Single Channel Recording of the Nuclear Pore Complex

描述（申请人提供）：细胞核是真核细胞的中心细胞器。细胞核和细胞质之间的分子运输仅通过核孔复合物（NPC）介导，所述核孔复合物穿透限定核隔室的双膜核膜。我们的长期目标是获得有关单个NPC中单个通道门控和蛋白质易位事件的实时信息。关于通过纳米大小的蛋白质机器的核质运输的信息，目前还不存在，将有助于我们对许多生物过程和疾病的全面理解，并将加速未来基因表达的药物调控。为了实现这一目标，我们将建立一种新的实验方法，通过提高扫描电化学显微镜（SECM）的空间和时间分辨率到纳米和微秒范围内的NPC的单通道记录。虽然SECM是一种非侵入性和定量测量膜渗透性的成熟方法，但通过纳米制造的SECM探针提高空间分辨率将使我们能够测量单个NPC渗透性。通过监测单个通道事件对单个NPC渗透率的影响，可以获得有关单个通道事件的实时信息，具有高时间分辨率。时间分辨的信息将使我们能够评估的传输模型，其有效性不能检查目前可用的实验数据，从而更全面地了解这一重要的传输过程。

DESCRIPTION (provided by applicant): The nucleus is the central organelle of eukaryotic cells. The molecular transport between the nucleus and the cytoplasm is mediated only through the nuclear pore complex (NPC), which perforates the double membraned nuclear envelope that defines the nuclear compartment. Our long-term objective is to obtain real-time information about individual channel-gating and protein-translocation events in the single NPC. The information about the nucleocytoplasmic transport through the nanometer-sized protein machine, which does not presently exist, will facilitate our comprehensive understanding of many biological processes and diseases, and will accelerate future pharmaceutical regulation of gene expression. To achieve the goal, we will establish a novel experimental approach for single channel recording of the NPC by improving spatial and time resolutions of scanning electrochemical microscope (SECM) to nanometer and microsecond ranges. While SECM is a well-established method for non-invasive and quantitative measurements of membrane permeability, improvement of the spatial resolution by nanofabricated SECM probes will enable us to measure the single NPC permeability. Real-time information about the individual channel events can be obtained by monitoring their effects on the single NPC permeability with high time resolution. The time-resolved information will allow us to evaluate the transport models whose validity can not be checked by currently-available experimental data, leading to more comprehensive understanding of this important transport process.