Identification of residues critical for Cu²⁺-mediated inhibition of glycine α1 receptors

Endogenous divalent cations Cu²⁺ and Zn²⁺ suppress the activity of glycine receptors (glyRs). Whereas residues critical for the effects of Zn²⁺ on glyRs have been identified, little is known about the determinants of Cu²⁺-mediated inhibition. In the present studies, we have assessed the potential commonality of Zn²⁺ and Cu²⁺-mediated inhibition of glyRs. Cu²⁺ potently inhibited recombinant human glycine α1 receptors, with an IC₅₀ of 4.1 ± 0.7 μM. Systematic mutation of extracellular histidine residues revealed that mutation H215A greatly reduced the inhibitory modulation by Cu²⁺. Substitution of H215 with C produced receptors with Cu²⁺ sensitivity similar to the wild type. Furthermore, modification of H215C with a thio-specific reagent, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), reduced Cu²⁺ sensitivity of H215C receptors. However, mutation of other extracellular histidine residues including H107 and H109, which are known inhibitory Zn²⁺coordination sites, failed to influence inhibition of glycine currents by Cu²⁺. Moreover, mutation to alanine of two threonine residues (T112, T133) critical for Zn²⁺ inhibition had no effect (T133A) or only partial inhibitory effects (T112A) on Cu²⁺-induced inhibition. The double mutation, T112A/H215A, caused greater effects on Cu²⁺-mediated inhibition than either mutation alone. In addition, the glycine currents recorded from T112A/H215A mutant receptors were significantly potentiated by low concentrations of Cu²⁺. Our results have identified critical determinants of Cu²⁺-mediated inhibition of glyRs. Moreover, we demonstrate for the first time a clear difference in residues responsible for Cu²⁺-mediated compared to Zn²⁺-mediated inhibition of glyRs.