MOLECULAR BASIS OF LOCAL ANESTHESIA

描述：(改编自申请人的摘要)该提案旨在 探讨局麻药对钠通道的作用机制 特别提到局麻药的抗心律失常特性 缺血发作后心肌钠通道的变化。这个 拟议研究的中心假设是局部麻醉剂 通过改变偶联来稳定钠通道的失活状态 在通道激活和失活之间，起变构作用 通道门控的效应器。这一假说与“调制”不同 “受体”假说在过去二十年中颇具影响力； 后者提出失活状态对局部具有更高的亲和力 麻醉剂比静息状态更好。策略是利用突变 修改快速失活和缓慢失活来检查分子决定因素 使用标准电生理电压的局部麻醉特性 钳制技术研究非洲爪哇卵母细胞或非洲爪哇卵母细胞中表达的通道 一种哺乳动物肾脏细胞系。它有3个具体目标：使用4种类型的 通过以下途径破坏大鼠骨骼肌快速失活的突变 不同的机制，将进行研究，以确定每种类型 改变依赖时间和电压的局部麻醉效果。突变 位于III-IV结构域间连接物上的疏水三联体(IFM)中 可能是失活的闩锁；结构域III胞质末端的残基 S4-S5和IV-S6可能是失活受体； IFM侧边的连接子；以及影响IV域的外部残基 激活-停用。不同大小、不同电荷和不同种类氨基酸 402位的疏水性将被取代，以确定如何 它们的性质会影响慢灭活与其他 门控过程，以及这种改变如何改变局部麻醉作用。 两个点突变，W402C，结构域I的色氨酸到半胱氨酸和T698M 域II，将用于研究以确定耦合相互作用如何 不同的门控过程会影响局部麻醉作用的差异 骨骼肌和心肌钠通道。这些突变都会改变 骨骼肌通道缓慢失活，并改变快速门控 通过不同的机制进行处理。

DESCRIPTION: (Adapted from the applicant's abstract) The proposal is to examine mechanisms of local anesthetic action on sodium channels with particular reference to the antiarrhythmic properties of local anesthetics in cardiac muscle sodium channels following an ischemic episode. The central hypothesis of the proposed studies is that local anesthetics stabilize inactivated states of the sodium channel by altering the coupling between channel activation and inactivation, functioning as allosteric effectors of channel gating. This hypothesis differs from the "modulated receptor" hypothesis which has been influential in the past two decades; the latter proposes that the inactivated state has a higher affinity for local anesthetics than the resting state. The strategy is to employ mutations that modify fast and slow inactivation to examine the molecular determinants of local anesthetic properties, using standard electrophysiologic voltage clamp techniques to study channels expressed either in Xenopus oocytes or in a mammalian kidney cell line. There are 3 specific aims: Using 4 types of mutations which disrupt fast inactivation in rat skeletal muscle by different mechanisms, studies will be carried out to determine how each type alters time- and voltage-dependent local anesthetic effects. The mutations are in the hydrophobic triplet (IFM) on the III-IV interdomain linker that may be the inactivation latch; residues on the cytoplasmic end of domain III S4-S5 and IV-S6 that may be the inactivation receptor; hinge residues of the linker lateral to IFM; and external residues in domain IV that influence activation-inactivation. Amino acids of different size, charge and hydrophobicity will be substituted at the 402 position to determine how their properties influence coupling between slow inactivation and other gating processes, and how such modifications alter local anesthetic action. Two point mutations, W402C, tryptophan to cysteine in domain I and T698M in domain II, will be used in studies to determine how coupling interactions among gating processes influence differences between local anesthetic action on skeletal muscle and cardiac sodium channels. These mutations both modify slow inactivation in skeletal muscle channels, and alter fast gating processes via different mechanisms.