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(A)R) subtypes. We report a direct interaction between the GABA(A)R 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(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R 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(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR ß subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR ß subunits with gephyrin with dissociation constants of 5.3 µm and 0.2 µm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.

Complex role of collybistin and gephyrin in GABAA receptor clustering.

Gephyrin and collybistin are key components of GABA(A) receptor (GABA(A)R) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA(A)R subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA(A)R α2 and α3 subunit intracellular M3-M4 domain (but not α1, α4, α5, α6, ß1-3, or γ1-3) with gephyrin. Curiously, GABA(A)R α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA(A)R α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA(A)R α3 interacts with gephyrin, GABA(A)R α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA(A)R α2 and collybistin or GABA(A)R α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA(A)R α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA(A)R α2 is capable of "activating " collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA(A)R α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA(A)R and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA(A)Rs, but not GlyRs or other GABA(A)R subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA(A)Rs containing the α2 subunit.