Local Anesthetic Interactions with Membranes

局麻药(LAS)为了达到药理作用的目的，与膜发生了密切的相互作用。钠通道和其他神经元蛋白的阻断似乎发生在穿透质膜的药物分子必须到达的位置。此外，对于周围神经阻滞，LAS必须穿透组成周围神经外膜的鞘膜，才能到达实际的神经细胞。因此，LAS的有效性和安全性都取决于其透膜能力。在这项应用中，我们致力于确定决定LA的各种因素的长期目标：膜相互作用，从而了解药物通过双层膜的渗透机制。五个特定的目标构成了实验方法的框架：1.测量LAS的平衡膜分配和膜渗透性；2.测量LAS在模拟膜区域的溶剂中的分子内成键；3.测量膜的“流动性”及其被LAS修饰。4.关联AIMS 1-3中的测量，以确定LA的疏水性、电荷、偶极矩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、偶极电势和生物(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.将对哺乳动物上皮膜测量AIMS 1-3中使用的LAS的渗透性和平衡结合，其中转运更类似于药物通过包裹神经组织的屏障。新的荧光光谱方法将被应用于测定分配系数、中性和带电LAS的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，使用已知被LAS拮抗的配体结合反应猝灭的蛋白质的荧光，将被并入由相同膜制成的囊泡中，用于连续检测LA内流并计算相应的渗透性。虽然这项提案的直接目标是了解乳酸-细胞膜的相互作用，但该理论和方法也可以应用于许多其他与细胞膜相互作用和渗透的药物。

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.