PHYSICS OF IONIC CHANNELS AND OTHER PROTEINS WITH AQUEOUS CAVITIES

这项工作的主题是，蛋白质，特别是那些设计 通过通道运输离子，对渗透压敏感 因为这些物质无法进入它们的水腔 我们有 利用施加渗透压的能力来测量 当蛋白质，特别是蛋白质， 跨膜离子通道打开或关闭。 在我们过去一年的成就中，我们确定 大约有20到40个水分子进入一个典型的钾 通道的鱿鱼巨大轴突时，它打开下电 刺激. 这些测量，或整个轴突的准备， 表明蛋白质结构中相当重要的电荷必须 在通道开放期间发生。 如此大的变化， 不同于通常想象的通道“门控”。 我们开发了一种分离囊泡的新方法 包含单个通道，并用于将这些通道并入 人工系统 这一发展使更多的控制 蛋白质重组到人工双层膜中， 单通道研究 在设计计算机方面已成功地投入了大量精力 硬件和软件允许高速数据采集， 记录通道电活动，同时分析 结果和把实验条件的变化， 计算机控制 该系统是个人计算机的一个原型 实验室使用，也使得更好的校正成为可能， 大型机设备，用于更复杂的数据分析（如需要） 是. 最后，我们开始研究酶活性的敏感性 渗透压，看看这种压力，自然发生在 活细胞，是细胞内活动的一部分。

The theme of this work is that proteins, especially those designed to transport ions across channels, are sensitive to osmotic stress from materials unable to enter their aqueous cavities. We have used the ability to exert osmotic stress to measure the amount of water that goes in or comes out when a protein, particularly a trans-membrane ionic channel opens or closes. Among our accomplishments during the past year, we have determined that some 20 to 40 water molecules enter a typical potassium channel of the squid giant axon when it opens under electrical stimulation. These measurements, or whole axon preparations, suggest that rather significant charges in protein structure must occur during channel opening. Such large changes are quite different from what is usually imagined to be channel "gating". We have developed a new method for separating out vesicles containing single channels and for incorporating these into artificial systems. This development is enabling more controlled protein reconstitution into artificial bilayer membranes for single-channel study. Major effort has been successfully expended in designing computer hardware and software to allow high-speed data acquisition while recording channel electrical activity, concurrently analyzing results and putting the variation of experimental conditions under computer control. This system, a prototype for personel computer laboratory use, also makes possible better correction with powerful mainframe equipment for more sophisticated data analyses if need be. Finally we have begun to look at the sensitivity of enzyme activity to osmotic stress to see how such stress, naturally occurring in living cells, is a part of intracellular activity.