Introduction

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Mibefradil is a new Ca²⁺ antagonist that is effective as an antianginal and antihypertensive agent (Noll and Lusher, 1998). Among Ca²⁺ channel blockers, mibefradil is distinctive in its favorable hemodynamic actions and lack of side effects that are frequently observed with other Ca²⁺ antagonists. At therapeutic concentrations, mibefradil reduces vascular resistance and heart rate without negative inotropy.

The favorable pharmacological profile of mibefradil and limited side effects appear to be related to selective block of T-type Ca²⁺ channels. Unlike other Ca²⁺ antagonists that are used clinically, mibefradil preferentially blocks T-type rather than L-type Ca²⁺ channels with 10- to 20-fold selectivity (Mehrke et al., 1994; Mishra and Hermsmeyer, 1994a; Ertel, and Ertel, 1997). Despite its desirable pharmacological and hemodynamic actions, mibefradil was removed from the market after it was shown to produce serious toxicity when taken in combination with a number of other drugs, including some H₁ antihistamine antagonists (Woosley, 1996).

Although a number of studies have been performed that characterize the effects of mibefradil on various Ca²⁺ channel subtypes and document its selective block of T-type channels, little is known about the effect of this drug on other types of ion-selective channels. In this regard, older Ca²⁺ antagonists, such as the dihydropyridines, that preferentially block L-type Ca²⁺ channels have also been shown to inhibit voltage-gated K⁺ channels, albeit at considerably higher concentrations (Hume, 1985; Nerbonne and Gurney, 1987; Mlinar and Enyeart, 1994).

We studied the inhibition of K⁺ channels by mibefradil in whole-cell and single-channel patch-clamp recordings from bovine adrenal zona fasciculata (AZF) cells. These cells express a novel ATP-activated K⁺ channel (I_AC) that sets the membrane potential and couples adrenocorticotrophic hormone (ACTH) receptor activation to depolarization-dependent Ca²⁺ entry and cortisol secretion (Mlinar et al., 1993; Enyeart et al., 1997). Mibefradil potently blocks I_AC K⁺ channels at concentrations below those required to inhibit T-type Ca²⁺ channels in the same cells (Gomora et al., 1999).

	Materials and Methods

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Tissue culture media, antibiotics, fibronectin, and FBS were obtained from Life Technologies (Grand Island, NY). Coverslips were from Bellco Glass (Vineland, NJ). Enzymes, MgATP, ACTH(1-24), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid were obtained from Sigma Chemical Co. (St. Louis, MO). Mibefradil was a gift from Hoffman La Roche (Basel, Switzerland).

Isolation and Culture of AZF Cells. Bovine adrenal glands were obtained from steers (age range, 1-3 years) within 30 min of slaughter at a local slaughterhouse. Fatty tissue was removed immediately, and the glands were transported to the laboratory in ice-cold PBS containing 0.2% dextrose. Isolated AZF cells were prepared as previously described (Enyeart et al., 1997). Cells were plated in Dulbecco's modified Eagle's medium/F-12+ in 35-mm dishes containing 9-mm² glass coverslips that had been treated with fibronectin (10 µg/ml) at 37°C for 30 min and then rinsed with warm, sterile PBS immediately before adding cells. Dishes were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Patch-Clamp Experiments. Patch-clamp recordings of K⁺ channel currents were made in the whole-cell and outside-out patch configurations. For both recording configurations, the standard pipette solution consisted of 115 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 20 mM HEPES, 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and 200 µM GTP, with pH buffered to 7.2 using KOH. For whole-cell and single-channel patch recordings, pipette solutions contained 5 -and 2 mM MgATP, respectively. Pipette [Ca²⁺] was 22 nM as determined using the Bound and Determined program (Brooks. and Storey, 1992). The external solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl, 2 mM MgCl₂, 10 mM HEPES, and 5 mM glucose, pH 7.4 using NaOH. All solutions were filtered through 0.22-µm cellulose acetate filters.

	Results

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Bovine AZF cells express two types of K⁺ currents: a voltage-gated, rapidly inactivating A-type current (I_A) and the noninactivating, ATP-activated current I_AC (Mlinar et al., 1993; Mlinar and Enyeart, 1993; Enyeart et al., 1997). I_AC consists of two components: an apparent instantaneous component and a time-dependent component (Enyeart et al., 1996). I_AC is only weakly voltage dependent with open probability (P_o) increasing by ~30% between voltages of 40 and +40 mV (Enyeart et al., 1997).

