Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Homi Bhabha National Institute, 1/AF Bidhannagar, Kolkata, India.
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Homi Bhabha National Institute, 1/AF Bidhannagar, Kolkata, India.
Biochim Biophys Acta Biomembr. 2021 May 1;1863(5):183568. doi: 10.1016/j.bbamem.2021.183568. Epub 2021 Jan 30.
KvAP is a tetrameric voltage-gated potassium channel that is composed of a pore domain and a voltage-sensing domain (VSD). The VSD is crucial for sensing transmembrane potential and gating. At 0 mV, the VSD adopts an activated conformation in both n-octylglucoside (OG) micelles and phospholipid membranes. Importantly, gating-modifier toxins that bind at S3b-S4 loop of KvAP-VSD exhibit pronounced differences in binding affinity in these membrane-mimetic systems. However, the conformational heterogeneity of this functionally-important sensor loop in membrane mimetics is poorly understood, and is the focus of this work. In this paper, we establish, using intrinsic fluorescence of the uniquely positioned W70 in KvAP-VSD and environment-sensitive NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine) fluorescence of the labelled S3b-S4 loop, that the surface charge of the membrane does not significantly affect the topology and structural dynamics of the sensor loop in membranes. Importantly, the dynamic variability of the sensor loop is preserved in both zwitterionic (POPC) and anionic (POPC/POPG) membranes. Further, the lifetime distribution analysis for the NBD-labelled residues by maximum entropy method (MEM) demonstrates that, in contrast to micelles, the membrane environment not only reduces the relative discrete population of sensor loop conformations, but also broadens the lifetime distribution peaks. Overall, our results strongly suggest that the conformational heterogeneity of the sensor loop is significantly altered in membranes and this correlates well with its environmental heterogeneity. This constitutes the first report demonstrating that MEM-lifetime distribution could be a powerful tool to distinguish changes in conformational heterogeneity in potassium channels with similar architecture and topology.
KvAP 是一种四聚体电压门控钾通道，由孔域和电压感应域（VSD）组成。VSD 对于感应跨膜电位和门控至关重要。在 0 mV 时，VSD 在正辛基葡糖苷（OG）胶束和磷脂膜中均采用激活构象。重要的是，与 KvAP-VSD 的 S3b-S4 环结合的门控调节剂毒素在这些膜模拟系统中表现出明显不同的结合亲和力。然而，该功能重要传感器环在膜模拟物中的构象异质性理解甚少，这是本工作的重点。在本文中，我们通过 KvAP-VSD 中独特位置的 W70 的本征荧光和标记的 S3b-S4 环的环境敏感 NBD（7-硝基苯并-2-氧杂-1,3-二唑-4-基乙二胺）荧光，建立了在膜中传感器环的拓扑结构和结构动力学不受膜表面电荷显著影响的结论。重要的是，传感器环的动态可变性在两性离子（POPC）和阴离子（POPC/POPG）膜中都得以保留。此外，最大熵法（MEM）对 NBD 标记残基的寿命分布分析表明，与胶束相比，膜环境不仅降低了传感器环构象的相对离散种群，而且拓宽了寿命分布峰。总体而言，我们的结果强烈表明，在膜中传感器环的构象异质性发生了显著改变，这与其环境异质性密切相关。这是第一个报告，证明 MEM 寿命分布可以成为一种强大的工具，用于区分具有相似结构和拓扑的钾通道构象异质性的变化。
