Mutations within the agonist-binding site convert the homomeric α1 glycine receptor into a Zn2+-activated chloride channel

Abstract

The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 μM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 μM) have an inhibitory effect. Mutational studies have localized the Zn 2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR α-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric α1- subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (α1R65A, α1R131A), or of the aromatic residue phenylalanine 207 by histidine (α1 F207H), converted the α1 GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the α1F207H GlyR disclosed an EC 50 value of 1.2 μM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the α1 subunit convert the inhibitory GlyR from a glycinegated into a selectively Zn 2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis. ©2008 Landes Bioscience.