Molecular determinants of cesium- and glycine-dependent glycine receptor activation

The neurotransmitter receptor for glycine (GlyR) is a ligand-gated chloride-permeable ion channel in the central nervous system, and dysregulation of this channel is associated with diverse neurological disorders. Cesium (Cs⁺) is an agonist of GlyRs, and recent atomistic molecular dynamic simulations suggested amino acids D141, E192, and D194 with negatively charged side chains as possible binding sites for Cs⁺. To test this hypothesis, we mutated these positions to code for alanine with a neutral hydrophobic side chain or lysine with a positively charged side chain. The correspondingly mutated GlyR channels were expressed in HEK293T cells and analyzed using whole-cell patch clamp electrophysiology. The results show that D141 mutations had no considerable effect on GlyR currents, E192K prolonged desensitization and D194 turned out to be critical for general (glycine- and Cs⁺-dependent) GlyR activation, which was not due to differentially reduced cell surface expression of all the investigated GlyR mutants. Thus, E192 and D194 are critically involved in cell surface GlyR activation by Cs⁺ and glycine.