Mechanism of Alkaline Gating in a Pentameric Ion Channel

Pentameric ligand-gated ion channels (pLGICs) are key mediators of electrochemical signal transduction in various organisms. Like many proteins involved in cellular signaling, they are modulated by a variety of environmental factors including pH and small molecules. However, the molecular mechanisms underlying pLGIC activation and modulation remain unclear. A promising model system in this family is the bacterial ion channel sTeLIC, which can be activated by alkaline pH, and for which we recently determined structures in multiple functional states. However, protonation changes and other pH-driven dynamics cannot be directly observed in these structures. Here, we used constant-pH molecular dynamics simulations and oocyte-electrophysiology recordings from engineered mutants to develop a comprehensive mechanistic model for pH sensing. Interestingly, critical residues include two Glu residues (E106, E160) located in the extracellular-vestibule and domain-interface regions of each subunit, where they mediate differential electrostatic interactions in closed versus open states. This work demonstrates the applicability of constant-pH methods to model dynamic processes in a multimeric membrane-embedded protein, and offers a detailed mechanism for pH sensing, likely extensible to human drug targets.