Hill R J, Duff H J, Sheldon R S
Department of Medicine, University of Calgary, Alberta, Canada.
Mol Pharmacol. 1989 Jul;36(1):150-9.
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.
使用[3H]蛙毒素A 20-α-苯甲酸酯（[3H]BTXB）结合实验评估了I类抗心律失常药物与心脏钠通道相关受体的状态依赖性结合。[3H]BTXB特异性结合并稳定钠通道的激活状态。奎尼丁（IC50 = 40 microM）和利多卡因[IC50 = 61 microM]抑制了[3H]BTXB与新鲜分离的大鼠心肌细胞上存在的钠通道的平衡结合。在奎尼丁和利多卡因存在下对[3H]BTXB结合进行的Scatchard分析揭示了两种明显的抑制模式。奎尼丁（33 microM）增加了KD，但对Bmax没有显著影响，而利多卡因（91 microM）降低了Bmax，但对KD没有显著影响。为了研究药物与激活态和非激活态的结合，我们利用了[3H]BTXB的状态特异性结合。增加[3H]BTXB解离速率（k-1）的药物必须与已经结合了[3H]BTXB的钠通道结合（即激活的通道）。因此，药物介导的k-1增加衡量了药物与激活态的结合。奎尼丁和利多卡因都增加了[3H]BTXB的k-1，表明药物与激活的钠通道结合并使其不稳定。然而，奎尼丁和利多卡因对激活通道的最小亲和力（KDact）估计分别为433和455 microM，这些浓度远高于平衡IC50值。变构降低[3H]BTXB结合速率（k+1）的药物必须与尚未结合[3H]BTXB的钠通道结合（即非激活通道）。因此，药物介导的k+1降低衡量了药物与非激活态的结合。奎尼丁和利多卡因降低了[3H]BTXB的k+1，表明药物与非激活的钠通道结合并使其稳定。奎尼丁和利多卡因对非激活通道的亲和力（KDnon）估计分别为10和35 microM，这些浓度接近平衡IC50值。奎尼丁和利多卡因明显不同的KDact和KDnon值表明奎尼丁和利多卡因与心脏钠通道上的I类受体存在状态依赖性结合。两种药物都使激活通道不稳定并使非激活通道稳定。Scatchard结果表明，奎尼丁和利多卡因可能具有变构抑制[3H]BTXB结合的不同机制。
