Characterization of the dynamic resting state of a pentameric ligand-gated ion channel by cryo-electron microscopy and simulations

Abstract

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological development in this family relies on high-quality structural data in multiple, subtly distinct functional states. However, structural data remain limited, particularly for the unliganded or resting state. Here we report cryo-electron microscopy structures of the Gloeobacter violaceus ligand-gated ion channel (GLIC) under resting and activating conditions (neutral and low pH). Parallel models were built either manually or using recently developed density-guided molecular simulations. The moderate resolution of resting-state reconstructions, particularly in the extracellular domain, was improved under activating conditions, enabling the visualization of residues at key subunit interfaces including loops B, C, F, and M2-M3. Combined with molecular dynamics simulations, the cryo-electron microscopy structures at different pH describe a heterogeneous population of closed channels, with activating conditions condensing the closed-channel energy landscape on a pathway towards gating.

Molecular Simulations of Hydrophobic Gating of Pentameric Ligand Gated Ion Channels: Insights into Water and Ions

“…A range of approaches have been applied including direct (long) simulations of 5-HT 3 R , and other pLGICs, e.g., GluCl, and the use of a range of enhanced sampling approaches, including, e.g., the use of strings and swarms of simulations , and of extensive cloud-based nonequilibrium simulations . A promising approach is to combine extended MD simulations initiated from multiple starting states as determined by, e.g., cryo-EM (seen, e.g, in a recent study of the bacterial channel GLIC) or NMR (as has recently been applied to gating modes of potassium channels). It is likely that such an approach, combined with, e.g., Markov state modeling, − will enable a rigorous integration of insights from structure-based simulations and from single-channel kinetic analysis and modeling.…”