Gating mechanism of the human α1β GlyR by glycine

Glycine receptors (GlyRs) are members of the Cys-loop receptors that constitute a major portion of neurotransmitter receptors in the human nervous system. GlyRs are found in the spinal cord and brain mediating locomotive, sensory and cognitive functions, and are targets for pharmaceutical development. GlyRs share a general gating scheme with Cys-loop receptor family members, but the underlying mechanism is unclear. Recent resolution of heteromeric GlyRs structures in multiple functional states identified an invariable 4:1 α:β subunit stoichiometry and provided snapshots in the gating cycle, challenging previous beliefs and raising the fundamental questions of how α and β subunit functions in glycine binding and channel activation. In addition, how a single glycine-bound extracellular domain conformation leads to structurally and functionally different open and desensitized states remained enigmatic. In this study, we characterized in detail equilibrium properties as well as the transition kinetics between functional states. We show that while all allosteric sites bind cooperatively to glycine, occupation of 2 sites at the α-α interfaces is necessary and sufficient for GlyR activation. We also demonstrate differential glycine concentration dependence of desensitization rate, extent, and its recovery, which suggests separate but concerted roles of ligand-binding and ionophore reorganization. Based on these observations and available structural information, we developed a comprehensive quantitative gating model that accurately predicts both equilibrium and kinetical properties throughout glycine gating cycle. This model likely applies generally to the Cys-loop receptor family and informs on pharmaceutical endeavors in function modulation of this receptor family.