Molecular Basis of Local Anesthesia

描述(由申请人提供)：钠(Na)通道是可兴奋细胞中的基本信号分子，也是局部麻醉剂(LA)的主要分子靶标。随着钠通道改变其构象状态，或“门”响应于膜去极化，药物-受体相互作用的强度增加~100倍，1这种现象被称为“使用依赖”。2尽管使用依赖是LA药物广泛疗效的基础，在引起细胞应激的病理生理条件下，这些状态依赖的结合相互作用也可能引起威胁生命的副作用，如心律失常或癫痫。3，4该计划支持的研究表明，缓慢失活，一种与持续或频繁的细胞去极化相关的门控构象状态，在促进使用依赖的LA作用方面发挥关键作用。然而，要将这种理解转化为避免毒副作用的策略，需要更好地理解细胞应激、钠通道门控和LA作用之间的三向机制联系。当神经细胞或心肌细胞受到病理生理应激，如缺血或创伤时，细胞内游离钙离子升高。我们已经确定了细胞内钙离子能够调节电压门控钠通道(NAYL)失活门控功能的两种机制，而且这两种机制都涉及C末端的区域。这些包括钙离子与EF-Hand结构域6的结合以及钙离子调节的钙调蛋白(CaM)与“IQ”基序的结合。7同时，我们的初步数据表明，提高细胞内游离钙离子通过增加缓慢的失活来增强使用依赖的LA作用。此外，我们发现EF-Hand或IQ基序的突变改变了这一钙离子调节的LA作用。相关研究表明，AYL C-末端IQ基序与至少两个具有潜在失活作用的Na通道胞质结构域直接相互作用：N-末端CaM结合区8和III-Fv结构域间连接子。9结合核磁共振波谱、定点突变和膜片钳电生理学等研究方法，我们将研究钙信号与LA作用之间的机制相互作用。特别是，我们将测试这一假设，即细胞内钙离子、CaM和LA试剂具有共同的结合作用，涉及钠通道C末端，这些结合作用在缓慢失活门控和依赖使用的LA作用过程中机械耦合。

DESCRIPTION (provided by applicant): Sodium (Na) channels are fundamental signaling molecules in excitable cells, and are principal molecular targets for the local anesthetic (LA) agents. As Na channels change their conformational state, or "gate" in response to membrane depolarization, the intensity of the drug-receptor interaction is increased ~ 100-fold,1 a phenomenon termed "use dependence."2 While use dependence underlies the broad therapeutic efficacy of LA agents, under pathophysiologic conditions causing cellular stress, these state-dependent binding interactions may also provoke life-threatening side effects, such as cardiac arrhythmias or seizures.3,4 Studies supported by this program have shown that slow inactivation, a gated conformational state associated with sustained or frequent cellular depolarization, plays a critical role in facilitating use-dependent LA action. However, translating this understanding into strategies to avoid toxic side effects requires an improved understanding of the three-way mechanistic linkage between cellular stress, Na channel gating, and LA action. Intracellular free Ca2+ rises when neuronal or cardiac cells are subjected to patholphysiologic stress, such as ischemia or traumatic injury. We have identified two mechanisms whereby intracellular Ca2+ is capable of modulating voltage-gated Na channel (Nayl) inactivation gating function, and both involve regions within the C-terminus. These include Ca2+ binding to an EF-hand domain6 and Ca2+-regulated binding of calmodulin (CaM) to an "IQ" motif.7 At the same time, our preliminary data show that raising intracellular free Ca2+ enhances use-dependent LA action by increasing slow inactivation. Further, we find that mutation of either the EF-hand or the IQ motif alters this Ca2+-regulated LA action. Related studies have shown the ayl C-terminal IQ motif interacts directly with at least two other Na channel cytoplasmic domains with potential roles in inactivation: an N-terminal CaM-binding region8 and the III-FV interdomain linker.9 Using a combination of methods informing structure and function, including NMR spectroscopy, site-directed mutagenesis, and patch-clamp electrophysiology of wild-type and mutated Na channels, we will examine the mechanistic interactions between Ca2+ signaling and LA action. In particular, we will test the hypothesis that intracellular Ca2+, CaM, and LA agents have shared binding interactions involving the Na channel C-terminus that are mechanistically coupled during slow inactivation gating and use-dependent LA action.