[论文解读] Pore lifetimes in cell electroporation: Complex dark pores?

James C. Weaver, P. Thomas Vernier|arXiv (Cornell University)|Aug 24, 2017

Microbial Inactivation Methods参考文献 18被引用 11

一句话总结

该论文挑战了电穿孔中瞬态纯脂质孔道的长期模型，提出此类孔道在脉冲后约2 μs内即告消失，依据是分子动力学模拟结果。论文主张，持久存在的复杂孔道（由脂质及其他分子组成）才是导致脉冲后离子与分子持续运输的原因，尽管这些孔道‘不可见’（结构未知），并提供了实验证据支持其存在，其寿命可达数百秒。

ABSTRACT

We review some of the basic concepts and the possible pore structures associated with electroporation (EP) for times after electrical pulsing. We purposefully give only a short description of pore creation and subsequent evolution of pore populations, as these are adequately discussed in both reviews and original research reports. In contrast, post-pulse pore concepts have changed dramatically. For perspective we note that pores are not directly observed. Instead understanding of pores is based on inference from experiments and, increasingly, molecular dynamics (MD) simulations. In the past decade concepts for post-pulse pores have changed significantly: The idea of pure lipidic transient pores (TPs) that exist for milliseconds or longer post-pulse has become inconsistent with MD results, which support TP lifetimes of only $\sim$100 ns. A typical large TP number during cell EP pulsing is of order $10^6$. In twenty MD-based TP lifetimes (2 us total), the TP number plummets to $\sim$0.001. In short, TPs vanish 2 us after a pulse ends, and cannot account for post-pulse behavior such as large and relatively non-specific ionic and molecular transport. Instead, an early conjecture of complex pores (CPs) with both lipidic and other molecule should be taken seriously. Indeed, in the past decade several experiments provide partial support for complex pores (CPs). Presently, CPs are "dark", in the sense that while some CP functions are known, little is known about their structure(s). There may be a wide range of lifetimes and permeabilities, not yet revealed by experiments. Like cosmology's dark matter, these unseen pores present us with an outstanding problem.

研究动机与目标

• 基于现代分子动力学（MD）模拟，重新评估电穿孔后脂质孔道的持久性。
• 解决实验观测到的长期后脉冲通透性与MD模拟预测的纯脂质孔道短寿命（约100 ns）之间的矛盾。
• 提出复杂孔道（由脂质和其他分子组成）可能构成持久通透性的基础，尽管其结构特征尚未被直接表征。
• 综合实验与模拟证据，表明存在寿命长、具有功能活性的孔道，且并非纯脂质构成。

提出的方法

• 分析分子动力学（MD）模拟结果，显示孔道能量景观中无显著破坏能垒，暗示孔道迅速塌陷。
• 比较MD预测的瞬态孔道（TP）寿命（约100 ns）与实验测得的后脉冲恢复时间（长达180 s），识别其中的不一致之处。
• 综合平面双分子层、细胞及脉冲序列实验的实验数据，显示通透性延迟增加及数秒内重新封闭的现象。
• 利用电导率、离子通量（Ca²⁺、丙啶橙）及钙黑荧光素外排测量，推断脉冲后孔道行为。
• 分析脉冲间隔为0.1 s的脉冲序列实验，推断孔道的恢复与再形成过程。
• 解读电生理与溶质转运数据，推断存在寿命长、非扩散性运输通路。

实验结果

研究问题

• RQ1 为何实验测量显示电穿孔后膜通透性可持续数秒，而MD模拟却预测纯脂质瞬态孔道的寿命仅为约100 ns？
• RQ2 何种结构与能量特征可使孔道持续存在，超越纯脂质瞬态孔道约2 μs的预测寿命？
• RQ3 如何调和实验观测到的重新封闭时间尺度（长达180 s）与MD模拟中未检测到可检测脂质孔道的现象？
• RQ4 有哪些证据支持非脂质性复杂孔道结构的存在，这些结构可能介导长期通透性？
• RQ5 主动运输机制或电泳效应在多大程度上贡献于后脉冲溶质转运，表明存在稳定的孔道样实体？

主要发现

• 分子动力学模拟显示，纯脂质瞬态孔道无显著自由能破坏屏障，导致其迅速塌陷，预测寿命约为100 ns。
• 估算约10⁶个瞬态孔道群体衰减至约0.001个所需总时间为约2 μs，表明脉冲后数微秒内即完全消失。
• 实验测得的膜重新封闭与恢复时间尺度范围为0.01 s至180 s，远超纯脂质孔道的预测寿命。
• 脉冲序列实验中，脉冲间隔为0.1 s时，可形成新孔道，表明孔道数量与膜电导率在100 ms内显著恢复。
• 在脉冲后100–1800 s的时间段内观察到通透性延迟增加，表明存在寿命长、非扩散性运输通路。
• 在无电场条件下溶质转运的证据表明，扩散与电泳共同作用于运输过程，暗示稳定、持久的孔道样结构在瞬态孔道寿命之外仍持续存在。