Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin.

Modulation of the concentration of postsynaptic GABA(A) receptors contributes to functional plasticity of inhibitory synapses. The gamma2 subunit of GABA(A) receptor is specifically required for clustering of these receptors, for recruitment of the submembrane scaffold protein gephyrin to postsynaptic sites, and for postsynaptic function of GABAergic inhibitory synapses. To elucidate this mechanism, we here have mapped the gamma2 subunit domains required for restoration of postsynaptic clustering and function of GABA(A) receptors in gamma2 subunit mutant neurons. Transfection of gamma2-/- neurons with the gamma2 subunit but not the alpha2 subunit rescues postsynaptic clustering of GABA(A) receptors, results in recruitment of gephyrin to postsynaptic sites, and restores the amplitude and frequency of miniature inhibitory postsynaptic currents to wild-type levels. Analogous analyses of chimeric gamma2/alpha2 subunit constructs indicate, unexpectedly, that the fourth transmembrane domain of the gamma2 subunit is required and sufficient for postsynaptic clustering of GABA(A) receptors, whereas cytoplasmic gamma2 subunit domains are dispensable. In contrast, both the major cytoplasmic loop and the fourth transmembrane domain of the gamma2 subunit contribute to efficient recruitment of gephyrin to postsynaptic receptor clusters and are essential for restoration of miniature IPSCs. Our study points to a novel mechanism involved in targeting of GABA(A) receptors and gephyrin to inhibitory synapses.

The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering.

Glycine receptors (GlyRs) and specific subtypes of GABA(A) receptors are clustered at synapses by the multidomain protein gephyrin, which in turn is translocated to the cell membrane by the GDP-GTP exchange factor collybistin. We report the characterization of several new variants of collybistin, which are created by alternative splicing of exons encoding an N-terminal src homology 3 (SH3) domain and three alternate C termini (CB1, CB2, and CB3). The presence of the SH3 domain negatively regulates the ability of collybistin to translocate gephyrin to submembrane microaggregates in transfected mammalian cells. Because the majority of native collybistin isoforms appear to harbor the SH3 domain, this suggests that collybistin activity may be regulated by protein-protein interactions at the SH3 domain. We localized the binding sites for collybistin and the GlyR beta subunit to the C-terminal MoeA homology domain of gephyrin and show that multimerization of this domain is required for collybistin-gephyrin and GlyR-gephyrin interactions. We also demonstrate that gephyrin clustering in recombinant systems and cultured neurons requires both collybistin-gephyrin interactions and an intact collybistin pleckstrin homology domain. The vital importance of collybistin for inhibitory synaptogenesis is underlined by the discovery of a mutation (G55A) in exon 2 of the human collybistin gene (ARHGEF9) in a patient with clinical symptoms of both hyperekplexia and epilepsy. The clinical manifestation of this collybistin missense mutation may result, at least in part, from mislocalization of gephyrin and a major GABA(A) receptor subtype.