Mutations within the agonist-binding site convert the homomeric alpha1 glycine receptor into a Zn2+-activated chloride channel.
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 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-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 (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis.
Grudzinska
Joanna
J
Abteilung Neurochemie, Max-Planck-Institut für Hirnforschung, Frankfurt am Main, Germany.
Schumann
Tanja
T
Schemm
Rudolf
R
Betz
Heinrich
H
Laube
Bodo
B
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002412:Cations; D018118:Chloride Channels / Q000737:chemistry*; D005260:Female; D006801:Humans; D007473:Ion Channels / Q000737:chemistry; D008024:Ligands; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018009:Receptors, Glycine / Q000378:metabolism*; D014982:Xenopus laevis; D015032:Zinc / Q000737:chemistry*
D002412:Cations; D018118:Chloride Channels; D007473:Ion Channels; D008024:Ligands; D018009:Receptors, Glycine; D015032:Zinc