Molecular basis of the γ-aminobutyric acid a receptor α3 subunit interaction with the clustering protein gephyrin

The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA A receptor (GABA AR) subtypes. We report a direct interaction between the GABA AR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA AR α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA AR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA ARs in recombinant systems. Interestingly, motifs responsible for interactions with GABA ARα2, GABA ARα3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA AR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA AR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μM and 0.2 μM, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc