Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, Ecole Normale Supérieure, Inserm U1024, 75230 Paris Cedex 05, France.
J Neurosci. 2011 Oct 12;31(41):14677-87. doi: 10.1523/JNEUROSCI.2001-11.2011.
The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-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 α1 subunit with an affinity of ∼20 μm, 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)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 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 α1 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 α1-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 α1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, thereby modulating the strength of synaptic inhibition.
大脑中大多数快速突触抑制是由苯二氮䓬敏感的 α1 亚基包含 GABA 型 A 受体 (GABA(A)Rs)介导的；然而，我们对神经元用来调节其突触积累的机制的了解还很初级。使用免疫沉淀，我们证明在培养的大鼠神经元中的抑制性突触中，GABA(A)Rs 和神经胶质蛋白紧密相关。在体外，我们揭示了神经胶质蛋白的 E 结构域通过该受体亚基的细胞内结构域内的残基 360-375，以约 20 μm 的亲和力直接与 α1 亚基结合。突变残基 360-375 会降低海马神经元中含 α1 的 GABA(A)Rs 在神经胶质蛋白阳性抑制性突触中的积累以及 mIPSCs 的幅度。我们还证明，神经胶质蛋白与 α1 亚基的亲和力受 Thr375 调节，Thr375 是一个假定的磷酸化位点。将 Thr375 突变为带负电荷的磷酸模拟氨基酸，会降低 α1 亚基与神经胶质蛋白的亲和力，从而降低受体在突触处的积累以及 mIPSCs 的幅度。最后，单颗粒跟踪显示，神经胶质蛋白特异性地降低含 α1 亚基的 GABA(A)Rs 的扩散，从而增加它们在这些结构处的限制。我们的结果表明，神经胶质蛋白与 α1 亚基的残基 360-375 的直接结合及其调节可能是 GABA(A)Rs 在突触部位稳定的重要决定因素，从而调节突触抑制的强度。
