Local Anesthetic Interactions with Membranes

局部麻醉药（LA）与膜密切相互作用，以达到其药理学目标。钠通道和其他神经元蛋白质的阻断似乎发生在药物分子必须到达的部位，这些药物分子已经渗透到和/或穿过质膜。 此外，对于周围神经阻滞，LA必须在它们到达实际神经细胞之前穿透构成周围神经的神经束膜的鞘膜。因此，LA的有效性和安全性取决于其膜穿透能力。 在本申请中，我们解决了确定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渗透的机制。 五个具体目标框架的实验方法：1。LA的平衡膜分配和膜渗透性的测量; 2.测量分子内键合的LA在溶剂中，模型膜区域; 3。测量膜的“流动性”和它的修改LAs。 4.将目标1-3中的测量结果关联起来，以确定LA疏水性、电荷、偶极矩和分子内键合对膜分配和渗透的相对贡献，以及LA：膜相互作用中膜流动性、表面电荷、胆固醇存在、偶极场电位和生物（组织）源的相对重要性。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.将针对哺乳动物上皮膜测量目的1-3中使用的LA的渗透性和平衡结合，其中转运更接近于药物穿过屏障鞘神经组织的通道。 新的荧光分光光度法将被应用于确定分配系数，膜结合的中性和带电的LA的深度，和泡囊膜吸附药物的pKa。一个新开发的报告系统，使用的荧光猝灭的配体结合反应已知被拮抗的LA的蛋白质，将被纳入囊泡由相同的膜，连续检测LA流入和计算相应的渗透性。 虽然该提议的直接目标集中于理解LA-膜相互作用，但是该理论和方法可以应用于与细胞膜相互作用并渗透细胞膜的许多其他药物。

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.