.¹² Under control solutions, the normalized amplitude of I_Ca,L on the 30th pulse (or the steady state level) relative to I_Ca,L on the first pulse (I_Ca-P30/I_Ca-P1) was 0.95±0.01 (n=21) at 0.5 Hz. Fig 5B shows that exposure to 0.3 µmol/L terfenadine resulted in a small tonic block, followed by a gradual decline in peak I_Ca,L during successive depolarizing pulses. The decreased I_Ca,L approached a steady state at the 30th pulse, indicative of use-dependent block. Interest-ingly, under these conditions, the ratio of I_{Ca-P1,with drug} to I_{Ca-P1,no drug} was similar during exposure to different concentrations of terfenadine, eg, 0.71±0.06 (n=3), 0.82±0.04 (n=7), and 0.85±0.05 (n=6) at 0.1, 0.3, and 1 µmol/L terfenadine, respectively (P>.1). In other experiments, in control conditions, when the same protocol was repeated and separated by a 2-minute rest, the relative amplitude of I_Ca,L elicited by the first pulse of the second train (I_Ca-P1,train2/I_Ca-P1,train1) was 0.97±0.01 (n=22). However, when the second train was performed 20 minutes after the first train, the ratio of I_Ca-P1,train2 to I_Ca-P1,train1 was 0.87±0.02 (n=5). Thus, the small tonic block observed in the presence of terfenadine at -70 mV might, in part, result from the time-dependent decline in peak I_Ca,L. In contrast, when membrane potential was held at -40 mV, tonic block was significantly increased. The ratio of I_{Ca-P1,with drug} to I_{Ca-P1,no drug} was 0.28±0.05 (n=6) and 0.16 (n=2) at 0.3 and 1.0 µmol/L terfenadine, respectively (ie, 72% and 84% tonic block, respectively). When the holding potential was changed to -90 mV, tonic block in the presence of 1.0 µmol/L terfenadine was <5%. Similarly, in another experiment, when a myocyte was voltage-clamped at -40 mV, 0.3 µmol/L terfenadine induced a tonic block of 68%, which was partially relieved to 51% if the second train of pulses was preceded by a 2-minute rest at -90 mV, followed by a 30-s return to -40 mV. These results demonstrate that changes in membrane potential can modulate terfenadine-induced block of I_Ca,L. Depolarized potentials favor association of terfenadine with its binding sites, whereas hyperpolarized potentials favor dissociation from its receptor. These results also support those illustrated in Fig 2, which show that terfenadine interacts with the inactivated state of Ca²⁺ channels with a higher affinity than it does with the rested state.

Fig 5 also shows that terfenadine induced a use-dependent block of peak I_Ca,L in a concentration-dependent manner. In a series of experiments, the apparent steady state use-dependent block was 38±6% (n=4), 57±2% (n=7), 89±1% (n=7), and 94% (n=1) in the presence of 0.1, 0.3, 1, and 5 µmol/L terfenadine, respectively. The terfenadine-induced use-dependent block of I_Ca,L could be well described by a double-exponential function (solid lines in Fig 5C).

We have previously shown that the steady state and tonic blocks of I_Ca,L by terfenadine are voltage dependent. To support the hypothesis that the terfenadine-induced voltage-dependent block of I_Ca,L results from its interaction with inactivated channels, depolarization such as holding at -40 mV would be expected to enhance the use-dependent block of I_Ca,L. We found that after a 12-minute equilibration in 0.3 µmol/L terfenadine, the degree of the use-dependent block was increased to 69±5% (n=6) compared with a 53±3% inhibition (n=6) at -70 mV. Conversely, when the holding potential was -90 mV, use-dependent blocks of I_Ca,L elicited by 0.01 and 1 µmol/L terfenadine were significantly attenuated to 7% from 17.6% block and to 44% from 89% inhibition at -70 mV, respectively. Thus, the voltage dependence of both tonic and use-dependent blocks is consistent with the preferential binding of terfenadine to the inactivated state of Ca²⁺ channels.

