Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, Oregon 97239, United States.
J Am Chem Soc. 2023 Aug 23;145(33):18529-18537. doi: 10.1021/jacs.3c05339. Epub 2023 Aug 14.
The potassium ion (K) configurations of the selectivity filter of the KcsA ion channel protein are investigated with two-dimensional infrared (2D IR) spectroscopy of amide I vibrations. Single C-O isotope labels are used, for the first time, to selectively probe the S1/S2 or S2/S3 binding sites in the selectivity filter. These binding sites have the largest differences in ion occupancy in two competing K transport mechanisms: soft-knock and hard-knock. According to the former, water molecules alternate between K ions in the selectivity filter while the latter assumes that K ions occupy the adjacent sites. Molecular dynamics simulations and computational spectroscopy are employed to interpret experimental 2D IR spectra. We find that in the closed conductive state of the KcsA channel, K ions do not occupy adjacent binding sites. The experimental data is consistent with simulated 2D IR spectra of soft-knock ion configurations. In contrast, the simulated spectra for the hard-knock ion configurations do not reproduce the experimental results. 2D IR spectra of the hard-knock mechanism have lower frequencies, homogeneous 2D lineshapes, and multiple peaks. In contrast, ion configurations of the soft-knock model produce 2D IR spectra with a single peak at a higher frequency and inhomogeneous lineshape. We conclude that under equilibrium conditions, in the absence of transmembrane voltage, both water and K ions occupy the selectivity filter of the KcsA channel in the closed conductive state. The ion configuration is central to the mechanism of ion transport through potassium channels.
钾离子（K）在 KcsA 离子通道蛋白的选择性过滤器中的构型通过酰胺 I 振动的二维红外（2D IR）光谱进行研究。首次使用单 C-O 同位素标记，选择性探测选择性过滤器中的 S1/S2 或 S2/S3 结合位点。这两个结合位点在两种竞争的 K 转运机制中离子占据率差异最大：软敲击和硬敲击。根据前者，水分子在选择性过滤器中的 K 离子之间交替，而后者则假设 K 离子占据相邻的位点。分子动力学模拟和计算光谱用于解释实验 2D IR 光谱。我们发现，在 KcsA 通道的闭合传导状态下，K 离子不占据相邻的结合位点。实验数据与软敲击离子构型的模拟 2D IR 光谱一致。相比之下，硬敲击离子构型的模拟光谱无法重现实验结果。硬敲击机制的 2D IR 光谱具有较低的频率、均匀的 2D 线形状和多个峰。相比之下，软敲击模型的离子构型产生的 2D IR 光谱在较高频率处具有单个峰，且线形状不均匀。我们得出结论，在平衡条件下，在没有跨膜电压的情况下，水和 K 离子都占据 KcsA 通道的选择性过滤器，处于闭合传导状态。离子构型是离子通过钾通道转运机制的核心。
