CRITICAL RESIDUES MEDIATING GLYCINE- AND CESIUM-DEPENDENT GLYCINE RECEPTOR ACTIVATION

Glycine receptors (GlyRs) are chloride permeable ligand-gated ion channels that play a crucial role in synaptic transmission in the CNS. In addition to glycine as the endogenous neurotransmitter, cesium (Cs⁺) has been shown to activate GlyRs, and our recently published study identified residue D194 as essential for GlyR activation at the cell surface. This residue is located within the N-terminal extracellular ligand-binding domain and is part of a cluster of amino acids (D141, E192, D194 as well as D91, D97, E110, D114, Y161) that have been predicted by recent atomistic molecular dynamic simulations to contribute to Cs⁺ binding.
Here, we mutated the negatively charged residues D91, D97, E110, D114 and Y161 to code for either alanine, introducing a neutral hydrophobic side chain, or lysine, introducing a positively charged side chain. Functional consequences of these mutations were assessed by patch-clamp recordings and surface GlyR staining in transfected HEK293T cells. We found that the substitutions D97A/K and Y161K resulted in a complete loss of receptor function in response to glycine and Cs⁺, whereas mutations at D91, E110, and D114 retained detectable current responses.
Notably, the Y161A mutant preserved partial Cs⁺-dependent GlyR responsiveness, indicating that a positive charge (K) at position Y161 is particularly detrimental to glycine-dependent GlyR activation. Importantly, all mutants that exhibited glycine-evoked currents also displayed Cs⁺-dependent receptor activity. Together, these findings suggest that Cs⁺-mediated GlyR activation relies on the same structural framework required for glycine-induced channel opening and does not represent an independent receptor activation mechanism.