• allosteric interactions;
  • gating;
  • glycine receptor chloride channel;
  • receptor structure and function;
  • substituted cysteine accessibility method


This study investigated the role of β subunits in the activation of αβ heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in α and β subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the α subunit M2–M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the β subunit. Furthermore, although the α subunit M2–M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding β subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between α and β subunit M2–M3 loops. In addition, a threonine[RIGHTWARDS ARROW]leucine mutation at the 9′ position in the β subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR β subunit is involved in receptor gating. In conclusion, this study demonstrates that β subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the α subunit.