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CELLULAR AND MOLECULAR
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic
Received for publication
March 7, 2007
Accepted
May 1, 2007.
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Abstract |
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= –0.60 for verapamil and a positive interaction factor = +0.24 for diltiazem. These results suggest that at physiological membrane potentials, the L-type calcium channel cannot be simultaneously occupied by a dihydropyridine agonist and antagonist, whereas it can simultaneously bind a dihydropyridine agonist and a nondihydropyridine antagonist. Generally, the effects of the drugs on the L-type calcium channel support a concept of a channel domain responsible for binding of calcium channel antagonists and agonists changing dynamically with the membrane voltage and occupancy of individual binding sites.
1 subunit of the channel (for review, see Hockerman et al., 1997a
; Zahradníková and Lacinová, 1998; Striessnig et al., 1998). In the transmembrane segments IIIS6 and IVS6 of the 1 subunit, several of the amino acids essential for the high-affinity binding of either drug type are shared by two or by all three binding sites (Hering et al., 1996; Schuster et al., 1996; Hockerman et al., 1997b; Kraus et al., 1998; Striessnig et al., 1998). This suggests that all three binding sites reside in close proximity.
The inhibition of calcium current by DHP, PAA, and BTZ calcium antagonists in all L-type calcium channel isoforms is dependent on channel state (for review, see McDonald et al., 1994). Channel affinity for all calcium antagonists increases with depolarization. DHP, PAA, and BTZ drugs differ by their use dependence, with DHPs being the least use-dependent, BTZs being intermediate, and PAAs being the most use-dependent. For the interaction of PAA and BTZ drugs with their binding sites, amino acids on the 1 subunit of the channel that play a role in channel inactivation are also important (Hering et al., 1997; Kraus et al., 1998; Motoike et al., 1999). Therefore, it was suggested that inactivation and use-dependent block share common molecular determinants (Hering et al., 1998).
Recent findings in chimeric channels (Berjukow et al., 1999) suggest important differences between BTZ drug binding to the skeletal and cardiac calcium channel isoforms. Indeed, the effects of diltiazem (Porzig and Becker, 1988; Kanda et al., 1997) and verapamil (Porzig and Becker, 1988) on binding of dihydropyridine antagonists in living cardiac myocytes differed from those in skeletal muscle. Noncompetitive interactions between the binding sites for DHP, BTZ, and PAA drugs were observed in binding studies with calcium channel antagonists on purified skeletal muscle membrane preparations (Glossmann et al., 1985; Striessnig et al., 1986). They have been interpreted as a result of a positive allosteric effect of BTZ drugs and a negative allosteric effect of the PAA drugs on the DHP binding site. In cardiac myocytes, both BTZ and PAA drugs displayed a positive allosteric effect on the DHP binding in polarized cells, whereas both drug types had a negative allosteric effect in depolarized cells. In addition, the effects of the DHP antagonist nitrendipine and the BTZ antagonist diltiazem on calcium currents through expressed Cav1.2 channels were shown to be additive at room temperature but synergic at elevated temperature (Kanda et al., 1998). These results suggest that in cardiac cells, not only binding of a single drug but also the character of mutual interactions between different drugs and the calcium channel depends on experimental conditions defining the state of the channel. There is only one study of the interaction between the DHP and PAA or BTZ drugs using direct recording of the calcium currents, that is, on functional calcium channels (Kanda et al., 1998). Yet, such studies can provide a deeper insight into the mechanism of drug action under physiological conditions, under which membrane potential periodically changes.
In the present work, we studied the inhibitory effects of different types of calcium antagonists in the presence of the dihydropyridine agonist Bay K8644. With the help of mathematical modeling, we characterized their mutual binding interactions. We conclude that the diversity of mutual DHP, PAA, and BTZ binding interactions conforms to an idea of a dynamic drug-binding domain within the calcium channel, which changes as a function of the channel state and occupancy of individual binding sites.
