High‐Throughput MicroED for Probing Ion Channel Dynamics

Ion channels play a crucial role in ion transport and are integral to fundamental physiological processes. Understanding channel structures is essential for elucidating the mechanisms of ion permeation and selectivity beyond simple computational simulations. Visualizing dynamics at high resolution, however, remains a significant challenge by structural techniques. In this study, high-throughput microcrystal electron diffraction (MicroED) is applied to explore the structural dynamics of two ion channels, the non-selective ion channel NaK and its mutant, NaK2CNG. This approach utilizes automated data collection and processing to capture distinct structural substates from a large number of microcrystals, offering a deeper understanding of ion channel mechanisms. From a subset of NaK structures, consistent sodium binding at specific sites is observed. In contrast, NaK2CNG appears more dynamic and undergoes dilation of the selectivity filter upon potassium binding. Further, the conduction state of NaK2CNG appears to be influenced by channel gating. Comparative analysis of these structures suggests that plasticity of the selectivity filter may contribute to the non-selectivity of these channels, potentially allowing dynamic control over ion passage. These studies demonstrate the potential to employ high-throughput MicroED as a technique to address persistent questions regarding ion channel permeation, complementing current computational molecular dynamics studies.