The Residence Time Of Gaba(A)Rs At Inhibitory Synapses Is Determined By Direct Binding Of The Receptor Alpha 1 Subunit To Gephyrin

The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive alpha 1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABA(A)Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the alpha 1 subunit with an affinity of similar to 20 mu M, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of alpha 1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the alpha 1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the alpha 1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of alpha 1-subunit-containing GABA(A)Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the alpha 1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, there by modulating the strength of synaptic inhibition.