Molecular determinants of glycine receptor αβ subunit sensitivities to Zn2+-mediated inhibition

Glycine receptors exhibit a biphasic sensitivity profile in response to Zn²⁺-mediated modulation, with low Zn²⁺ concentrations potentiating (< 10 μ m ), and higher Zn²⁺ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn²⁺-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) α1 subunit compared to either GlyR α2 or α3 subtypes. Swapping the divergent histidine (H107) residue in GlyR α1, which together with the conserved H109 forms part of an intersubunit Zn²⁺-binding site, for the equivalent asparagine residue present in GlyR α2 and α3, reversed this phenotype. Co-expression of heteromeric GlyR α1 or α2 with the ancillary β subunit yielded receptors that maintained their distinctive sensitivities to Zn²⁺ inhibition. However, GlyR α2β heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR α2 homomer. Comparative studies to elucidate the specific residue in the β subunit responsible for this differential sensitivity revealed instead threonine 133 in the α1 subunit as a new vital component for Zn²⁺-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated β subunit could indeed affect Zn²⁺-mediated inhibition but only from one side of the intersubunit Zn²⁺-binding site, equivalent to the GlyR α1 H107 face. This strongly suggests that the α subunit is responsible for Zn²⁺-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn²⁺-binding site where H109 and T133 are located.