Functional characterization of the voltage-gated bacterial sodium channel NaChBac using lanthanide-based resonance energy transfer (LRET)

电压门控离子通道是在所有可兴奋细胞中产生和传播动作电位所必需的。它们由四个同源结构域组成，每个结构域包含六个跨膜片段。尽管通道的不同部分已被证明在通道门控中发挥作用，似乎经历了一些构象变化对电压的响应，但对这些变化的性质知之甚少。在我们的项目中，我们打算通过基于稀土的共振能量转移(LRET)技术来研究最近克隆的六个跨膜片段电压依赖的细菌Na+通道。我们计划使用蛋白质中的EF-Hand来研究重建通道中的距离。EF-Hand抓住供体荧光团Tb，它是由于靠近Tb的激发色氨酸残基的敏化而发出的。通过确定亚单元间和亚单元内的距离作为电压的函数，可以有效地测量电压敏感区域附近的电压相关运动。用膜片钳技术分析通道的门控特性和通透性将为我们提供有关通道功能的额外信息。这两种技术的结合是电压门控离子通道生物物理检测的有力工具，因为在通道门控过程中发生的构象变化可以被可视化，从而建立了膜片钳测量的电生理特性变化与这些变化的来源之间的联系。

Voltage-gated ion channels are essential for Generation and propagation of action potentials in all excitable cells. They are made up of four homologous domains, each of which contains six transmebrane segments. Although various parts of channels have been shown to play a role in channel gating, seemingly undergoing some conformational changes in response to voltage, little is known about the nature of these changes. In our project, we intend to investigate the recently cloned six transmembrane segment voltage-dependent bacterial Na+-channel by the lanthanidebased resonance energy transfer (LRET) technique. We plan to study distances in reconstituted channel using an EF-hand in the protein. The EF-hand grabs the donor fluorophore Tb which emits owing to sensitization from an excited tryptophan residue close to the Tb. Voltage-dependent movements near the voltage-sensing regions can be effectively measured by determining intersubunit and intrasubunit distances as a function of voltage. Analysis of gating properties and permeation of the channel by the patch-clamp technique will provide us with an additional information about channel functioning. Combination of these two techniques is a powerful tool for biophysical examination of voltage-gated ion channels, because the conformational alterations occuring during channel gating can be visualized, establishing the link between changes in electrophysiological properties measured by patch-clamp and the source of these changes.