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

Yuxuan Zhuang,Rebecca J Howard,Björn O Forsberg,Marta Carroni,Erik Lindahl,Linnea Yvonnesdotter,Urška Rovšnik

doi:10.26508/lsa.202101011

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 side chain 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 allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

Highlights

Pentameric ligand-gated ion channels are major mediators of fast synaptic transmission in the mammalian nervous system and serve a variety of biological roles across evolution [1]
Gloeobacter violaceus ligandgated ion channel (GLIC) has been thoroughly documented as a proton-gated ion channel, conducting currents in response to low extracellular pH with half-maximal activation around pH 5 [8]
Overall map quality improved at lower pH, local resolution in the transmembrane domain (TMD) remained high relative to the extracellular domain (ECD) (Fig 1C and D)

Summary

Introduction

Pentameric ligand-gated ion channels are major mediators of fast synaptic transmission in the mammalian nervous system and serve a variety of biological roles across evolution [1]. The extracellular domain (ECD) of each subunit contains β-strands β1–β10, with the characteristic Cys- or Pro-loop [3] connecting β6–β7, and loops A–F enclosing a canonical ligand-binding site [4] at the interface between principal and complementary subunits. The transmembrane domain (TMD) contains α-helices M1–M4, with M2 lining the channel pore, and an intracellular domain of varying length (2–80 residues) inserted between M3 and M4. Extracellular agonist binding is thought to favor subtle structural transitions from resting to intermediate or “flip” states [2], opening of a transmembrane pore [5], and in most cases, a refractory desensitized phase [6]. A detailed understanding of pentameric channel biophysics and pharmacology depends on high-quality structural templates in multiple functional states. High-resolution structures can be biased by stabilizing measures such as ligands, mutations, and crystallization, leaving open questions as to the wild-type activation process

Methods

Results

Conclusion