BASIC MECHANISMS OF ION CHANNEL FUNCTION

这项建议旨在继续就基本机制开展工作， 离子通道蛋白的工作原理，以及它们在细胞内转运离子的能力。 门控方式与它们的基本分子结构有关。 的 这项研究的重点是三种不同的离子通道：高， 电导Ca 2+激活的哺乳动物肌肉K+通道，A型K+ 通道编码的Shaker基因的果蝇，和电压门控的Cl- 加州鱼雷电风琴的频道。 我们将探索 Ca 2+激活的K+通道的传导孔重构成平面 双层膜，通过检查Ba 2+离子的阻断和相互作用 与肽Charybdotoxin，和定点突变， 肽。 我们将尝试纯化和重组Shaker K+通道 在杆状病毒感染的昆虫细胞系中高水平表达后， 作为首次尝试进行蛋白质水平的生化分析， 频道 最后，我们将完成鱼雷Cl-的纯化， 通道，以启动一个突出的成员的分子分析， 电压依赖性阴离子通道家族。 的公众利益 该项目位于离子通道蛋白的中心位置， 电信号的过程。研究人员现在才刚刚开始 一窥这些重要蛋白质的分子结构， 项目将有助于这一努力。

This proposal seeks to continue work on the fundamental mechanisms by which ion channel proteins work, and on how their ability to transport ions in a gated fashion is related to their underlying molecular structures. The focus of this research is on three different ion channels: the high- conductance Ca2+-activated K+ channel from mammalian muscle, the A-type K+ channel encoded by the Shaker gene of Drosophila, and the voltage-gated Cl- channel from the electric organ of Torpedo californica. We will probe the conduction pore of the Ca2+-activated K+ channel reconstituted into planar bilayer membranes by examining blocking by Ba2+ ion and by the interaction with the peptide charybdotoxin, and of site-directed mutants of this peptide. We will attempt to purify and reconstitute the Shaker K+ channel after high-level expression in the baculovirus-infected insect cell line, as a first attempt to carry out protein-level biochemical analysis of this channel. Finally, we will complete the purification of the Torpedo Cl- channel in order to launch a molecular analysis of a prominent member of the family of voltage-dependent anion channels. The general interest in this project lies in the central place of ion channel proteins in the process of electrical signalling. Researchers are now just beginning to glimpse the molecular architecture of these important proteins, and this project will contribute to that effort.