Jacob Tija C, Bogdanov Yury D, Magnus Christopher, Saliba Richard S, Kittler Josef T, Haydon Philip G, Moss Stephen J
Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
J Neurosci. 2005 Nov 9;25(45):10469-78. doi: 10.1523/JNEUROSCI.2267-05.2005.
The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABAA receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABAA receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABAA receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABAA receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABAA receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABAA receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses.
快速突触抑制的功效严重依赖于GABAA受体在抑制性突触处的积累，而这一过程仍知之甚少。在这里，我们使用经N端细胞外嗜酸性pH荧光蛋白报告基因修饰的受体亚基，研究了细胞表面GABAA受体的动态变化。在海马神经元中，发现含有pH荧光蛋白标记亚基的GABAA受体聚集在突触部位，也表现为弥漫性的突触外染色。通过将光漂白后荧光恢复（FRAP）测量与FM4-64标记的活跃突触前终末的实时成像相结合，很明显，与突触外的对应物相比，聚集的突触受体在细胞表面的移动速率显著更低。为了研究这种限制的基础，我们使用RNA干扰来抑制桥连蛋白的表达，桥连蛋白是一种已被证明可调节GABAA受体在突触部位积累的蛋白质。然而，桥连蛋白是控制受体簇的实际形成、它们的稳定性，还是仅仅是受体细胞表面数量的全局调节因子，仍然未知。抑制桥连蛋白的表达并没有改变神经元细胞表面表达的GABAA受体的总数，但显著减少了受体簇的数量。实时成像显示，在没有桥连蛋白的情况下形成的簇与对照神经元中的簇相比，移动性明显更高。总之，我们的结果表明，与突触外的对应物相比，突触GABAA受体的横向移动水平更低，并表明桥连蛋白在减少GABAA受体的扩散、促进它们在抑制性突触处的积累方面具有特定作用。
