Direct binding of GABAA receptor β2 and β3 subunits to gephyrin

GABAergic transmission is essential to brain function, and a large repertoire of GABA type A receptor (GABAAR) subunits is at a neuron's disposition to serve this function. The glycine receptor (GlyR)‐associated protein gephyrin has been shown to be essential for the clustering of a subset of GABAAR. Despite recent progress in the field of gephyrin‐dependent mechanisms of postsynaptic GABAAR stabilisation, the role of gephyrin in synaptic GABAAR localisation has remained a complex matter with many open questions. Here, we analysed comparatively the interaction of purified rat gephyrin and mouse brain gephyrin with the large cytoplasmic loops of GABAAR α1, α2, β2 and β3 subunits. Binding affinities were determined using surface plasmon resonance spectroscopy, and showed an ~ 20‐fold lower affinity of the β2 loop to gephyrin as compared to the GlyR β loop–gephyrin interaction. We also probed in vivo binding in primary cortical neurons by the well‐established use of chimaeras of GlyR α1 that harbour respective gephyrin‐binding motifs derived from the different GABAAR subunits. These studies identify a novel gephyrin‐binding motif in GABAAR β2 and β3 large cytoplasmic loops. GABAA receptor (GABAAR) α‐subunits were recently shown to interact with gephyrin, which contributes to postsynaptic receptor stabilisation. We show that the C‐terminal parts of cytoplasmic loops between transmembrane domains 3 and 4 of GABAAR β2 and β3 subunits also interact with gephyrin. Compared with the GlyR β loop, the binding of GABAAR β2 and β3 loops to gephyrin showed a twenty fold lower affinity, which is in the range of the KD values that were determined for GABAAR α‐subunits. The identified gephyrin‐binding sequences of β2 and β3 subunits also mediate postsynaptic localisation of corresponding GlyR α1‐GABAAR β chimaeras in cortical neurons. Thus, at least five different GABAAR subunits can interact with gephyrin.