Time Course of Block Development
Verapamil or D-600 inhibits I_Ca,L by interacting with either the open¹⁰ or inactivated^{8 11} state. Terfenadine-induced inhibition of I_Ca,L in response to depolarizing pulses can result from its interaction with either the open state or inactivated state of Ca²⁺ channels. To distinguish between these two possibilities, we used a double-pulse protocol to define the time course of block development before and during exposure to terfenadine (see inset in Fig 6). Myocytes were given a conditioning depolarization pulse to +10 mV from a holding potential of -70 mV, with a variable duration from 10 ms to 1.5 or 3.1 s in 50- or 300-ms increments, respectively. The conditioning pulse was followed by a return to the holding potential for 150-ms and a fixed 200-ms test pulse to +10 mV. A 15-s interval was allowed to minimize use-dependent effects. The 150-ms return to the holding potential was long enough to allow full recovery of drug-free channels from inactivation but short enough to allow only minimal recovery of drug-bound channels from the blockade induced by terfenadine (see the following section). The block development during the conditioning pulse was determined from the decline in I_Ca,L elicited by the test pulse.

View larger version (36K): [in this window] [in a new window]   Figure 6. Time course of terfenadine-induced block development during a depolarizing pulse. Kinetics of block development in the presence of terfenadine was determined using the pulse protocol shown in the inset. The duration of a conditioning voltage pulse from a holding potential of -70 to +10 mV was varied from 10 ms to 1.5 or 3.1 s. After the conditioning pulse, a 150-ms return to the holding potential was allowed for recovery of drug-free channels and was followed by a 200-ms test pulse to +10 mV to evaluate the amount of unblocked channel activity during the conditioning depolarization. The protocol was applied once every 15 s. The current amplitude of each test pulse was normalized to that in response to the first conditioning pulse in the absence or presence of terfenadine (IPX/IP1). In control conditions, an increase in the duration of depolarization during conditioning pulses caused a slow inactivation of ICa,L (, n=22). This slow inactivation was fit by a single exponential function with a time constant of 862±32 ms (solid line). Terfenadine elicited a concentration-dependent block of ICa,L (, 0.1 µmol/L; , 0.3 µmol/L; and , 1.0 µmol/L; n=4 or 5). The time course of block development by 0.1 and 0.3 µmol/L terfenadine was best fit by a single exponential (solid line), whereas that for 1.0 µmol/L terfenadine was best fit by a biexponential function (solid lines).

Fig 6 shows the time course of block development induced by three concentrations of terfenadine; relative peak I_Ca,L elicited by the test pulse (I_Ca-P2X/I_Ca-P1) was plotted as a function of the duration of the conditioning pulse. In the absence of terfenadine, an increase in duration of the conditioning pulse to 3.1 s resulted in a 38% decrease of peak I_Ca,L in response to the test pulse (open squares, Fig 6), suggesting a slow inactivation. The time course of this slow inactivation was described by a single-exponential process with a time constant of 862±32 ms (n=22). Exposure to terfenadine caused a more progressive decline in the amplitude of I_Ca,L during the test pulse by enhancing both the rate and the degree of block development in a concentration-dependent manner. Fig 6 shows that when the duration of the conditioning pulse increased up to 1 s, 1 µmol/L terfenadine almost completely blocked I_Ca,L. Because of the slow inactivation of I_Ca,L occurring in the absence of drug, currents measured in the presence of terfenadine were normalized by the control in each cell to eliminate the contribution of the slow inactivation to the terfenadine action. After the correction, terfenadine (1 µmol/L) resulted in a 95.1% inhibition of I_Ca,L with a _f of 42.1±5.4 ms and a _s of 224±30 ms (n=5), whereas 0.1 and 0.3 µmol/L terfenadine caused 62% (n=4) and 82.3% (n=5) inhibition, respectively. These results suggest that the development of channel block during prolonged depolarizing pulses by terfenadine results from its preferential binding to inactivated states of Ca²⁺ channels. The Table shows the calculation of the apparent K_di for the interaction of terfenadine with inactivated channels from the steady state level of block and the rate of block development (1/_s).