In whole-cell recordings, I_AC is present initially at low density but grows dramatically over a period of minutes, provided that ATP or other nucleotides are present at millimolar concentrations in the recording pipette (Enyeart et al., 1997). The absence of time and voltage-dependent inactivation allow I_AC to be easily isolated for measurement in whole-cell recordings using either of two voltage-clamp protocols. When voltage steps of 300-ms duration were applied from a holding potential of 80 mV to a test potential of +20 mV, I_AC was measured selectively near the end of a voltage step where the rapidly inactivating A-type current had completely inactivated (Fig. 1A, left traces). Alternatively, I_AC was selectively activated with an identical voltage step, after a 10-s prepulse to 20 mV had fully inactivated the A-type current (Fig. 1A, right traces).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Time- and concentration-dependent inhibition of IAC K+ current by mibefradil. Whole-cell K+ currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of 80 mV with or without 10-s prepulses to 20 mV that inactivate A-type K+ current. After IAC reached a stable amplitude, cells were superfused with mibefradil at concentrations ranging from 0.1 to 5 µM. A, K+ current records made with standard pipette solution supplemented with 5 mM MgATP and 200 µM GTP with (right) or without (left) 10-s prepulses to 20 mV. Numbers correspond to currents immediately after initiation of whole-cell recording (1); after IAC reached a maximum amplitude (2); and after inhibition by 0.1 µM (3), 1 µM (4), or 5 µM (5) mibefradil. B, IAC amplitudes recorded with () or without () depolarizing prepulses are plotted against time. Mibefradil was superfused as indicated. Numbers correspond to currents as in A. C, inhibition curve. Fraction of unblocked IAC current is plotted against mibefradil concentration. Data were fit with an equation of the form: I/Imax = 1/[1 + (B/IC50)X], where B is the mibefradil concentration, IC50 is the concentration that reduces IT by 50%, and X is the Hill coefficient. Values are mean ± S.E. of indicated number of determinations.

Mibefradil applied to AZF cells externally through bath perfusion inhibited both noninactivating I_AC and rapidly inactivating I_A currents in AZF cells. Of these two currents, I_AC was more potently inhibited. The rapidly inactivating I_A current was also inhibited by mibefradil at ~10-fold higher concentrations.

In the experiment illustrated in Fig. 1, I_AC K⁺ current grew to a stable amplitude over a 15-min period before the cell was superfused with mibefradil at concentrations between 0.1 and 5 µM. Over this range of concentrations, I_AC was preferentially inhibited in a concentration-dependent manner (Fig. 1, A and B). Inhibition was partially reversible with washing (Fig. 1B). Overall, mibefradil inhibited I_AC current with an IC₅₀ value of 0.50 µM (Fig. 1C). The inhibition of I_AC by mibefradil was insensitive to changes in holding potential. Mibefradil (0.5 µM) was equally effective at inhibiting I_AC, activated from holding potential of 80 or 40 mV (data not shown).

ACTH (100 pM) selectively and completely suppresses the expression of I_AC in whole-cell recordings, allowing the rapidly inactivating A-type current to be studied in isolation (Mlinar et al., 1993; Enyeart et al., 1996; Fig. 2A). Under these conditions, mibefradil inhibits I_A with an IC₅₀ value of 4.65 µM (Fig. 2, A and B). Inhibition of I_A by mibefradil was slowly reversible. Washing with control saline reduced I_A inhibition by 65.5 ± 9.3% (n = 6) after 15 to 20 min.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Inhibition of voltage-gated IA K+ current by mibefradil. IAC K+ current was selectively inhibited by superfusing the cell with 200 pM ACTH for 5 min. IA was activated by voltage steps to +20 mV, applied at 30-s intervals from a holding potential of 80 mV. A, IA current records in control saline and after steady-state block by mibefradil at 1, 5, and 10 µM as indicated. B, inhibition curve. Fraction of unblocked IA current is plotted against mibefradil concentration. Data were fit with an equation of the form: I/Imax = 1/[1 + (B/IC50)X], where B is the mibefradil concentration, IC50 is the concentration that reduces IT by 50%, and X is the Hill coefficient. Values are mean ± S.E. of indicated number of determinations.

Block of Unitary I_AC Currents by Mibefradil. Mibefradil inhibited unitary I_AC K⁺ currents recorded from excised outside-out patches without reducing the amplitude of the single-channel current. Figure 3 shows unitary currents recorded from an outside-out patch in response to depolarizing steps to +30 mV from a holding potential of 40 mV where I_A channels are inactivated (Mlinar and Enyeart, 1993). Under these conditions, a single type of K⁺ channel was typically present in the membrane patch. After recording currents in control saline, unitary currents were recorded at several different mibefradil concentrations.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of mibefradil on unitary IAC K+ currents. Unitary IAC currents were recorded in the outside-out configuration using pipettes containing standard solution supplemented with 2 mM MgATP. Voltage steps to +30 mV were applied at 10-s intervals from a holding potential of 40 mV. Each amplitude histogram was constructed from idealized channel openings obtained from unitary currents recorded in response to 80 to 96 separate voltage steps of 400-ms duration applied at 0.25 Hz. Unitary current amplitudes were distributed into bins of 0.15-pA width. Traces and corresponding amplitude histograms in control saline, in the presence of 1, 5, and 10 µM mibefradil and 7 min after return to control saline (wash) as indicated. The continuous line in the histograms represents the fit of Gaussian distributions to the data. Currents were filtered at a cutoff frequency of 2 kHz and sampled at 5 kHz. Similar results were obtained in each of eight outside-out patches.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effect of mibefradil on IAC channel Po and dwell times. The effect of mibefradil on IAC K+ channel Po and mean open (o) and closed (c1 and c2) time constants was determined from experiments such as those in Fig. 3. A, Po was calculated by integrating the area of the current traces and dividing this value by the product N · î · T, where N is the number of functional channels in the patch, î is the unitary current, and T is total recording time. Each Po value was calculated from channel openings recorded in response to 16 voltage steps to +30 mV. B, dwell time histograms with logarithmic time axes and square-root ordinates. Open and closed events were accumulated in bins whose width was proportional to the logarithm of the event duration. Open time histograms were fit (continuous line) with single exponentials (o), and the closed time histograms were fit with two exponentials (c1 and c2). o values for control, mibefradil, and wash were 1.73, 1.51, and 1.63 ms. Respective c1 and c2 values were 1.56 and 17.88 ms (control), 4.09 and 70.60 ms (mibefradil), and 2.40 and 23.52 ms (wash). Events of <0.5 ms in duration were disregarded.

