LIPOSOME-PATCH RECORDING OF RECONSTITUTED ION CHANNELS

这项建议的目的是研究 几种离子通道蛋白的分子结构和功能行为 来自电活性电池。三个离子通道将成为 哺乳动物肌肉钙激活钾通道(CAK)的研究 神经，来自鱼雷电浆的电压依赖的氯离子通道，以及 哺乳动物肌肉的兴奋性膜Na+通道。单通道 分析将应用于所有三个渠道，这三个渠道都将被研究 在由脂质重建的模型膜和纯化的膜或 蛋白质组分。所有提出的实验都是为了解决 关于观测到的单通道的潜在分子机制的问题 行为。 具体地说，我建议从功能上纯化CAK通道蛋白 活性形式，使用新发现的针对此的蝎子毒素 频道。此外，钙离子激活通道的机制 将研究这一机制，以及从 不同的哺乳动物来源。同样，我将开发一种分析方法，基于 脂质体中的氯离子通量，以检测是否存在氯离子通道 洗涤剂--鱼雷非神经性面膜的增溶提取物。 这里的目标将是执行隔离这一点的开始步骤 蛋白。最后，探讨了钠离子通道与钙离子通道的相互作用机制。 生物碱毒素，如藜芦碱和巴曲霉毒素，将在#年进行研究。 平面双层膜。

The intention of this proposal is to study the relationships between molecular structure and functional behavior of several ion channel proteins from electrically active cells. Three ion channels will be the objects of study: the Ca++-activated K+ channel (CaK) from mammalian muscle and nerve, the voltage-dependent Cl- channel from Torpedo electroplax, and the excitable membrane Na+ channel from mammalian muscle. Single-channel analysis will be applied to all three channels, which will all be studied in model membranes reconstituted from lipids and purified membrane or protein fractions. All experiments proposed are designed to address questions of the underlying molecular mechanisms of observed single-channel behaviors. Specifically, I propose to purify the CaK channel protein in a functionally active form, using a newly discovered scorpion toxin directed against this channel. In addition, the mechanism by which Ca++ activates the channel will be studied, as will modifications of this mechanism in channels from different mammalian sources. Likewise, I will develop an assay, based on Cl- fluxes in liposomes, to detect the presence of the Cl- channel in detergent-solubilized extracts of Torpedo noninnervated-face membranes. The object here will be to carry out the beginning steps for isolating this protein. Finally, the mechanisms of interaction of the Na+ channel with alkaloid toxins, such as veratridine and batrachotoxin, will be studied in planar bilayer membranes.