View this table: [in this window] [in a new window]   Table 1. Calculated Dissociation Constant for Interaction With Inactivated Channels

In contrast to the absence or low concentrations of drug, the rate of block development at high concentrations of terfenadine was best fit by a biexponential function. Terfenadine (1 µmol/L) caused a 10% decrease in I_Ca,L on the first test pulse following the first 10-ms conditioning pulse (solid circles, Fig 6). Note that when held at -70 mV in the absence of terfenadine, the time constant of I_Ca,L recovering from its rapid inactivation is 22.4 ms (indicating that I_Ca,L would be expected to completely recover after the 150-ms rest at -70 mV). Therefore, at high concentrations of terfenadine, the initial 10% decrease in I_Ca,L following a 10-ms prepulse suggests that terfenadine also interacts with the open state of the channels under this condition.

Kinetics of Recovery From Terfenadine-Induced Block of I_Ca,L
Results in Fig 6 show, in the presence of 1 µmol/L terfenadine after a 1.5-s depolarizing pulse, almost no drug-free channel recovered from inactivation after the 150-ms rest at -70 mV. Thus, we increased the duration of the interval to examine the kinetics of recovery from the terfenadine-induced steady state inhibition of I_Ca,L using the double-pulse protocol shown in the inset of Fig 7. A myocyte was first given a 2-s depolarizing pulse to +10 mV from a holding potential of -70 mV to develop either a steady state inactivation in the absence of drug or a steady state block of Ca²⁺ channels in the presence of 0.3 and 1 µmol/L terfenadine. This fixed 2-s prepulse was followed by a return to the holding potential for varied durations from 10 ms to 6 s and then by a 200-ms test pulse to +10 mV. Fig 7 shows the time course of fractional recovery of I_Ca,L in response to the test pulse after various durations of recovery time at -70 mV. In control solutions, recovery of I_Ca,L from the long slow inactivation can be described by a two-exponential process composed of a large fast component (A_f of 0.57±0.02 and _f of 48±4 ms) and a small slow component (A_s of 0.28±0.01 and _s of 1.87±0.12 s). A_f and A_s reflect recovery from the rapid inactivation and slow inactivation of Ca²⁺ channels, respectively. After a 15-minute equilibration with 0.3 µmol/L terfenadine, the recovery of I_Ca,L at -70 mV was significantly slowed but could still be described by a two-exponential function. Terfenadine shifted the recovery process to a small fast component with A_f of 0.08±0.01 and _f of 81±1 ms and a large slow component with A_s of 0.81±0.01 and _s of 3.84±0.1 s. These results suggest that in the presence of terfenadine, the fast component may result from recovery of drug-free Ca²⁺ channels from channel inactivation, whereas the slow component is mainly due to dissociation of terfenadine from drug-bound channels.

View larger version (21K): [in this window] [in a new window]   Figure 7. Kinetics of recovery of ICa,L from inactivation and from terfenadine (TFD)-induced inhibition. Kinetics of recovery of ICa,L was determined using the pulse protocol shown in the inset. In the control condition, the 2-s conditioning depolarizing pulse resulted in a slow inactivation following the initial rapid inactivation of ICa,L. An increase in the interval between the conditioning and the test pulse allowed recovery of ICa,L () that was best fit by a biexponential with a f of 48 ms and Af of 0.57 and a s of 1.9 s and As of 0.28. In the presence of 0.3 µmol/L TFD, during the 2-s conditioning pulse TFD elicited a steady state block of ICa,L (see Fig 6). An increase in the interval between the conditioning and the test pulse allowed recovery of ICa,L from TFD-induced block ( and ) that was also best fit by a biexponential with a f of 81 ms and Af of 0.08 and a s of 3.8 s and As of 0.81.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Terfenadine, a nonsedating H₁ receptor antagonist, has a structure similar to that of the diphenylalkylamines. Present data show that terfenadine has inhibitory effects on cardiac L-type Ca²⁺ channels that are similar to those of the phenylalkylamine class of Ca²⁺ channel blockers. We show that terfenadine elicits (1) a steady state inhibition and tonic and use-dependent block of I_Ca,L, all of which are modulated by membrane potential, (2) a decrease in the rate of I_Ca,L inactivation, and (3) a shift of steady state inactivation to negative potentials.

