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，是局部麻醉剂（LA）的主要分子靶点。随着Na通道改变其构象状态，或响应膜去极化的“门”，药物-受体相互作用的强度增加约100倍，1这种现象称为“使用依赖性”。“2虽然使用依赖性是LA药物广泛疗效的基础，但在引起细胞应激的病理生理条件下，这些状态依赖性结合相互作用也可能引起危及生命的副作用，如心律失常或心律失常。3，4该项目支持的研究表明，缓慢失活，与持续或频繁的细胞去极化相关的门控构象状态，在促进使用依赖性LA作用方面发挥关键作用。然而，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、Na通道门控和LA作用之间的三向机制联系。当神经元或心脏细胞受到病理生理应激，如缺血或创伤性损伤时，细胞内游离Ca 2+升高。我们已经确定了两种机制，细胞内Ca 2+能够调节电压门控Na通道（Nayl）失活门控功能，都涉及C-末端内的区域。这些包括Ca 2+与EF-手结构域的结合6和钙调蛋白（CaM）与“IQ”基序的Ca 2+调节结合。7同时，我们的初步数据表明，提高细胞内游离Ca 2+通过增加缓慢失活增强了使用依赖性LA作用。此外，我们发现EF-手或IQ基序的突变改变了这种钙离子调节的LA行动。相关研究表明，ayl C-末端IQ基序直接与至少两个其他Na通道胞质结构域相互作用，在失活中具有潜在作用：N-末端CaM结合区8和III-FV结构域间接头9。使用告知结构和功能的方法的组合，包括野生型和突变的Na通道的NMR光谱、定点诱变和膜片钳电生理学，我们将研究Ca 2+信号传导和LA作用之间的机械相互作用。特别是，我们将测试的假设，细胞内钙离子，钙调素，和LA剂有共同的结合相互作用，涉及钠通道C-末端的机械耦合过程中缓慢失活门控和使用依赖性LA行动。

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.