Mindell J A, Zhan H, Huynh P D, Collier R J, Finkelstein A
Department of Physiology, Albert Einstein College of Medicine, Bronx, NY 10461.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5272-6. doi: 10.1073/pnas.91.12.5272.
The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two alpha-helices, lying within the membrane, connected by a short interhelical loop of four amino acids (residues 349-352). To investigate the validity and implications of this model, we singly mutated each of these amino acids to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and added to the trans compartment sulfhydryl-specific reagents [methanethiosulfonate derivatives (MTS-ER)] that introduce a positive or negative charge to reacted cysteines. The introduction of a positive charge at residue 351 or 352 (through the MTS-ER reactions) resulted in a step decrease in single-channel conductance, whereas the introduction of a negative charge resulted in a step increase. The opposite sign of these effects indicates the predominantly electrostatic nature of the phenomenon and implies that residues 351 and 352 lie close to the channel entrance. The same reactions at residue 350 resulted in very little change in channel conductance but instead changed the character of the natural rapid flickering of the channel between open and closed states to one in which the channel spent more time in the closed state; this may have resulted from the group introduced at position 350 acting as a tethered channel blocker. The MTS derivatives had no effect on channels containing a cysteine at position 349, suggesting that this residue faces away from the channel entrance. We propose that the step changes in conductance or flickering pattern result from the chemical reaction of one MTS-ER molecule with one cysteine, and thus a bimolecular chemical reaction is being witnessed at the single molecule level. From the distribution of waiting times between the appearance (i.e., the opening) of a channel and the step change in its conductance or flickering pattern, we can calculate a pseudo-first-order rate constant, which can then be converted to a second-order rate constant, for the chemical reaction.
白喉毒素通道被认为是其T结构域的同型寡聚体，其中每个亚基由两个α螺旋组成，位于膜内，通过四个氨基酸的短螺旋间环（残基349 - 352）相连。为了研究该模型的有效性和意义，我们将这些氨基酸逐一突变为半胱氨酸，在平面脂质双分子层中用突变的T结构域蛋白形成通道，并向跨膜区添加对巯基具有特异性的试剂[甲硫基磺酸盐衍生物（MTS - ER）]，这些试剂会给反应的半胱氨酸引入正电荷或负电荷。在残基351或352处引入正电荷（通过MTS - ER反应）导致单通道电导呈阶梯式下降，而引入负电荷则导致阶梯式增加。这些效应的相反符号表明该现象主要是静电性质的，并意味着残基351和352靠近通道入口。在残基350处进行相同反应导致通道电导变化很小，但却将通道在开放和关闭状态之间自然的快速闪烁特性改变为通道在关闭状态花费更多时间的特性；这可能是由于在350位引入的基团起到了拴系通道阻滞剂的作用。MTS衍生物对在位置349含有半胱氨酸的通道没有影响，这表明该残基面向远离通道入口的方向。我们提出，电导或闪烁模式的阶梯式变化是由一个MTS - ER分子与一个半胱氨酸的化学反应引起的，因此在单分子水平上见证了一个双分子化学反应。根据通道出现（即开放）与其电导或闪烁模式的阶梯式变化之间的等待时间分布，我们可以计算出一个伪一级速率常数，然后将其转换为该化学反应的二级速率常数。
