Binding-site mutations in the α1 subunit of the inhibitory glycine receptor convert the inhibitory metal ion Cu into a positive modulator

The divalent cation copper (Cu²⁺) has been shown to inhibit chloride currents mediated by the inhibitory glycine receptor (GlyR). Here, we analyzed Cu²⁺ inhibition of homo- and hetero-oligomeric GlyRs expressed in Xenopus oocytes. No significant differences in Cu²⁺ inhibitory potency were found between α1, α2 and α3 GlyRs as well as heteromeric α1β receptors. Furthermore, GlyR α1 mutations known to reduce inhibition or potentiation of GlyR currents by Zn²⁺ had no effect on Cu²⁺ inhibition. However, Cu²⁺ was found to competitively antagonize glycine binding, suggesting that Cu²⁺ binds at the agonist-binding site. Mutations within the glycine-binding site of the GlyR α1 subunit reduced the inhibitory potency of Cu²⁺ and led to an up to 4-fold potentiation of glycine-elicited currents by Cu²⁺. Molecular dynamics simulation suggests this to be due to increased Cu²⁺ binding energies. Our data show that GlyR binding-site mutations can convert inhibitors of agonist binding into highly effective positive modulators.