High-Throughput MicroED for Probing Ion Channel Dynamics - PubMed (original) (raw)

High-Throughput MicroED for Probing Ion Channel Dynamics

Marc J Gallenito et al. Adv Sci (Weinh). 2025 Aug.

Abstract

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.

Keywords: Ion Channels; MicroED; ion permeation; ion selectivity; membrane proteins; structural biology.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Schematic flow of high‐throughput MicroED.

Figure 2

MicroED structures of NaK and NaK2CNG. A) Alignment of tetrameric NaK structures. Sodium B) and potassium D) binding sites along the selectivity filter and representative sodium C) and potassium E) binding sites from all structures.

Figure 3

Interaction of Na+ and K+ with the selectivity filter. Tally of ions found in NaK A) and NaK2CNG B). C) Electron density maps of the three types of binding configuration of Na in S3: planar with V64 carbonyls, square antiprism with carbonyls of T63, V64, and planar with T63 carbonyls. The distances between Na+ (purple) and the oxygens (red) are shown. E) Density maps of the coordination of K+ (red) in S2/S3 and their corresponding HOLE profile D,F). Opposite subunits are shown for ease.

Figure 4

Gating at the restriction site. HOLE profiles of all NaK A) and representative NaK2CNG B), SF sites, and constriction site are labelled. C) K+ ions found within the conduction route of NaK2CNG with significant pore radius changes, labelled as conduction 1–4. D) Alignment of four representative NaK2CNG highlighting F91. Electron density maps of F91 of conduction 1 E) and conduction 4 F).

References

1. Hille B. Ion Channels of Excitable Membranes Sinauer, Sunderland: 2001.
1. Zaydman M. A., Silva J. R., Cui J., Chem. Rev. 2012, 112, 6319. -PMC -PubMed
1. Cooper E. C., Jan L. Y., Proc. Natl. Acad. Sci. USA 1999, 96, 4759.
1. Doyle D. A., Cabral J. O. M., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R., Science 1979, 280, 69. -PubMed
1. Zhou Y., Morais‐Cabral J. H., Kaufman A., MacKinnon R., Nature 2001, 414, 43. -PubMed

High-Throughput MicroED for Probing Ion Channel Dynamics - PubMed (original) (raw)