Effect of Mibefradil on I_AC Dwell Times. Dwell time analysis of unitary I_AC currents showed that under control conditions, I_AC channel kinetics could be described by a single open time constant (_o) and two closed time constants (_c1, _c2) that differ by approximately one order of magnitude. Mibefradil increased both closed time constants but did not significantly alter the mean open time. In the experiment illustrated in Fig. 4B, mibefradil (1 µM) increased _c1 and _c2 from 1.56 and 17.9 ms to 4.09 ms and 70.6 ms, respectively. By comparison, the open time constant in control saline (_o = 1.73 ms) did not differ significantly from that determined in the presence of 1 µM mibefradil (_o = 1.51 ms). The effect of mibefradil on closed time constants was reversed on washing with control saline (Fig. 4B). Overall, mibefradil (1 µM) increased _c1 and _c2 from control values of 2.30 ± 0.18 and 27.9 ± 4.7 ms (n = 9) to 4.32 ± 0.61 and 62.50 ± 13.80 ms (n = 5), respectively. In contrast, in the presence of 1 µM mibefradil, _o was 1.25 ± 0.07 ms (n = 9 compared with a control value of 1.47 ± 0.07 ms (n = 9).

	Discussion

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In this study, it was discovered that the T-type Ca²⁺ channel antagonist mibefradil potently blocks ATP-activated I_AC K⁺ channels in AZF cells. The inhibition of I_AC K⁺ channels by mibefradil was voltage independent and, at the single-channel level, appeared to reduce channel P_o through stabilization of closed states. At 10-fold higher concentrations, mibefradil also inhibited voltage-gated, rapidly inactivating A-type K⁺ channels.

Block of Ca²⁺ and K⁺ Channels by Mibefradil. Mibefradil inhibits I_AC currents recorded in response to voltage steps from 80 with an IC₅₀ value of 0.50 µM, a concentration 2-fold lower than that required to inhibit T-type Ca²⁺ channels in the same cells under similar conditions (IC₅₀ = 1.0 µM; Gomora et al., 1999). Mibefradil inhibits T currents in other cells, including cerebellar neurons and vascular smooth muscle cells, with potency similar to that observed in AZF cells (Mishra and Hermsmeyer, 1994b; McDonough and Bean, 1998). In cells, including mouse spermatozoa, thyroid C cells, and rat sensory neurons, slightly higher IC₅₀ values have been reported for T channel inhibition (Mehrke et al., 1994; Arnoult et al., 1998; Todorovic and Lingle, 1998). The relative potency of mibefradil as a T channel blocker is complicated because the drug displays prominent voltage and use dependence (see later). Regardless, in well-polarized cells, mibefradil blocks I_AC-type K⁺ channels at concentrations lower than those required to block T-type Ca²⁺ channels in a variety of cells.

Mechanism of Mibefradil Inhibition of I_AC K⁺ Channels. Mibefradil-mediated inhibition of T-type Ca²⁺ channels shows prominent voltage and use dependence (McDonough and Bean, 1998; Gomora et al., 1999). As a result, its potency increases markedly in response to sustained or repeated depolarizations. According to the "modulated receptor hypothesis," voltage- and use-dependent block occurs when drugs preferentially bind to channels that have been opened or inactivated by depolarization (Hille, 1977; Hondeghem and Katzung, 1977).

Mechanism of Toxicity. Despite its beneficial hemodynamic effects, mibefradil has been withdrawn from the market due to toxicity associated with its use. The inhibition of cytochrome P-450 3A4 enzyme by mibefradil may result in numerous toxic drug interactions. Notably, when mibefradil is used in combination with certain antihistamines, such as astemizole, the Q-T interval is prolonged and ventricular arrhythmias may occur. The cellular mechanism probably involves inhibition of K⁺ channels in the myocardium by elevated concentrations of the antihistamine (Woosley, 1996). The ability of mibefradil to potently block some types of K⁺ channels suggests that in addition to altering the pharmacodynamics of antihistamines, the cardiovascular toxicity produced by the combination of these drugs could be due to their combined direct inhibition of K⁺ channels in the heart.