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Materials and Methods |
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). Approximately 30 min after administration of heparin (5000 U/kg i.p.), rats were anesthetized by intraperitoneal injection of 100 mg/kg pentobarbital sodium. The hearts were rapidly dissected and retrogradely perfused with oxygenated solutions at 37°C. The perfusion started with Tyrode's solution (135 mM NaCl, 5.4 mM KCl, 5 mM MgCl2, 10 mM HEPES, 0.33 mM NaH2PO4, and 1 mM CaCl2, pH 7.3) applied for 5 min. It continued with a nominally Ca2+-free Tyrode's solution (free Ca2+ of 5 µM) for 5 min, and it completed with the enzyme solution (Collagenasa cruda; ÚSOL, Prague, Czech Republic; 180 µg/ml dissolved in Tyrode's solution with free Ca2+ set to 20 µM). After 5 min of enzymatic perfusion, the left ventricle and septum were dissected, and the tissue was further incubated up to 5 min in the enzyme solution. The tissue was then minced, placed into 4 ml of the stopping solution (106 mM CH3SO3K, 1 mM EGTA, 22 mM taurine, 22 mM glucose, 2.4 mM MgSO4, and 8 mM K2HPO4, pH 7.3), gently stirred, and triturated. The cell suspension was filtered through a nylon mesh and centrifuged at 25g for 5 min. The cells were washed twice by 4 ml of the stopping solution, and they were stored in Petri dishes at room temperature for use within 1 to 4 h after isolation.
Calcium Current Measurements. The L-type calcium current (ICa) was recorded using the standard whole-cell patch-clamp technique (Hamill et al., 1981). The patch-clamped myocyte was placed into the perfusion channel (volume 50 µl) allowing rapid (<1s) and complete exchange of external solutions. The sodium channel current was inhibited using a –50-mV holding potential and 20 µM TTX in the external solution, and potassium currents were blocked by replacement of K+ ions with Cs+ in both the external and internal solution. Calcium channels were kept in maximally phosphorylated state by including 50 µM cAMP to the internal solution and 10 µM IBMX (a membrane-permeable phosphodiesterase inhibitor) to the external solution. The standard protocol for recording calcium currents consisted of a train of 70-ms voltage pulses to 0 mV, elicited every 3 s from a holding potential of –50 mV. All experiments were carried out at room temperature (22–24°C).
Solutions for Patch-Clamp Recordings. The external solution contained 135 mM NaCl, 5.4 mM CsCl, 10 mM HEPES, 5 mM MgCl2, 0.33 mM NaH2PO4, 1 mM CaCl2, 0.02 mM TTX, and 0.01 mM IBMX, pH adjusted to 7.3 with NaOH. The internal solution contained 135 mM CsCH3SO3, 10 mM CsCl, 10 mM HEPES, 1 mM EGTA, 3 mM MgSO4, 3 mM ATP, and 0.05 mM cAMP, pH adjusted to 7.3 with CsOH.
Drugs. IBMX was from Sigma-Aldrich (St. Louis, MO). TTX was from Sigma-Aldrich or Alomone Labs (Jerusalem, Israel). The calcium channel drugs were from the following sources: (±)-Bay K8644 (Calbiochem, Lucerne, Switzerland), D-cis-diltiazem (Lachema, Brno, Czech Republic), and (±)-verapamil (Sigma-Aldrich). Felodipine was kindly provided by Astra Hässle (Mölndal, Sweden). All other chemicals were of analytical grade. All solutions were made using double-distilled water. Solutions of dihydropyridines were prepared by dilution of 50 mM (Bay K8644) and 10 mM (felodipine) stock solutions of the drugs in ethanol in the extracellular solution at the beginning of each experiment (the final ethanol concentration, <0.01%, was without effect on calcium currents).
Data Acquisition and Analysis. Whole-cell currents were measured with the EPC-7 amplifier (List Electronics, Darmstadt, Germany), filtered at 2 kHz, and digitized at 5 kHz by the LabMaster interface (Scientific Solutions, Mentor, OH) using commercial software (pClamp version 5.5.1; Molecular Devices, Sunnyvale, CA). Series resistance of approximately 2.5 M
was compensated electronically by approximately 70% of its value. The capacitance charging current was canceled in part electronically and in part with an on-line subtraction procedure.
The results are expressed as mean ± S.E. The values of fitted parameters are given with the mean ± S.E. of the fit. Statistical significance was estimated using the Student's t test. The significance of differences between the quality of fit by individual models was tested using the F-test. A difference was considered significant if p < 0.05.
Quantitative Description of Dose-Response Data. The relationships between concentration of drugs and the peak calcium current was described using dose-response equations derived from the law of mass action. It has to be noted, however, that the dissociation constants of the drugs are voltage-dependent; thus, the equations describe the steady state rather than equilibrium. Therefore, we use the term "pseudoequilibrium models". The models that involve simultaneous binding of Bay K8644 and the calcium antagonist were defined as "noncompetitive", because they do not distinguish whether the ligands induce conformational changes of the channel, i.e., modulate each other's binding sites allosterically, or whether they hinder each other's binding/unbinding sterically. The inhibition of calcium current by calcium antagonists was described by the following dose-response equation:
 | (1) |
is the peak calcium current in the presence of an inhibitory drug B, ICa,p is the peak calcium current in the absence of the drug, KB is the dissociation constant, [B] is the concentration of the drug, and nH is the Hill slope of the dose response.
