Plant Membrane Biophysics, Technische Universität Darmstadt, Darmstadt, Germany.
Department of Structural Biology, Christian-Albrechts-University, Kiel, Germany.
Channels (Austin). 2024 Dec;18(1):2402749. doi: 10.1080/19336950.2024.2402749. Epub 2024 Oct 9.
Kcv channels from plant viruses represent the autonomous pore module of potassium channels, devoid of any regulatory domains. These small proteins show very reproducible single-channel behavior in planar lipid bilayers. Thus, they are an optimum system for the study of the biophysics of ion transport and gating. Structural models based on homology modeling have been used successfully, but experimental structural data are currently not available. Here we determine the size of the cytosolic pore entrance by studying the blocker kinetics. Blocker binding and dissociation rate constants ranging from 0.01 to 1000 ms were determined for different quaternary ammonium ions. We found that the cytosolic pore entrance of Kcv must be at least 11 Å wide. The results further indicate that the residues controlling a cytosolic gate in one of the Kcv isoforms influence blocker binding/dissociation as well as a second gate even when the cytosolic gate is in the open state. The voltage dependence of the rate constant of blocker release is used to test, which blockers bind to the same binding site.
植物病毒的 Kcv 通道代表钾通道的自主孔模块，没有任何调节域。这些小蛋白在平面脂质双层中表现出非常可重复的单通道行为。因此，它们是研究离子传输和门控的生物物理学的最佳系统。基于同源建模的结构模型已被成功使用，但目前尚无实验结构数据。在这里，我们通过研究阻滞剂动力学来确定细胞溶质孔入口的大小。对于不同的季铵离子，确定了从 0.01 到 1000 ms 的阻滞剂结合和解离速率常数。我们发现 Kcv 的细胞溶质孔入口必须至少为 11 Å 宽。结果进一步表明，控制 Kcv 同工型中一个细胞溶质门的残基不仅影响阻滞剂的结合/解离，而且影响第二个门，即使细胞溶质门处于打开状态。阻滞剂释放速率常数的电压依赖性用于测试哪种阻滞剂结合到相同的结合位点。
