Ehrensperger Marie-Virginie, Hanus Cyril, Vannier Christian, Triller Antoine, Dahan Maxime
Laboratoire Kastler Brossel, Centre National de la Recherche Scientifique UMR8552, Ecole normale supérieure, Université Pierre et Marie Curie-Paris 6, 75005 Paris, France.
Biophys J. 2007 May 15;92(10):3706-18. doi: 10.1529/biophysj.106.095596. Epub 2007 Feb 9.
The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types--able or unable to bind gephyrin--gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.
支架蛋白gephyrin已知可在突触处锚定甘氨酸受体（GlyR），并参与神经元膜中突触和突触外GlyR之间的动态平衡。在此，我们研究了共转染了带有YFP标签的gephyrin和带有细胞外myc标签的GlyR亚基的细胞中这种相互作用的特性。在HeLa细胞和年轻神经元中，使用单粒子追踪实时跟踪单个用量子点标记的GlyR进出gephyrin簇的情况。对两种GlyR亚基类型（能够或不能结合gephyrin）的扩散特性进行分析，从而了解GlyR与其支架蛋白的结合状态。我们的结果表明，GlyR的一大部分可以与gephyrin簇外的少数gephyrin分子相连。这强调了支架蛋白在突触外膜中的作用，并支持了gephyrin-gephyrin相互作用在突触处GlyR稳定中的作用。还对控制簇内和簇外GlyR平衡的动力学参数进行了表征。在簇内，我们鉴定出了GlyR的两个亚群，它们在受体和支架蛋白之间具有不同程度的稳定性。
