Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations

Abstract

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and sidechain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1-β2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones, and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results support a dissection of protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

“…Atomic force microscopy images have also revealed a mixture at low pH, including states that predominated under neutral resting conditions (24). We have also recently used cryoelectron microscopy of GLIC to reconstruct multiple particle classes at pH 3, including a dominant state with an expanded ECD and closed pore, and a minority class intermediate between resting and open crystal structures (7). Due in part to its low conductance in single-channel recordings (35), the open probability of GLIC has not been well established.…”

“…This could suggest some channels (e.g., GLIC) might be able to exist in conformations not yet observed experimentally, or that features such as crystal packing, experimental conditions, or the liquid nitrogen freezing might have biased the conformational distribution. Indeed, the vast majority of GLIC structures to date contain an activated ECD conformation; the few resting-state structures available were reported to notably lower resolution (6,7). Biophysical techniques including electron paramagnetic resonance spectroscopy (11,23) and atomic force microscopy (24) have suggested GLIC can sample additional states outside the crystalline context, emphasizing the importance of alternative structural approaches to model pLGIC conformational cycling in solution.…”

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

“…Atomic force microscopy images have also revealed a mixture at low pH, including states that predominated under neutral resting conditions [20]. We have also recently used cryo-electron microscopy of GLIC to reconstruct multiple particle classes at pH 3, including a dominant state with an expanded ECD and closed pore, and a minority class intermediate between resting and open crystal structures [7]. Due in part to its low conductance in single-channel recordings [31], the open probability of GLIC has not been well established.…”

“…The proton-gated cation channel GLIC, from the prokaryote Gloeobacter violaceus, has been a popular model system for structure-function studies in this superfamily [3]; it accounts for 40% of all pLGIC structures in the Protein Data Bank. This channel is pH-gated, and its structure has been solved at both high and low pH in apparent closed and open states, respectively [4,5,6,7]. Additional states have been determined in the presence of engineered mutations or lipids, possibly representing gating intermediates [8,9,10,11,12].…”

“…GLIC) might be able to exist in conformations not yet observed experimentally, or that features such as crystal packing, experimental conditions or the liquid nitrogen freezing might have biased the conformational distribution. Indeed, the vast majority of GLIC structures to date contain an activated ECD conformation; the few resting-state structures available were reported to notably lower resolution [6,7]. Biophysical techniques including electron paramagnetic resonance spectroscopy [19,11] and atomic force microscopy [20] have suggested GLIC can sample additional states outside the crystalline context, emphasizing the importance of alternative structural approaches to model pLGIC conformational cycling in solution.…”