Activation of calcium current by the agonist Bay K8644 was described by the following equation:
 | (2) |
is the peak calcium current in the presence of an activating drug, KA is the dissociation constant, [A] is the concentration of the drug, and is the maximal relative increase of the calcium current ICa,p induced by the agonist.
The inhibition of calcium current by calcium antagonists in the presence of a constant concentration of the agonist Bay K8644 was described by the following dose-response equation:
 | (3) |
is the peak calcium current in the presence of both drugs, 
is the apparent dissociation constant of the inhibitory drug, and the remaining parameters were defined previously.
The combined effect of Bay K8644 and a competing calcium antagonist was described by the following equation:
 | (4) |
The combined effect of Bay K8644 and a noncompetitive calcium antagonist was described by the following equation:
 | (5) |
The dose-response curves of the agonist and the antagonist applied alone as well as those of the antagonist at all studied agonist concentrations were simultaneously approximated by the appropriate model. Thus, all data contained in panels A and B of one of Figs. 4, 5, 6 constituted a single data set for the fitting procedure. Fitting was performed in Origin version 5.1 (OriginLab Corp., Northampton, MA).
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Results |
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The effect of the DHP felodipine on calcium currents in the absence and the presence of Bay K8644 is illustrated in Fig. 1, A and B. The inhibitory efficiency of felodipine was strongly depressed by Bay K8644. The concentration of felodipine necessary for inhibition of ICa,p to 50% of the control was increased by an order of magnitude (Table 1). A 60-s sojourn at –80 mV led to almost complete reversal of felodipine inhibition (Table 2). However, the activating effect by Bay K8644 persisted at this membrane potential. Still notably, inhibition of ICa by felodipine was substantially slowed down in the presence of Bay K8644 (Table 3).
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Similar but less pronounced effects of Bay K8644 were observed in experiments with verapamil (Fig. 2, A and B). The design of the experiments was the same as with felodipine. Again, the inhibitory efficiency of verapamil and the rate of ICa,p inhibition by verapamil were significantly reduced (Tables 1 and 3). However, recovery of the peak ICa amplitude after 1-min hyperpolarization was less prominent for verapamil than for felodipine (Table 2).
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; Uehara and Hume, 1985; Méry et al., 1996). Fitting the experimental dose dependencies with the appropriate dose-response equations (see Materials and Methods) provided the apparent dissociation constants given in Table 1. The corresponding values of the Hill coefficients (Table 1), both in the presence and the absence of Bay K8644, were not significantly different from nH = 1. The effect of Bay K8644 was manifested as a shift in the apparent inhibitory constant of the calcium antagonists. The 300 nM Bay K8644 concentration induced pronounced increases in , 15 times for felodipine and three times for verapamil, but a two-time decrease of for diltiazem.
Quantitative Description of the Interactions. The quantitative models of the concentration dependence of ICa,p in the simultaneous presence of two drugs are derived in the Supplemental Material for both competitive and noncompetitive mechanisms. The parameters of the best fits are given in Table 4. The summarized dose-response data for felodipine and Bay K8644 are shown in Fig. 4 together with the best-fitting competitive and cooperative models. It is apparent that both models provided very good fits, which were not significantly different (p = 0.5). Nevertheless, the competitive model is preferred, because the cooperative model, besides yielding higher errors of fitted parameters, has one additional parameter—interaction factor , the introduction of which did not decrease the 
2 value in this case. The values of KA, KB, and obtained by the competitive model (Table 4) were not significantly different from those obtained by fitting the dose-response curves of felodipine and Bay K8644 applied alone (Table 1).
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Analogous data for verapamil are given in Fig. 5. In this case, the two models provided substantially different fits to the data. The cooperative model with a negative value of = –0.60 ± 0.06 was superior to the competitive model, as judged by the 2 value (F-test; p < 0.0001) and standard errors of the fitted parameters. The systematic error introduced by the competitive model was apparent especially at low concentrations of verapamil and Bay K8644. The values of KA, KB, and obtained by the cooperative model (Table 4) were not significantly different from those obtained by fitting of the dose-response curves for verapamil and Bay K8644 applied alone (Table 1) whereas analogous parameters of the competitive model differed substantially.
