LIPOSOME-PATCH RECORDING O RECONSTITUTED ION CHANNELS

本提案的目的是研究 几种离子通道蛋白的分子结构和功能行为 电活性细胞。 三个离子通道将成为 研究：哺乳动物肌肉Ca++激活的K+通道（CaK）， 神经，来自电鳐的电压依赖性Cl-通道，以及 来自哺乳动物肌肉的可兴奋膜Na+通道。 单通道 分析将适用于所有三个渠道，这将是所有研究 在由脂质和纯化膜重构的模型膜中， 蛋白质级分。 所有提出的实验旨在解决 观察到的单通道的潜在分子机制的问题 行为。 具体地说，我建议纯化CaK通道蛋白， 活性形式，使用一种新发现的蝎子毒素针对这一点 频道 此外，Ca++激活通道的机制 将进行研究，因为将修改这一机制的渠道， 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Cl-通量，以检测脂质体中Cl-通道的存在。 鱼雷无神经支配的面部膜的洗涤剂溶解提取物。 这里的目标是执行隔离此 蛋白 最后，讨论了Na+通道与细胞膜的相互作用机制。 生物碱毒素，如藜芦碱和蟾毒，将在 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.