State-Dependent Block
The mechanism underlying the terfenadine-induced inhibition of I_Ca,L can be interpreted using the modulated-receptor model described for the inhibition of Na⁺ channels by amine local anesthetics¹⁴ (see the following model diagram). Terfenadine (pK_a 10) is a highly lipophilic tertiary amine, which exists in the charged form at physiological pH.¹⁵ Therefore, terfenadine can access its binding sites in Ca²⁺ channels via either hydrophobic or hydrophilic pathways. The present study offers evidence for the state-dependent block of I_Ca,L by terfenadine. Terfenadine (0.3 µmol/L) elicits an 18% tonic block of I_Ca,L and reduces channel availability by 9% at -70 mV (Fig 4A). Taking time-dependent inhibition of Ca²⁺ channels into consideration, the data suggest that terfenadine can block Ca²⁺ channels by binding to both the rested and inactivated state. Terfenadine-induced tonic block in the rested state could result from its entry to binding sites through a hydrophilic pathway. Interaction of terfenadine with the rested state of ion channels has also been observed with the cloned human delayed rectifier K⁺ channel.¹⁶

The steady state inhibition (52%) and use-dependent block (53%) elicited by 0.3 µmol/L terfenadine at -70 mV suggest that the degree of interaction of terfenadine with the inactivated state is greater than that with the rested state. We then estimated apparent dissociation constants for terfenadine binding to the rested and inactivated states of Ca²⁺ channels. Assuming a one-to-one relationship between terfenadine binding and response and taking 5% tonic block at -80 mV into account, 89% of the Ca²⁺ channels are available at 0.3 µmol/L terfenadine (see Fig 4A), and the estimated rested-state dissociation constant for terfenadine is 2.4x10^-6 mol/L. The results of terfenadine-induced block development during long depolarizing pulses give an apparent K_di of 60 nmol/L (see Table). The value of K_di can also be calculated from an equilibrium equation: B=B_max/(1+K_d/[D]), where [D] is the concentration of drug, K_d is the dissociation constant, and B {1-[(I_P2X/I_P1)_{with drug}/(I_P2X/I_P1)_{no drug}]} and B_max represent the block of I_Ca,L by a given and a maximally effective concentration of terfenadine, respectively. We used values for steady state levels of the time-dependent block (ie, 62%, 82.3%, and 95.1% inhibition of I_Ca,L elicited by 0.1, 0.3, and 1.0 µmol/L terfenadine, respectively) to perform the double-reciprocal or Lineweaver-Burk plot. When plotted as 1/B versus 1/[terfenadine], the apparent K_di was calculated to be 59 nmol/L with a B_max of 96.4% (linear coefficient, r²=.99), a value comparable to the estimated K_di shown in the Table. These results suggest that terfenadine binds to inactivated Ca²⁺ channels with an 40-fold greater affinity compared with the rested channels. This proposed preferential binding of terfenadine to inactivated Ca²⁺ channels is supported by results showing that the terfenadine-induced use-dependent block of I_Ca,L is demolished by (1) a decrease in stimulation frequency, (2) a reduction in the duration of repetitive depolarizing pulses, and (3) hyperpolarization of the membrane potential.

In addition to decreasing peak I_Ca,L, terfenadine increases _f and _s and thus results in a crossover phenomenon (inset of Fig 1A). The crossover phenomenon has been discussed previously using a model for open-state blockers of Na⁺ channels¹⁷ and a model for open- and/or closed-state block of K⁺ channels.¹⁸ In the first model, after entering the open state, blockers rapidly interact with the channel (OO*) and consequently shorten channel open time. Then blockers dissociate rapidly to allow the channel entry into the inactivated state (O*OI), thereby delaying the inactivation process. However, our data show that terfenadine decreases the rate of activation of I_Ca,L only at 1 µmol/L, suggesting that open-state block of I_Ca,L occurs only at high concentrations. Additionally, the use-dependent block and the absence of the fast component of recovery from blockade by 1 µmol/L terfenadine suggest that rapid dissociation from open channels does not occur.

The second model proposes that the drug binds to closed and open channels with different kinetics and affinities. Voltage-dependent tonic and use-dependent block of I_Ca,L suggest that terfenadine strongly associates with its binding sites in Ca²⁺ channels at depolarized potentials (II*). According to the second model, it is also likely that at subthreshold potentials, terfenadine binds to a closed state right before activation of the channels (or preactivated state, C₄C₄*) with a relatively high affinity compared with its binding to the early closed state (C₁C₁*). After the first pulse, terfenadine is trapped in the closed state because of its slow dissociation rate. In response to the second pulse, the trapped molecules together with newly associated terfenadine substantially reduce peak I_Ca,L and delay channel entry into the inactivation state. This possibility is consistent with the terfenadine-induced biphasic change in apparent steady state inactivation (Fig 4A) and voltage-dependent tonic block (ie, <5% block at -90 mV and 18% at -70 mV in the presence of 0.3 µmol/L terfenadine).

