The Molecular Basis of Local Anesthesia

描述（来自申请人摘要的逐字描述）钠（Na）通道， 局部麻醉剂的主要靶标，改变它们的构象状态 响应于膜电位。去极化增加了 药物与受体的相互作用增加了近100倍。虽然这一现象令人担忧， 局部麻醉剂的广泛疗效，这些电压依赖性 相互作用还引起危及生命的副作用（心律失常， 癫痫发作）。阐明药物-受体相互作用的动态调节 将补充确定局部麻醉剂结合域的新数据，并将 改进这些化合物的设计。 许多不同的钠通道位点介导内在构象 变化（快速和缓慢失活）也调节局部麻醉作用， 与最初的提议（调节受体假说）一致， 局部麻醉剂结合亲和力由构象状态控制。一 推论是，局部麻醉剂，当结合时，应该诱导 Na通道占据更高亲和力的构象状态。最近的工作 揭示了内在钠通道构象变化涉及相当大的 分子运动改变了孔隙的结构我们建议 局部麻醉剂作为变构效应物发挥作用，诱导内在的 孔中的构象变化，类似于配体对变构的影响， 内切酶 我们的研究将检查门控构象特性的 含有工程化半胱氨酸残基的异源表达的Na通道 使用膜片钳方法。使用甲硫基磺酸盐试剂，我们将检查 缓慢失活如何改变氨基酸的巯基可及性 反应于局部麻醉剂结合的外孔区域中的残基。在 此外，我们将研究最早阶段与 去极化引起的阻滞和带电S4段的运动， 将这些节段的运动与外层的构象变化联系起来， 毛孔最后，我们将利用二硫键捕获方法来限制 局部麻醉阻滞引起的域间距离变化， 解释这些使用一个新的分子模型的钠通道孔。我们的研究 将阐明钠通道孔的动态结构，并将 有助于设计更有针对性和毒性更低的局部麻醉剂。

DESCRIPTION (Verbatim from the applicant's abstract) Sodium (Na) channels, the principle target of local anesthetic agents, change their conformational state in response to membrane potential. Depolarization increases the intensity of the drug-receptor interaction nearly 100-fold. While this phenomenon imderlies the broad therapeutic efficacy of local anesthetics, these voltage-dependent interactions also provoke life-threatening side effects (arrhythmias, seizures). Clarifying this dynamic modulation of the drug-receptor interaction will complement new data identifying local anesthetic binding domains, and will improve the design of these compounds. A number of distinct Na channel loci that mediate intrinsic conformational changes (fast and slow inactivation) also modulate local anesthetic action, consistent with the original proposal (the Modulated Receptor Hypothesis) that local anesthetic binding affinity is governed by conformational state. A corollary prediction is that local anesthetics, when bound, should induce the Na channel to occupy higher-affinity conformational state(s). Recent work reveals that intrinsic Na channel conformational changes involve sizeable molecular motions that modify the architecture of the pore. We propose that local anesthetics function as allosteric effectors to induce intrinsic conformational changes in the pore, analogous to ligand effects on allosteric enzymes. Our studies will examine the gating conformational properties of heterologously-expressed Na channels containing engineered cysteine residues using patch-clamp methods. Using methanethiosulfonate reagents, we will examine how slow inactivation changes the sulfhydryl accessibility of amino acid residues in the outer pore region in response to local anesthetic binding. In addition, we will examine the linkage between the earliest phase of depolarization-induced block and motion of the charged S4 segments, with a view to linking motion of these segments to conformational changes in the outer pore. Finally, we will utilize a disulfide trapping approach to constrain the interdomain distance changes induced by local anesthetic block, and will interpret these using a new molecular model of the Na channel pore. Our studies will illuminate the dynamic structure of the Na channel pore, and will facilitate the design of more targeted and less toxic local anesthetic agents.