Characterizing Functional States of a Model Ligand-Gated Ion Channel by Cryo-Electron Microscopy

Abstract

Pentameric ligand-gated ion channels are important mediators of electrochemical signal transduction in the brain and other systems. Recent progress in protein biochemistry and structural biology has shed light on topology and possible gating mechanisms in these channels, yet the functionally relevant endpoints of activation and modulation remain unclear. In order to obtain alternative structural insights, we optimized protein purification, reconstitution, and grid preparation conditions for structure determination of a prokaryotic ion channel using cryo-electron microscopy (EM). We prepared samples at various presumed distributions of active and inactive states, with the goal of quantifying distinct classes within individual cryo-EM grids. Modification of carbon support, glow-discharging vapor, and blotting time were critical to improving particle distribution and orientation, and molecular simulations were harnessed to validate structural models. This work promises to inform methods development in cryo-EM of membrane proteins, and to populate the gating landscape in this important ion channel family.