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Trapping and accumulation of inhibitory receptors at synapses
Inhibitory GABA-A and glycine receptors are clustered at synapses via key interactions between the receptors, a ‘scaffolding’ protein known as gephyrin and an ‘exchange factor’ called collybistin. Using a multidisciplinary approach, I have mapped the binding sites for the glycine receptor β subunit and collybistin to the C-terminal ‘MoeA homology domain’ of gephyrin, and shown that mutants of collybistin prevent gephyrin and associated GABA-A and glycine receptors from being correctly located to synapses (Harvey et al 2004, J Neurosci 24: 5816-5826). The importance of collybistin for synaptic function was further defined by work in collaboration with Prof Heinrich Betz (Max-Planck-Institute for Brain Research, Frankfurt, Germany) that produced a knockout mouse line missing the collybistin protein (Papadopoulos et al 2007, EMBO J 26: 3888-3899). Another collaboration with Dr Vera Kalscheuer (Max-Planck Institute for Molecular Genetics, Berlin, Germany) revealed a chromosomal translocation disrupting the human collybistin gene in a patient with intellectual disability (Kalscheuer et al 2009, Hum Mutat 30: 61-68). My recent work demonstrated a direct interaction of the GABA-AR α2 and α3 subunits with gephyrin. Curiously, GABA-AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA-AR α2 also overlap at the start of the gephyrin E domain. This suggests that whilst GABA-AR α3 interacts with gephyrin, GABA-AR α2, collybistin and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA-AR α2 and collybistin or GABA-AR α2 and gephyrin were strengthened in the presence of gephyrin or collybistin, respectively. Interestingly, GABA-AR α2 was capable of ‘activating’ collybistin isoforms harbouring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA-AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (G55A) that causes X-linked intellectual disability and seizures (Saiepour et al 2010, J Biol Chem 285: 29623-29631). Work characterizing the interactions of the GABA-AR α1 and α3 subunits with gephyrin was performed in collaboration with Prof Stephen Moss (Tufts University, Boston, USA), Dr Verena Tretter and Prof Werner Sieghart (University of Vienna, Austria). We demonstrated that the gephyrin E-domain directly binds to the α1 subunit mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABA-A receptors at gephyrin-positive inhibitory synapses and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by T375, a putative phosphorylation site. Mutation of T375 to a phosphomimetic amino acid decreases the affinity of the α1 subunit for gephyrin, receptor accumulation at synapses and the amplitude of mIPSCs. These results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are important determinants for the stabilization of GABA-A receptors at synaptic sites (Mukherjee et al 2011, J Neurosci 31: 14677-14687). We also located critical determinants of the direct interaction between the GABA-A receptor α3 subunit and gephyrin. In addition, isothermal titration calorimetry revealed a 27-fold difference in the interaction strength between GABA-A receptor 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 GABA-A receptor α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses (Tretter et al 2011, J Biol Chem 286: 37702-37711).
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