Comparison With the Phenylalkylamine Class of Ca²⁺ Channel Blockers
The mechanism underlying the inhibition of cardiac I_Ca,L by phenylalkylamines has been extensively studied.^{8 10 11 19} Verapamil and D-600 (pK_a 8.6), which exist in the charged form at pH 7.4, produce a substantial use-dependent block of I_Ca,L with little tonic block development.^{8 10 11 19} The use-dependent block induced by verapamil or D-600 has been proposed to be open-state block¹⁰ or inactivated-state block.^{8 11} Similar to these phenylalkylamines, terfenadine (pK_a 10), a charged but highly lipophilic amine at pH 7.4, elicits the state-dependent block of Ca²⁺ channels. However, terfenadine possesses an additional phenyl group, which makes it more hydrophobic. The increased hydrophobicity allows terfenadine to diffuse from the membrane to its receptors and to interact with resting channels. This may account for the small terfenadine-induced tonic block and its voltage dependence. It is likely that terfenadine combines the effects of the charged and lipophilic forms by binding to Ca²⁺ channels in the resting, inactivated, and open states with different kinetics via both the hydrophilic and hydrophobic pathways in the modulated-receptor theory (see the model diagram) as proposed by Uehara and Hume¹¹ for D-600.

Mechanism for Cardiotoxicity of Terfenadine
When used in patients with asthma, terfenadine is capable of inhibiting I_Ca,L of bronchial smooth muscle and thereby causing relaxation of the bronchioles. However, terfenadine therapy has been associated with cardiac arrhythmias, such as torsade de pointes, life-threatening ventricular tachycardia, and sudden death.¹ This cardiotoxic effect of terfenadine has been attributed to its inhibition of I_K.^{2 3 4} Terfenadine-induced block of I_K in cat ventricular myocytes has an IC₅₀ of 0.15 µmol/L,² whereas in cloned human I_K the IC₅₀ is 0.37 µmol/L.^{4 16} Interestingly, the present study shows that terfenadine inhibits I_Ca,L with an IC₅₀ of 0.14 µmol/L, a potency similar to that for inhibition of I_K. Thus, it is likely that both I_K and I_Ca,L are diminished at clinically relevant terfenadine concentrations (0.01 to 0.1 µmol/L).^{1 16} More important, terfenadine binds to the inactivated state of Ca²⁺ channels with a high affinity of 60 nmol/L. This suggests that under pathophysiological conditions, such as ischemia, terfenadine interacts with depolarized or injured tissue more tightly than with normal hearts, consequently inducing a greater block of I_Ca,L. Inhibition of cardiac I_Ca,L can result in decreases in pacemaker activity, atrioventricular nodal conduction velocity, and contractility, which may account for observed syndromes such as sudden death.

	Selected Abbreviations and Acronyms

 

f, s = fast and slow time constants of ICa,L inactivation or ICa,L recovery from inactivation 4-AP = 4-aminopyridine Af, As = amplitude scalars for fast and slow components of recovery from inactivation Cm = cell membrane capacitance d = steady state activation f = steady state inactivation G/Gmax = relative conductance ICa,L = L-type Ca2+ channel current I-V = current-voltage IK = delayed rectifier K+ current k = slope factor Kdi = dissociation constant for interaction with inactivated state of channels NMDG = N-methyl-D-glucamine P (as subscript) = pulse TEA = tetraethylammonium chloride TFD = terfenadine Vh = half-maximum activation or inactivation potential

	Acknowledgments

 
This study was supported in part by the American Heart Association, Arkansas Affiliate, Inc, and the American Health Assistance Foundation. We thank Dr Randall L. Rasmusson for his helpful, insightful discussion and review. We also wish to express our appreciation to Meei-Yueh Liu for her excellent technical assistance.