The data for diltiazem are plotted in Fig. 6. The dose-response data could not be reasonably approximated by the competitive model, but they were well fitted by the cooperative model with a positive value of the interaction factor . The competitive model deviated from the data at all concentrations of diltiazem as well as of Bay K8644; therefore, it could not be accepted (F-test; p < 0.0001). The values of KA, KB, and obtained by the cooperative model (Table 4) were not significantly different from those obtained by fitting of the dose-response curves for diltiazem and Bay K8644 applied alone (Table 1), whereas analogous parameters of the competitive model differed substantially.
The experimentally estimated values of for the three studied calcium antagonists in the presence of 300 nM Bay K8644 are compared in Table 1 to the theoretical values, calculated from parameters of the competitive model for felodipine and from parameters of the cooperative model for verapamil and diltiazem. The theoretical values were calculated from the parameters given in Table 4, using eq. 4 for competitive interaction and eq. 5 for cooperative interaction, by setting the value of agonist concentration to 300 nM. The quality of description of the data by the model is underlined by the similarity of experimentally estimated and theoretical values, which were not significantly different from each other.
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Discussion |
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Competition between an activating and an inhibiting dihydropyridine has not been previously demonstrated. In binding studies, it is not possible to control the gating state of the channel, and the DHP agonists are known to inhibit calcium channel activity at depolarized potentials (Sanguinetti et al., 1986; Hadley and Hume, 1988; Kamp et al., 1989). In the presented electrophysiological experiments, Bay K8644 induced a very pronounced decrease in the apparent affinity of the dihydropyridine antagonist felodipine. A model assuming that Bay K8644 and felodipine cannot simultaneously bind to the calcium channel, that is, a pure competitive mechanism, could quantitatively account for this decrease. However, our results do not strictly imply that both Bay K8644 and the inhibitory dihydropyridines bind to identical binding site. The "competitive" shape of the concentration dependence implies only that both drugs cannot bind to their binding sites simultaneously, with no implication about the exact place of binding. Therefore, it is possible that different conformations of the channel expose different binding sites, one that has a high affinity to inhibitory and the other to activatory dihydropyridines, such as predicted by homology modeling (Zhorov et al., 2001). In this context, it has been noted that the agonist/antagonist action of dihydropyridines might be determined by their orientation with respect to the SIII/SIV interface of the calcium channel (Yamaguchi et al., 2003). Different voltage dependence of the action of dihydropyridine antagonists and agonists is well known (Kamp et al., 1989; Wei et al., 1989). In the presented experiments, this difference was demonstrated as the reversal of felodipine inhibition but persistent Bay K8644 activation of the channel at –80 mV (Fig. 2). Results of others, such as dual agonist and antagonist action of certain dihydropyridines measured in single-channel experiments (Hess et al., 1984; Kokubun and Reuter, 1984; Brown et al., 1986), and the presence of amino acids differently regulating the binding of agonist and antagonist dihydropyridines (Mitterdorfer et al., 1996; Schuster et al., 1996; Ito et al., 1997) further favor this mechanism.
Although a lot is known about the mutual interactions between the dihydropyridine binding site on one hand and phenylalkylamine or benzothiazepine binding site on the other hand, these studies have been traditionally performed in biochemically isolated (and often purified) sarcolemmal membrane preparation from skeletal muscle (Glossmann et al., 1985; Striessnig et al., 1986). In these experiments, diltiazem enhanced and verapamil decreased the affinity of the channel to bind dihydropyridines by noncompetitive mechanisms, which were regarded allosteric. In the comparative study on equilibrium binding of felodipine (Minarovic and Meszaros, 1998), the interactions between the binding sites for DHP, BTZ, and PAA drugs were qualitatively similar in both, skeletal and cardiac membranes. Brauns et al. (1997) observed in solubilized skeletal muscle preparation that dihydropyridines decrease the association rate for BTZ antagonists, but they do not affect equilibrium binding. On the basis of these observations, it was suggested that the interaction between the two drug sites on the calcium channel has a large steric component.
The data on polarized membrane vesicles or cardiac myocytes with a defined membrane potential are scarce. Both verapamil (Porzig and Becker, 1988) and diltiazem (Porzig and Becker, 1988; Kanda et al., 1997) enhanced the affinity of dihydropyridines to L-type calcium channels in polarized cells, whereas they had inhibitory effect on dihydropyridine binding in depolarized cells (Porzig and Becker, 1988). The main difference between verapamil and diltiazem was in the magnitude of the effects: Although in polarized cells the effect of diltiazem was more pronounced than that of verapamil, it was the opposite in depolarized cells; here, the effect of verapamil prevailed.
