Department of Mechanical Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, TN 37996, USA.
Department of Mechanical Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, TN 37996, USA.
Biochim Biophys Acta Biomembr. 2024 Dec;1866(8):184386. doi: 10.1016/j.bbamem.2024.184386. Epub 2024 Sep 27.
We show that voltage alone can inactivate alamethicin channels, which has been previously observed for monazomycin and suzukacillin channels. The voltage required to trigger inactivation is above the potential to form channels, and, like with channel activation, this threshold reduces with increasing peptide concentration and membrane fluidity. Since similar monazomycin channels inactivate via channel break up and translocation, we hypothesized that inactivation of alamethicin channels occurs via the same mechanism. Our data prove this hypothesis to be true through two experiments. First, we show that inactivation of channels at positive voltages when peptides are supplied to only the cis side correlates to new channel activity on the trans side at negative potentials. This result indicates translocation of alamethicin peptides occurs only during voltage-induced inactivation. Second, we measured the ratio of steady-state (with inactivation) to ideal (without inactivation) conductance versus voltage for membranes with equal amounts of alamethicin on both sides and used these values to quantify alamethicin flux. Plotting flux versus steady-state conductance across multiple alamethicin concentrations shows a single linear dependence, signifying that translocated peptides originate from active channels that break up under prolonged voltage. Given the frequent use of alamethicin as model ion channels, these results add important understanding of their kinetic responses when subjected to prolonged, high voltages.
我们证明，电压本身可以使 alamethicin 通道失活，这在 monazomycin 和 suzukacillin 通道中已有先前观察。引发失活所需的电压高于形成通道的电位，并且与通道激活一样，该阈值随着肽浓度和膜流动性的增加而降低。由于类似的 monazomycin 通道通过通道破裂和易位失活，我们假设 alamethicin 通道的失活通过相同的机制发生。我们的数据通过两个实验证明了这一假设是正确的。首先，我们表明，当仅在 cis 侧提供肽时，在正电压下失活的通道与在负电位下 trans 侧的新通道活性相关。这一结果表明，alamethicin 肽的易位仅发生在电压诱导的失活过程中。其次，我们测量了两侧具有等量 alamethicin 的膜的稳态（具有失活）与理想（无失活）电导与电压的比值，并使用这些值来量化 alamethicin 通量。在多个 alamethicin 浓度下绘制通量与稳态电导的关系图显示出单一的线性依赖性，表明迁移的肽来自在延长的电压下破裂的活性通道。鉴于 alamethicin 经常被用作模型离子通道，这些结果增加了对它们在长时间、高电压下的动力学反应的重要理解。
