PHYSICS OF IONIC CHANNELS AND OTHER PROTEINS WITH AQUEOUS CAVITIES

研究了S. Aureus α-毒素通道用“噪音”检测 在几种不同的pH下进行分析以测量质子的动力学 结合到通道内的可电离位点。 认识到这 结合遵循一级动力学，并注意到质子结合 在沟道电导中产生阶跃变化， 负责任的可电离位点具有5.5的PK， 解离速率常数分别为8 × 10 ~（-9）和10 ~（-5）。 这些 这些值意味着可电离的残基可自由地接近 水相和活性基团是组氨酸，谷氨酸 酸或天冬氨酸。 通过将离子通道暴露于不同浓度的中性聚乙二醇， 分子量，我们已经能够推广水的使用- 可溶性聚合物来探测通道结构。 排除的大溶质 膜浴溶液产生一种渗透功， 不同的电导状态，功正比于 这些状态的聚合物不可接近的水空间。逐步 较小的聚合物开始渗透通道空间， 降低沟道电导并施加相应降低的 有效渗透压 我们发现，渠道，一种 最终的分子筛，似乎排除聚合物的基础上， 它们的旋转体积从回转半径计算（而不是 通常用于测量通道的聚合物横截面或直径 尺寸）。 在丙甲霉素的一个特殊情况下，聚合物探测揭示了一个 每个连续步骤的固定（约3，000 A-3）水体积变化 在五种不同的导电状态之间。 因此，这些国家是 电导率几乎相同的导电单元的集合， 比半径逐渐变大的孔更大。

The kinetic of S. Aureus alpha-toxin channels were examined by "noise" analysis at several different pH's to measure the kinetics of proton binding to ionizable sites within the channel. Recognizing that this binding follows first-order kinetics, and noting that proton binding creates a step change in channel conductance one sees that the responsible ionizable sites have a pk of 5.5 and that the association and dissociation rate constants are 8x10-9 and 10-5 respectively. These values imply that the ionizable residues are freely accessible to the aqueous phase and that the active groups are either histidines, glutamic acids or aspartic acids. By exposing ionic channels to neutral polyethyleneglycols of different molecular weight, we have been able to generalize the use of water- soluble polymers to probe channel structure. Large excluded solutes in membrane- bathing solutions create an osmotic work of transition between different conductance states, a work proportional to the difference in polymer-inaccessible aqueous spaces of these states. Progressively smaller polymers begin to penetrate the channel space, to cause a decrease in channel conductance and to exert a correspondingly reduced effective osmotic pressure. We find that the channel, a kind of ultimate molecular sieve, appears to exclude polymers on the basis of their rotational volume calculated from radius of gyration (rather than the polymer cross-section or diameter typically used to gauge channel size). In a particular case of alamethicin, polymer probing reveals a fixed (about 3,000 A-3) aqueous volume change with each successive step between the five different conductance states. These states, then, are collections of conducting units of nearly the same conductance rather than pores of successively larger radius.