Mindell J A, Silverman J A, Collier R J, Finkelstein A
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
J Membr Biol. 1994 Jan;137(1):45-57. doi: 10.1007/BF00234997.
The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membrane depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.
白喉毒素（DT）在脂质双分子层膜中形成的通道的电导率强烈依赖于pH值。我们之前已经表明，该蛋白的一个61个氨基酸的区域，记为TH8-9，足以形成具有与全毒素通道相同的pH依赖性电导率特性的通道。该区域中的一个残基，天冬氨酸352，决定了单通道电导率对跨膜pH值的所有依赖性，而另一个残基，谷氨酸349，则没有影响。在此，我们报告，在TH8-9区域其余的七个带电荷残基中，改变H322、H323、H372和R377电荷的突变对单通道电导率的影响最小；然而，谷氨酸326、327或362的突变则显著影响单通道电导率及其对顺式pH值的依赖性。此外，谷氨酸362可从膜的顺式和反式两侧滴定，表明该残基位于通道内；然而，顺式质子比反式质子更容易接近它。这些结果与之前提出的TH8-9区域的跨膜拓扑结构一致，并提出了一个DT通道的几何模型。