The results of the present study, obtained in the physiological range of membrane potentials using repetitive stimulation, revealed important differences between the action of verapamil and diltiazem. These differences could be conveniently quantified by a single interaction factor, which attained a negative value in the case of the PAA verapamil and a positive value in the case of the BTZ diltiazem (Table 4). From a kinetic point of view, the differences between the PAA and BTZ type of antagonists were also prominent. Although the effective on-rate of verapamil binding decreased when the channel was occupied by Bay K8644, no significant change in the on-rate of inhibition by diltiazem in the presence of the dihydropyridine agonist was observed. As the apparent affinity of diltiazem is increased due to its positive cooperativity with Bay K8644, it can be inferred that the effective rate of dissociation of diltiazem from the channel is decreased when the channel is occupied by Bay K8644.
In our study, the cells were held at a membrane potential of –50 mV for 3 s, and then they were depolarized to 0 mV for 70 ms. The stimulation protocol was repetitive, so that a steady state between the population of drug-free and differently drug-bound channels was established shortly. The dissociation constants of the drugs determined in the present work therefore are apparent constants, in which affinities of different channel states to the drugs are weighted according to their probability distribution, as was observed before by several laboratories (Bean, 1984; Méry et al., 1996).
To give molecular interpretation to our findings, we extended the guarded modulated receptor model of the L-type calcium channel (Hering et al., 1998; Berjukow et al., 1999) to incorporate the simultaneous action of an agonist and a non-DHP antagonist. These authors have shown the importance of both the preferential drug binding to inactivated channels and the slow rate of drug unbinding from noninactivated channels in the phenomenon of use dependence. In the depolarized state, drugs have high affinity to the channel, because of the high rate of drug binding in the open state and of drug trapping in the inactivated state. Unbinding of the drug occurs mainly upon recovery from inactivation. We propose that in polarized cells, occupation of the DHP site hinders unbinding of the PAA/BTZ drugs from the calcium channel, and vice versa, thereby effectively increasing drug affinity and accounting for the positive interaction coefficient. Thus, the rate constants from the ternary complexes are decreased in the closed states of the channel. In depolarized cells, when the channels reside in the open state(s), the interaction of DHP and of the BTZ or PAA drugs with their respective binding sites is sterically hindered. As a result, the association rate constants of the ternary complex channel-DHP drug-non-DHP drug are lower than the rate constants of binary drug-channel complexes in the open state(s) of the channel, effectively decreasing drug affinity and accounting for a negative interaction coefficient. In contrast, no ternary complex can be formed in the presence of an agonist and antagonist dihydropyridine.
This model explains why DHP and BTZ/PAA decrease each other's affinity in depolarized cells, whereas they increase each other's affinity in polarized cells (at –50 mV) as observed by Porzig and Becker (1988). The difference in the action of verapamil and diltiazem in the presence of Bay K8644 can be then attributed to their different use dependence (Lee and Tsien, 1983; Uehara and Hume, 1985; for review, see McDonald et al., 1994), i.e., to the different weight of the rate constants of drug binding to the open channel and drug unbinding from the recovering channel. It is conceivable that steady-state inhibition by diltiazem (the less use-dependent compound) is more dependent on the interaction of the drug with the closed and recovering channel. In the presence of verapamil (the more use-dependent compound), the steady-state inhibition is more dependent on the rate constants of drug binding to the open channel.
The interaction between DHP and BTZ/PAA takes place within the small space to which their binding sites are confined at the 1 subunit of the channel (Lipkind and Fozzard, 2003; Yamaguchi et al., 2003). Therefore, the interaction between the drugs and Bay K8644 probably has a strong steric component, as suggested for verapamil by Kraus et al. (1998). The enhancement of diltiazem action by Bay K8644 at the expense of decreased off-rates of drugs in the ternary complex might be also interpreted by a steric model, such as that used for describing the interaction between alcuronium and N-methyl scopolamine at the cardiac muscarinic acetylcholine receptors (Proska and Tucek, 1994). Our findings suggest that the domain responsible for binding of calcium channel antagonists and agonists within the 1 subunit of the L-type calcium channel dynamically changes as a function of channel state and occupancy by these drugs, thus giving importance to its structural changes during gating.
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Acknowledgements |
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ová for technical assistance. |
Footnotes |
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skumu a vvoja. The research of A.Z. was supported in part by an International Research Scholar award from the Howard Hughes Medical Institute. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: DHP, dihydropyridine; PAA, phenylalkylamine; BTZ, benzothiazepine; Bay K8644, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester; ICa, calcium current; TTX, tetrodotoxin; IBMX, isobutylmethylxantine.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Ivan Zahradník, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovak Republic. E-mail: ivan.zahradnik{at}savba.sk
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