Figure 2: Identification of short GABAAR-derived peptides as gephyrin-binding probes.
From: Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin
(a) Peptide-array-based alanine-scan of the gephyrin core-binding site within the GABAAR α3 subunit. Residues shown in pink are conserved in the GABAAR α3 and GlyR β subunits. Gephyrin binding to peptides was detected by chemiluminescence of conjugated horseradish peroxidase. Shown are the relative averaged intensities of six peptide sets together with their standard deviations (error bars). Notably, T367A and T374A increase the gephyrin affinity, whereas an alanine exchange of residues 368–373 as well as residue 376 reduces gephyrin binding. (b) ITC analysis of gephyrin binding to GABAAR α3 and GlyR β/GABAAR α3 chimeric peptides. Peptide sequences and respective GephE affinities are shown. Peptides containing the seven N-terminal residues of the GlyR β peptide display a potentiated gephyrin affinity. N-terminal elongation reduces the affinity, whereas C-terminal elongation has an affinity-enhancing effect. (c) Short GABAAR-derived peptides were sufficient to retain native gephyrin. Pull-down of native gephyrin from whole mouse brain lysate using immobilized short GABAAR-derived peptides. Immunedetection of gephyrin reveals that peptides with a length of 9–11 residues (α9-α11) were sufficient for gephyrin binding, whereas an octamer (α8) did not display binding. The complete blot is shown in Supplementary Fig. 1.
