Francis J, Burnham W M
Department of Pharmacology and Bloorview Epilepsy Program, University of Toronto, Ontario, Canada.
Mol Pharmacol. 1992 Dec;42(6):1097-103.
The voltage-dependent sodium channel has been proposed as a specific target for the actions of the anticonvulsant drug phenytoin. Working at 0-4 degrees, we previously reported the existence of specific [3H]phenytoin binding sites in rat brain membranes. In the present study, the binding of [3H]phenytoin was assessed at 22 degrees, a temperature favorable to the binding of sodium channel ligands. At 22 degrees, the site had a Kd of 1.5 microM, which is in the relevant therapeutic concentration range for anticonvulsant activity (1-10 microM), and a calculated Bmax of 4.5 pmol/mg of protein, which is similar to previous estimates of sodium channel concentration in brain membranes. In competition experiments, specific [3H]phenytoin binding was found to be inhibited by drugs that interact with the sodium channel, including antiarrhythmics, local anesthetics, anticonvulsants, and site-specific neurotoxins (the steroidal alkaloid activators, beta-scorpion venoms, and brevetoxin-3). Diazepam, used clinically in the management of tonic-clonic status epilepticus, and flunarizine, a calcium channel blocker with anticonvulsant activity, potentiated [3H]phenytoin binding at micromolar concentrations. Other drugs and ligands, including neurotransmitters, neuromodulators, and ligands for other ion channels, had no effect. Depolarization with KCl showed [3H]phenytoin binding to be voltage sensitive. Experiments with batrachotoxin (a specific site 2 toxin) and anticonvulsants demonstrated that the interactions between these compounds and the [3H]phenytoin binding site are allosteric in nature. These results provide direct evidence that phenytoin interacts with the voltage-dependent sodium channel and indicate that such interactions take place at therapeutic concentrations. They support previous proposals, based on toxin-binding and electrophysiological studies, that the therapeutic effects of phenytoin result from a selective inhibition of voltage-dependent sodium flux.
电压依赖性钠通道已被认为是抗惊厥药物苯妥英钠作用的一个特定靶点。我们之前在0 - 4摄氏度的条件下开展研究，报告了大鼠脑膜中存在特异性的[3H]苯妥英结合位点。在本研究中，[3H]苯妥英的结合情况是在22摄氏度下评估的，该温度有利于钠通道配体的结合。在22摄氏度时，该位点的解离常数（Kd）为1.5微摩尔，处于抗惊厥活性的相关治疗浓度范围内（1 - 10微摩尔），计算得出的最大结合容量（Bmax）为4.5皮摩尔/毫克蛋白质，这与之前对脑膜中钠通道浓度的估计相似。在竞争实验中，发现与钠通道相互作用的药物，包括抗心律失常药、局部麻醉药、抗惊厥药和位点特异性神经毒素（甾体生物碱激活剂、β-蝎毒和短裸甲藻毒素-3）可抑制特异性的[3H]苯妥英结合。临床上用于治疗强直阵挛性癫痫持续状态的地西泮以及具有抗惊厥活性的钙通道阻滞剂氟桂利嗪，在微摩尔浓度下可增强[3H]苯妥英的结合。其他药物和配体，包括神经递质、神经调质以及其他离子通道的配体，均无作用。用氯化钾进行去极化显示[3H]苯妥英的结合具有电压敏感性。用蛙毒素（一种特异性的位点2毒素）和抗惊厥药进行的实验表明，这些化合物与[3H]苯妥英结合位点之间的相互作用本质上是变构的。这些结果提供了直接证据，表明苯妥英与电压依赖性钠通道相互作用，并表明这种相互作用发生在治疗浓度下。它们支持了基于毒素结合和电生理研究的先前观点，即苯妥英的治疗作用源于对电压依赖性钠通量的选择性抑制。
