Molecular Basis of Local Anesthesia

描述（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（LA）药物的主要分子靶点。当钠离子通道改变其构象状态或“门”以响应膜去极化时，药物受体相互作用的强度增加了约100倍，这种现象称为“使用依赖”。虽然使用依赖性是LA药物广泛治疗效果的基础，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，如心律失常或癫痫发作该计划支持的研究表明，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作用中起着关键作用。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作用之间的三方机制联系。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Ca2+升高。我们已经确定了细胞内Ca2+能够调节电压门控Na通道（Nayl）失活门控功能的两种机制，并且都涉及c端内的区域。这包括Ca2+与EF-hand结构域的结合以及Ca2+调节的钙调蛋白（CaM）与“IQ”基元的结合同时，我们的初步数据表明，提高细胞内游离Ca2+通过增加缓慢失活来增强使用依赖性的LA作用。此外，我们发现EF-hand或IQ基序的突变都会改变Ca2+调节的LA作用。相关研究表明，ayl c端IQ基序与至少两个其他Na通道细胞质结构域直接相互作用，并可能在失活中发挥作用：n端cam结合区8和III-FV结构域间连接器9利用结构和功能方面的综合研究方法，包括核磁共振波谱、定点诱变和野生型和突变Na通道的膜片钳电生理学，我们将研究Ca2+信号和LA作用之间的机制相互作用。特别是，我们将测试细胞内Ca2+， CaM和LA剂在Na通道c端有共同的结合相互作用的假设，这些相互作用在缓慢失活门控和使用依赖的LA作用过程中是机械耦合的。

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.