Profiling Gephyrin – GABA\(_A\)/Glycine Receptor Binding Using Peptide Libraries

γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the mammalian brain. They assemble as hetero- as well as homopentamers and are targeted to functionally diverse synapse types by intracellularly associated proteins such as gephyrin. Multiple subunits have been shown to be binding partners of gephyrin, but only two subunit-gephyrin interactions – namely GABAA receptor α3 subunit and glycine receptor β subunit – were structurally resolved, leaving the details of the molecular interplay of gephyrin with the remaining 24 receptor subunits unresolved. Here, I employed peptide microarrays to systematically characterize the interactions between the primary synaptic GlyR α1-4, β and GABAAR α1-6 receptor subunits and gephyrin. Receptor subunit binding sites were mapped in array format using the isolated gephyrin E domain, its point variants as well as native gephyrin within lysates. The resulting data establish a hierarchy of GABAA and glycine receptor subunit binding to gephyrin and hint towards the involvement of subunit specific as well as overlapping gephyrin interaction surfaces and further propose an unexpected contribution to gephyrin-mediated recruitment through alpha subunits of the glycine receptor. Thereby, this study contributes to our molecular understanding of alternative recruitment of inhibitory receptors, as well as the formation, maintenance, and plasticity of the inhibitory synapse.