VOLTAGE DEPENDENT SODIUM CHANNEL--PLANAR LIPID BILAYER

这项拟议研究的长期目标是了解 离子运动的分子特征 跨膜通道。这一目标将通过 电压依赖性钠通道的研究 掺入到规定组成的平面脂质双层中。离子 渗透、药理修饰和电压激活 (GATING)，将被审查，特别强调 胞外和胞内表面的固定电荷 蛋白质和宿主双层在调节通道功能中的作用。 离子进入通道的机制将被研究以 确定靠近通道入口的负电荷是否 具有引导离子进入的生理功能。离子 由巴曲霉毒素修饰的通道的通透性和堵塞， 藜芦碱、拟除虫菊酯杀虫剂和特定于组的 将对修改进行比较，以试图澄清为什么 单通道电导降低的通道具有 降低了离子选择性。 将使用基团特定的修饰和蛋白水解性切割 要修改渠道入口，并检查两者之间的关系 胍类毒素结合部位与胞外通道 入口。毒素诱导的通道关闭的动力学将是 检查以确定是否由胍类毒素诱导 通道关闭以两步事件的形式发生，其中通道 毒素后的构象变化彻底闭合 有约束力的。 单通道的静态电压激活将是 检查以确定门控行为受以下因素影响的程度 脂质表面电荷，并进一步定义不对称性 胞外和胞内的表观表面电荷密度 渠道的表面。 我们将研究影响选通的缓慢“模式改变”。这个 目的是刻画一些固定构象 在一个完整的膜蛋白中发生的波动，以及 以确定是否可以完全考虑模式更改 通过激活的中点电势的离散移位 曲线。

The long-term goal of the proposed research is to understand molecular characteristics of ion movement through transmembrane channels. This goal will be pursued through studies on voltage-dependent sodium channels that are incorporated into planar lipid bilayers of defined composition. Ion permeation, pharmacological modification, and voltage activation (gating), will be examined, with special emphasis on the role of fixed charges at the extra- and intracellular surface of the protein and host bilayer in modulating channel function. The mechanism of ion entry into the channels will be studied to determine whether negative charges close to the channel entrance have a physiological function as guides for ion entry. Ion permeability and block of channels modified by batrachotoxin, veratradine, pyrethroid insecticides, and group-specific modification will be compared in an attempt to clarify why channels that have a decreased single-channel conductance have a decreased ion selectivity. Group-specific modification and proteolytic cleavage will be used to modify the channel entrance and examine the relation between the guanidinium toxin binding site and the extracellular channel entrance. The kinetics of toxin-induced channel closures will be examined to determine whether the guanidinium toxin-induced channel closures occur as a two-step event, where the channel is closed thorough a conformational change subsequent to toxin binding. The stationary voltage-activation of single channels will be examined to define to what extent gating behavior is affected by lipid surface charges, and to further define the asymmetry in the apparent surface charge density at the extra- and intracellular surfaces of the channel. The slow "mode changes" that affect gating will be studied. The aim is to characterize some of the stationary conformational fluctuations that occur in an integral membrane protein, as well as to determine whether the mode changes can be fully accounted for by discrete shifts in the midpoint potential of the activation curves.