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Our purpose was to assess the structural and physicochemical determinants of the binding of tocainide and several of its homologs to the class I antiarrhythmic drug receptor associated with rat cardiac sodium channels. The homologs were chosen to assess the contributions of substituents of the aryl ring and the arylamine link on drug binding. Drug affinity was measured with a radioligand binding assay using [3H]Batrachotoxin A 20 alpha-Benzoate and freshly isolated cardiac myocytes. The affinities of the homologs were compared to determine the relationship between the affinity for the receptor and the physicochemical and structural properties of the parent drug. The contributions to the free energy of binding were determined with the Gibb's equation delta G = -RT In (1/Ki). Hydrophobic interactions are important at most sites. Meta substituents on the aryl ring and substituents on the link each interact hydrophobically with the receptor and contribute about 0.3 kcal/mol of carbon. The hydrophobic pocket near the link binding site accommodates at least six carbons. A para methoxy substituent reduces the free energy of tocainide binding by 43%. This profound reduction in the free energy of binding might be due to anomolously high aqueous solubility of alkyl aryl ethers. Longer alkoxy chains contribute 1.09 kcal/mol of carbon to the binding energy. Ortho substituents contribute little to binding specificity. These findings support a notion of a complex drug receptor with hydrophilic and hydrophobic domains that recognize specific moieties on class I antiarrhythmic drugs.

This study assessed whether class I antiarrhythmic drugs allosterically inhibit [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTXB) binding to sodium channels on freshly isolated rat cardiac myocytes. All class I drugs tested inhibited equilibrium [3H]BTXB binding in a concentration-dependent manner. Scatchard analysis showed that disopyramide, flecainide, transcainide, lidocaine and amiodarone reduced [3H]BTXB maximum binding (Bmax) whereas procainamide, mexiletine, quinidine, quinine, tocainide, propafenone, encainide and O-demethylencainide increased [3H]BTXB KD but had little effect on Bmax. Kinetic [3H]BTXB binding assays were used to assess the mechanism by which class I drugs inhibit [3H]BTXB binding. Drugs that increase the unidirectional dissociation rate constant (k-1) of [3H]BTXB probably bind to sodium channels to which [3H]BTXB is already bound. Although all class I drugs increased the k-1 of [3H]BTXB, they did so weakly and at concentrations above the IC50 values of the drugs. Thus, drug binding to [3H]BTXB-bound channels does not appear to be the predominant mechanism underlying their ability to inhibit [3H]BTXB binding. Conversely, drugs which allosterically decrease the unidirectional association rate constant (K+1) of [3H]BTXB probably bind to channels to which [3H]BTXB is not already bound. All class I drugs decreased the k+1 of [3H]BTXB, indicating drug binding to [3H]BTXB-free channels. The estimated affinities of drugs for [3H]BTXB-free channels correlated closely with the IC50 values of these drugs (r = 0.94, P < .001), suggesting that this effect is a common and major determinant in their ability to inhibit [3H]BTXB binding. The results are discussed in light of electrophysiologic theories of class I antiarrhythmic drug action.

The structural and physicochemical determinants of binding of lidocaine and several of its aminoalkyl homologs to specific sites associated with the sodium channel were assessed using a radioligand assay and freshly isolated rat cardiac myocytes. The two series of closely related lidocaine homologs that were studied were composed, first, of homologs differing in the length of the link between the arylamide and amine domains of the molecule and, second, of homologs differing in the number of carbons attached to the terminal amine. Drug affinity was measured with a radioligand binding assay, using [3H]batrachotoxinin A 20 alpha-benzoate and freshly isolated cardiac myocytes. The affinities of the homologs were then compared with the pKa values, partition coefficients, distribution coefficients, and molecular structure of the homologs, to determine the relationship between the affinity for the receptor and the physicochemical and structural properties of the drug. Optimal binding was obtained with a link between the arylamide and amine domains that was two carbons in length. The affinity of the drug for the receptor was optimal with four or more amino-terminal carbons, and the precise arrangement of the carbons was not important. Each of the amino-terminal carbons independently contributed 0.3 kcal of free energy of binding, suggesting that the carbons dissolve in a hydrophobic pocket. The evolving picture of a drug structure that is optimal for receptor binding is one of a compound with a two-carbon arylamide-amine link and four or more amino-terminal carbons.

Radiolabeled neurotoxins have been used to study the structure and function of sodium channels. We studied the binding of [3H] batrachotoxinin A 20 alpha-benzoate [( 3H]BTX-B) to specific sites on sodium channels on rat cardiac myocytes. Calcium-tolerant myocytes were prepared by collagenase dispersion of adult rat hearts and were 75-83% viable. As with the nerve channel, specific binding of [3H]BTX-B to its receptor site was seen only in the presence of sea anemone toxin (ATX). The affinity of ATX for its binding sites may be estimated from its concentration-dependent stimulatory effect on [3H]BTX-B binding. These results suggest that, in the presence of 5.4 mM KCl, the myocytes have two affinities for ATX with estimated dissociation constants of 0.52 microM and 12.9 microM. Depolarization of the myocytes with either 65 mM KCl or 0.1 mM BaCl2 results in the loss of the 0.52 microM component, suggesting that it is voltage sensitive. The 0.52 microM and 12.9 microM components have maximal binding capacities corresponding to 4 and 11 sites/micron 2 of myocyte surface area, respectively. Scatchard analysis of [3H]BTX-B binding in the presence of ATX demonstrates a single class of sites with a KD of 25-35 nM. These results demonstrate that [3H]BTX-B can be used as a radioligand probe of the adult rat sodium channel and will facilitate a biochemical approach to the study of the interaction between antiarrhythmic drugs and the sodium channel.