	Footnotes

 
Reprint requests to Shi Liu, PhD, Department of Medicine, Division of Cardiology, University of Arkansas for Medical Sciences, 4301 W Markham St, Mail Slot 532, Little Rock, AR 72205.

Received November 7, 1996; accepted May 6, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Woosley RL. Cardiac actions of antihistamines. Annu Rev Pharmacol Toxicol. 1996;36:233-252.[Medline] [Order article via Infotrieve]
2. Woosley RL, Chen Y-W, Freiman JP, Gillis RA. Mechanism of the cardiotoxic actions of terfenadine. JAMA. 1993;269:1532-1536.[Abstract]
3. Ming Z, Nordin C. Terfenadine blocks time-dependent Ca²⁺, Na⁺, and K⁺ channels in guinea pig ventricular myocytes. J Cardiol Pharmacol. 1995;26:761-769.[Medline] [Order article via Infotrieve]
4. Rampe D, Wible B, Brown AM, Dage RC. Effects of terfenadine and its metabolites on a delayed rectifier K⁺ channel cloned from human heart. Mol Pharmacol. 1993;44:1240-1245.[Abstract]
5. Zhang MQ, Caldirola P, Timmerman H. Calcium antagonism and structure-affinity relationships of terfenadine, a histamine H1 antagonist, and some related compounds. J Pharm Pharmacol. 1993;45:63-66.[Medline] [Order article via Infotrieve]
6. Liu S, Schreur KD. G protein-mediated suppression of L-type Ca²⁺ current by interleukin-1 in cultured rat ventricular myocytes. Am J Physiol. 1995;268:C339-C349.[Abstract/Free Full Text]
7. Hamill OP, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 1981;391:85-100.[Medline] [Order article via Infotrieve]
8. Sanguinetti MC, Kass RS. Voltage-dependent block of calcium channel current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel antagonists. Circ Res. 1984;55:336-348.[Abstract/Free Full Text]
9. Bean BP. Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state. Proc Natl Acad Sci U S A. 1984;81:6388-6392.[Abstract/Free Full Text]
10. Lee KS, Tsien RW. Mechanism of calcium channel blockade by verapamil, D-600, diltiazem and nitrendipine in single dialysed heart cells. Nature. 1983;302:790-794.[Medline] [Order article via Infotrieve]
11. Uehara A, Hume JR. Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells. J Gen Physiol. 1985;85:621-647.[Abstract/Free Full Text]
12. Hryshko LV, Bers DM. Ca current facilitation during postrest recovery depends on Ca entry. Am J Physiol. 1990;259:H951-H961.[Abstract/Free Full Text]
13. Crumb WJ Jr, Clarkson CW. Characterization of cocaine-induced block of cardiac sodium channels. Biophys J.. 1990;57:589-599.[Abstract/Free Full Text]
14. Hille B. Local anesthetics: hydrophilic and hydrophobic pathways for the drug receptor reactions. J Gen Physiol. 1977;69:497-515.[Abstract/Free Full Text]
15. Badwan AA, Alkaysi HN, Owais LB, Salem MS, Arafat TA. Terfenadine. In: Florey K, ed. Analytical Profiles of Drug Substances. San Diego, Calif: Academic Press Inc; 1990:627-662.
16. Roy M-L, Dumaine R, Brown AM. HERG, a primary human ventricular target of the nonsedating antihistamine terfenadine. Circulation. 1996;94:817-823.[Abstract/Free Full Text]
17. Wang DW, Nie L, George AL Jr, Bennett PB. Distinct local anesthetic affinities in Na⁺ channel subtypes. Biophys J. 1996;70:1700-1708.[Abstract/Free Full Text]
18. Rasmusson RL, Zhang Y, Campbell DL, Comer MB, Castellino RC, Liu S, Strauss HC. Bi-stable block by 4-aminopyridine of a transient K⁺ channel (Kv1 4) cloned from ferret ventricle and expressed in Xenopus oocytes. J Physiol (Lond). 1995;485:59-71.[Medline] [Order article via Infotrieve]
19. Pelzer D, Trautwein W, McDonald TF. Calcium channel block and recovery from block in mammalian ventricular muscle treated with organic channel inhibitors. Pflügers Arch. 1982;394:97-105.

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