Local Anesthetic Interactions with Membranes

局部麻醉剂（LAs）与膜密切相互作用，以达到其药理目标。钠离子通道和其他神经元蛋白的阻断似乎发生在药物分子穿透和/或穿过质膜必须到达的部位。此外，对于周围神经阻滞，LAs在到达实际的神经细胞之前，必须穿透构成周围神经会神经膜的鞘膜。因此，LAs的有效性和安全性取决于其膜穿透能力。在这个应用程序中，我们解决了确定决定LA膜相互作用的各种因素的长期目标，从而了解药物通过双层膜渗透的机制。五个具体目标框架的实验方法：LAs平衡膜分配和膜透性的测定2. 模拟膜区溶剂中LAs分子内键的测量3. 膜“流动性”的测量及其LAs修饰。4. 将Aims 1-3中的测量方法联系起来，以确定LA疏水性、电荷、偶极时刻和分子内键对膜分配和渗透的相对贡献，以及膜流动性、表面电荷、胆固醇的存在、偶极场电位和生物（组织）源对LA：膜相互作用的相对重要性。LA的结构和膜的组成都将被系统地改变以操纵这些不同的因素。5. Aims 1-3中使用的LAs的渗透性和平衡结合将在哺乳动物上皮膜上进行测量，其运输更类似于药物穿过神经组织的屏障。将采用新的荧光光谱法测定中性和带电LAs的分配系数、膜结合深度以及囊泡膜吸附药物的pKa。一种新开发的报告系统，利用一种蛋白质的荧光，这种荧光被已知被LAs拮抗的配体结合反应猝灭，将被纳入由相同膜制成的囊泡中，用于连续检测LA内流并计算相应的通透性。虽然这个建议的直接目标集中在理解LA-膜相互作用，理论和方法可以应用于许多其他药物与细胞膜相互作用和渗透。

Local anesthetics (LAs) interact intimately with membranes in order to reach their pharmacological targets. Blockade of sodium channels and of other neuronal proteins appears to occur at sites that must be reached by drug molecules that have penetrated into and/or through the plasma membrane. In addition, for peripheral nerve block, LAs must penetrate the ensheathing membranes composing the peripheral nerve's perineurium, before they reach the actual nerve cells. Therefore, both the efficacy and safety of LAs depend on their membrane-penetrating abilities. In this application we address the long-term objectives of identifying the various factors that determine LA: membrane interactions and thereby understanding the mechanism of drug permeation through bilayer membranes. Five Specific Aims frame the experimental approach: 1. Measurements of equilibrium membrane partitioning and of membrane permeability of LAs; 2. Measurements of intramolecular bonding of LAs in solvents that model membrane regions; 3. Measurements of membrane "fluidity" and of its modification by LAs. 4. Correlate the the measures in Aims 1-3 in order to determine the relative contributions of LA hydrophobicity, charge, dipole moment and intramolecular bonding to membrane partitioning and to permeation, and the relative importance of the membrane fluidity, surface charge, presence of cholesterol, dipolar field potential and biological (tissue) source, for LA:membrane interactions. Both the structure of the LA and the composition of the membranes will be systematically altered to manipulate these various factors. 5. Permeability and equilibrium binding of the LAs used in Aims 1-3 will be measured for mammalian epithelial membranes, where transport more closely resembles the passage of drugs across the barriers ensheathing nerve tissue. Novel spectrofluorometric methods will be applied to determine partition coefficients, the depth of membrane binding of neutral and charged LAs, and the pKa of vesicle membrane-adsorbed drug. A newly developed reporter system, using the fluorescence of a protein that is quenched by ligand binding reactions known to be antagonized by LAs, will be incorporated within vesicles made of the same membranes, for continuous detection of LA influx and calculation of the corresponding permeability. Although the immediate goal of this proposal focuses on understanding LA- membrane interactions, the theory and the methods can be applied to many other drugs that interact with and permeate cellular membranes.