PHYSICS OF IONIC CHANNELS AND OTHER PROTEINS WITH AQUEOUS CAVITIES

这项工作的主题是蛋白质，特别是那些设计的蛋白质 通过通道运输离子，对渗透胁迫很敏感 来自无法进入其水腔的物质。我们有 利用施加渗透胁迫的能力来测量 当蛋白质进入或流出的水，尤指 跨膜离子通道打开或关闭。 在过去一年取得的成就中，我们确定 大约有20到40个水分子进入一个典型的钾 乌贼巨型轴突在电刺激下打开时的通道 刺激。这些测量，或者说整个轴突准备， 表明蛋白质结构中相当大的电荷必须 发生在通道打开期间。这么大的变化是相当大的 不同于通常想象中的频道“门控”。 我们开发了一种分离水泡的新方法。 包含单个通道，并将这些通道合并到 人工系统。这一发展使更多的受控 蛋白质在人工双层膜中的重组 单通道研究。 在设计计算机方面已经成功地投入了大量的精力 支持高速数据采集的硬件和软件 记录通道电活动，并发分析 结果，并将实验条件的变化放在 电脑控制。该系统是个人计算机的一个原型 实验室使用，也使更好的校正成为可能 大型机设备，用于在需要时进行更复杂的数据分析 是.。 最后，我们已经开始研究酶活性的敏感性。 以渗透胁迫来观察这种胁迫是如何自然发生在 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.