Identification of critical sites for glycine- and cesium-dependent activation of the RNA-edited glycine receptor α3L.

Glycine receptors (GlyRs) are chloride permeable ligand-gated ion channels that play an important role in synaptic transmission in the central nervous system. Besides glycine as neurotransmitter, cesium (Cs+) was shown to activate homomeric GlyRs with α2 and α3 subunits, and a recent study revealed the position D194 as essential for activation of RNA-edited GlyR α3185L at the cell surface. This position in the N-terminal extracellular ligand-binding domain belongs to a series of amino acid positions (D141, E192, D194 and D91, D97, E110, D114, Y161) predicted to be involved in Cs+ binding. In the present study, we mutated the negatively charged amino acids D91, D97, E110, and D114 as well as the neutral polar amino acid Y161 to code for alanine with a neutral hydrophobic side chain or lysine with a positively charged side chain and performed patch-clamp analysis as well as surface GlyR staining using transfected HEK293T cells. We found that substitutions D97A/K and Y161K resulted in a complete loss of receptor function in response to glycine or Cs+, whereas mutations at D91 and E110 retained detectable current responses, with E110A/K mainly affecting glycine-evoked currents. Y161A retained at least some Cs+-dependent GlyR responsiveness at concentrations above 5 mM, indicating that a positive charge (K) at position Y161 is detrimental to glycine-dependent GlyR activation. However, as no mutant was identified that responded to glycine while lacking Cs+-evoked activity, these findings suggest that Cs+-mediated GlyR activation involves the same structural framework required for glycine-induced channel opening.