The determinants of stereospecific binding of type I antiarrhythmic drugs to specific sites associated with the sodium channel were assessed using rat cardiac myocytes. The asymmetric carbon atoms of stereoisomers may be located at two sites within type I drugs. The structure of these drugs can be schematically illustrated as Aromatic-C1-link-C2-Amine, where C1 and C2 represent potentially asymmetric carbon atoms. We used enantiomeric pairs with either C1 or C2 asymmetric carbon atoms to assess the importance of conformation at these sites to drug binding. The affinities of enantiomers of seven sodium channel blockers were measured with a radioligand binding assay using [3H]batrachotoxinin benzoate [( 3H]BTXB) and freshly isolated cardiac myocytes. The enantiomers inhibited [3H]BTXB binding in a dose-dependent manner, with a mean Hill number of 1.0 +/- 0.1. The ratios of affinities [IC50 of (+)-isomer/IC50 of (-)-isomer] were, for the C1 pairs: quinidine, 0.29; cinchonidine, 0.55; disopyramide, 1.11; RAC 109, 5.33; and for C2 pairs: flecainide, 1.03; mexiletine, 2.15; tocainide, 3.01. The stereospecific differences in drug binding suggest that the orientations of both the aromatic and the amine groups to the rest of the drug molecule are important determinants of drug binding to the cardiac sodium channel. This also suggests the presence of at least two stereospecific domains within the binding sites for type I antiarrhythmic drugs.

The antiarrhythmic action of type I antiarrhythmic drugs may be mediated via binding of the drugs to a receptor associated with the cardiac sodium channel. This suggested that the effects of type I drugs might be stereospecific. We measured the effect of the tocainide stereoisomers (which have stereospecific antiarrhythmic effects) on conduction time and on radioligand binding to the cardiac sodium channel. The concentration-dependent effects of the individual enantiomers of tocainide on interventricular conduction time measured during constant rate ventricular pacing at 350 msec were assessed in 47 isolated perfused rabbit heart preparations. Significant increases (p less than 0.05) in conduction time occurred for both R-(-)-tocainide (75 microM, 10 +/- 5 msec) and S-(+)-tocainide (150 microM, 4 +/- 1 msec). R-(-)-Tocainide was more potent than the S-(+)-tocainide in prolonging conduction time (p less than 0.05). This stereospecific prolongation of conduction time suggested a stereospecific interaction with the sodium channel. The affinities of the enantiomers for the channel were measured with a radioligand binding assay using [3H]batrachotoxinin benzoate and freshly isolated cardiac myocytes. Both enantiomers inhibited [3H]batrachotoxin benzoate binding, but the IC50 (+/- SD) values were different: R-(-)-tocainide 184 +/- 8 microM; S-(+)-tocainide, 546 +/- 37 microM (p less than 0.003). Tocainide isomers are stereospecific in terms of prolonging conduction time and in binding to the sodium channel. The stereospecific electrophysiologic effects of tocainide may result from binding to a receptor associated with the cardiac sodium channel.

The state-dependent binding of class I antiarrhythmic drugs to a receptor associated with the cardiac sodium channel was assessed using [3H]batrachotoxinin A 20-alpha-benzoate [( 3H]BTXB) binding. [3H]BTXB binds specifically to and stabilizes activated states of the sodium channel. Quinidine (IC50 = 40 microM) and lidocaine [IC50 = 61 microM) inhibited equilibrium [3H]BTXB binding to sodium channels present on freshly isolated rat cardiac myocytes. Scatchard analysis of [3H]BTXB binding in the presence of quinidine and lidocaine revealed two apparent patterns of inhibition. Quinidine (33 microM) increased the KD but had no significant effect on the Bmax, whereas lidocaine (91 microM) reduced the Bmax but had no significant effect on the KD. To address drug binding to activated and nonactivated states, we exploited the state-specific binding of [3H]BTXB. Drugs that increase the rate of dissociation (k-1) of [3H]BTXB must bind to sodium channels to which [3H]BTXB is already bound (i.e., activated channels). Therefore, drug-mediated increases in k-1 measure drug binding to activated states. Both quinidine and lidocaine increased the k-1 of [3H]BTXB, indicating drug binding to and destablization of activated sodium channels. However, the minimal affinities of quinidine and lidocaine for activated channels (KDact) were estimated to be 433 and 455 microM, respectively, concentrations much higher than the equilibrium IC50 values. Drugs that allosterically decrease the rate of association (k+1) of [3H]BTXB must bind to sodium channels to which [3H]BTXB is not already bound (i.e., nonactivated channels). Therefore, drug-mediated decreases in k+1 measures drug binding to nonactivated states. Quinidine and lidocaine decreased the k+1 of [3H]BTXB, indicating drug binding to and stablization of nonactivated sodium channels. The affinity of quinidine and lidocaine for nonactivated channels (KDnon) was estimated to be 10 and 35 microM, respectively, concentrations close to the equilibrium IC50 values. The markedly different KDact and KDnon values for both quinidine and lidocaine indicate state-dependent binding of quinidine and lidocaine to the class I receptor on the cardiac sodium channel. Both drugs destabilize activated channels and stabilize nonactivated channels. The Scatchard results suggest that quinidine and lidocaine may have different mechanisms of allosteric inhibition of [3H]BTXB binding.