Department of Chemistry, Institute of Biochemistry, University of Cologne, Cologne 50674, Germany.
École Normale Supérieure, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Institute of Biology (IBENS), Paris Sciences et Lettres Research University, Paris 75005, France.
eNeuro. 2018 Feb 19;5(1). doi: 10.1523/ENEURO.0042-17.2018. eCollection 2018 Jan-Feb.
The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 3-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells.
突触传递的效能取决于突触处神经递质受体的数量。这些受体的募集取决于与受体特定结合序列相互作用的突触后支架分子的可用性。在抑制性突触中，网格蛋白（gephyrin）是主要的支架蛋白，通过β亚基（β-环）的细胞质环介导异源甘氨酸受体（GlyRs）的积累。这种结合涉及高亲和力和低亲和力相互作用，但这种双模态结合的分子机制及其对 GlyR 在突触处稳定的影响仍然未知。我们使用定量生化工具和高密度单分子跟踪技术在培养的大鼠脊髓神经元中研究了这个问题。可以确定高亲和力结合位点，并表明它依赖于β-环核心网格蛋白结合基序的 3 螺旋 C 末端的形成。该位点在塑造核心基序方面发挥结构作用，并代表网格蛋白对 GlyR 突触限制的主要贡献。N 端侧翼序列通过占据网格蛋白上新鉴定的结合位点来促进低亲和力相互作用。尽管亲和力低，但该结合位点在调节受体的流动性方面发挥着调节作用。总之，GlyR β-环侧翼核心结合位点的序列差异调节了受体与网格蛋白的亲和力及其在突触处的捕获。我们的实验方法因此弥合了受体-支架相互作用的热力学方面和活细胞中功能性受体稳定之间的差距。
