Department of Chemistry , University of Pittsburgh , 219 Parkman Avenue , Pittsburgh , Pennsylvania 15260 , United States.
Center for Environmentally Beneficial Catalysis, Department of Chemical and Petroleum Engineering , University of Kansas , 1501 Wakarusa Drive , Lawrence , Kansas 66047 , United States.
Anal Chem. 2019 Apr 16;91(8):5446-5454. doi: 10.1021/acs.analchem.9b00796. Epub 2019 Apr 3.
The nuclear pore complex (NPC) solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell to play important biological and biomedical roles. However, it is not well-understood chemically how this biological nanopore selectively and efficiently transports various substances, including small molecules, proteins, and RNAs by using transport barriers that are rich in highly disordered repeats of hydrophobic phenylalanine and glycine intermingled with charged amino acids. Herein, we employ scanning electrochemical microscopy to image and measure the high permeability of NPCs to small redox molecules. The effective medium theory demonstrates that the measured permeability is controlled by diffusional translocation of probe molecules through water-filled nanopores without steric or electrostatic hindrance from hydrophobic or charged regions of transport barriers, respectively. However, the permeability of NPCs is reduced by a low millimolar concentration of Ca, which can interact with anionic regions of transport barriers to alter their spatial distributions within the nanopore. We employ atomic force microscopy to confirm that transport barriers of NPCs are dominantly recessed (∼80%) or entangled (∼20%) at the high Ca level in contrast to authentic populations of entangled (∼50%), recessed (∼25%), and "plugged" (∼25%) conformations at a physiological Ca level of submicromolar. We propose a model for synchronized Ca effects on the conformation and permeability of NPCs, where transport barriers are viscosified to lower permeability. Significantly, this result supports a hypothesis that the functional structure of transport barriers is maintained not only by their hydrophobic regions, but also by charged regions.
核孔复合体（NPC）仅介导真核细胞的核质和细胞质之间的分子运输，以发挥重要的生物学和生物医学作用。然而，人们还不太清楚这种生物纳米孔如何通过富含高度无序重复的疏水性苯丙氨酸和甘氨酸与带电氨基酸的运输屏障，选择性和有效地运输各种物质，包括小分子、蛋白质和 RNA。在此，我们采用扫描电化学显微镜来成像和测量 NPC 对小分子氧化还原探针的高渗透性。有效介质理论表明，测量的渗透性受探针分子通过充满水的纳米孔扩散易位控制，而不会受到运输屏障的疏水区或带电区的空间或静电阻碍。然而，NPC 的渗透性会被低毫摩尔浓度的 Ca 降低，Ca 可以与运输屏障的阴离子区域相互作用，从而改变其在纳米孔内的空间分布。我们采用原子力显微镜证实，与生理 Ca 亚毫摩尔浓度下真实存在的约 50%纠缠、25%凹陷和 25%“堵塞”构象相比，NPC 的运输屏障在高 Ca 水平下主要是凹陷（约 80%）或纠缠（约 20%）。我们提出了一个关于 Ca 对 NPC 构象和渗透性同步影响的模型，其中运输屏障的粘性增加会降低渗透性。重要的是，该结果支持了这样一个假设，即运输屏障的功能结构不仅由其疏水区维持，而且由带电区维持。
