Sodium channel inhibitor drugs can exert their effect by either blocking, or modulating the channel. The extent of modulation versus channel block is crucial regarding the therapeutic potential of drug candidates. Modulation can be selective for pathological hyperactivity, while channel block affects vital physiological function as much as pathological activity. Previous results indicated that riluzole, a drug with neuroprotective and antiepileptic effects, may have a unique mechanism of action, where modulation is predominant, and channel block is negligible. We studied the effects of riluzole on rNa_V1.4 channels expressed in HEK cells. We observed that inhibition by riluzole disappeared and reappeared at a rate that could not be explained by association/dissociation dynamics. In order to verify the mechanism of non-blocking modulation, we synchronized photolabeling with the voltage clamp protocol of patch-clamp experiments. Using this method, we could bind a photoreactive riluzole analog covalently to specific conformations of the channel. Photolabeling was ineffective at resting conformation, but effective at inactivated conformation, as judged from persisting modulated gating after removal of unbound photoactive drug from the solution. Mutation of the key residue of the local anesthetic binding site (F1579A) did not fully prevent ligand binding and inhibition, however, it eliminated most of the modulation caused by ligand binding. Our results indicate that riluzole binds with highest affinity to the local anesthetic binding site, which transmits inhibition by the unique non-blocking modulation mechanism. Our results also suggest the existence of one or more additional binding sites, with lower affinity, and different inhibition mechanism.

中文翻译：

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构象选择性光标记揭示的钠通道无阻滞抑制机制

钠通道抑制剂药物可以通过阻断或调节通道发挥作用。调制对通道阻滞的程度对于候选药物的治疗潜力至关重要。调节对于病理性活动过度可能是选择性的，而通道阻滞对重要的生理功能的影响与病理活动一样多。先前的结果表明，具有神经保护和抗癫痫作用的药物利鲁唑可能具有独特的作用机制，其中调节作用占主导地位，而通道阻滞作用可以忽略不计。我们研究了利鲁唑对rNa _V的影响在HEK细胞中表达的1.4个通道。我们观察到，利鲁唑的抑制作用消失并以无法用缔合/解离动力学解释的速率重新出现。为了验证无阻塞调制的机制，我们将光标记与膜片钳实验的电压钳协议同步。使用这种方法，我们可以将光反应性利鲁唑类似物与通道的特定构象共价结合。根据从溶液中除去未结合的光敏药物后持续的调节门控判断，光标记对静止构象无效，但对失活构象有效。局麻药结合位点的关键残基（F1579A）的突变不能完全阻止配体的结合和抑制，但是，它消除了由配体结合引起的大部分调节作用。我们的结果表明，利鲁唑以最高的亲和力与局麻药结合位点结合，从而通过独特的非阻滞调节机制传递抑制作用。我们的结果还表明存在一个或多个其他结合位点，具有较低的亲和力和不同的抑制机制。