Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures.
Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures.
复制标题
作者：
Al Awabdh S;Gupta-Agarwal S;Sheehan DF;Muir J;Norkett R;Twelvetrees AE;Griffin LD;Kittler JT
星形胶质细胞GLT-1（或EAAT 2）是清除突触谷氨酸的主要谷氨酸转运蛋白。虽然在神经元表面的神经递质受体的扩散动力学是很好的理解，少得多的是已知的关于星形胶质细胞膜的亚细胞结构域的转运蛋白的表面贩运。在这里，我们使用活细胞成像来研究在解离和脑切片培养物中调节星形胶质细胞中GLT-1表面扩散的机制。使用GFP-时间推移成像，我们表明GLT-1形成稳定的簇，这些簇在谷氨酸处理后以转运蛋白活性依赖性方式快速可逆地分散。光漂白后的荧光恢复和使用量子点的单粒子追踪显示，聚集的GLT-1比扩散的GLT-1更稳定，谷氨酸盐增加了星形胶质细胞膜中的GLT-1表面扩散。有趣的是，在大脑中表达的两种主要的GLT-1亚型，GLT-1a和GLT-1b，都被发现在基础条件下相对于突触是稳定的，GLT-1b更是如此。GLT-1表面迁移率在激活的突触附近增加，神经元活动的改变可以双向调节两种GLT-1亚型的动力学。总之，这些数据表明，星形胶质细胞GLT-1表面的流动性，通过其运输活动，在神经元放电，这可能是一个关键的过程，形成谷氨酸清除和谷氨酸能突触传递调制。GLIA 2016;64：1252-1264
The astrocytic GLT‐1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live‐cell imaging to study the mechanisms regulating GLT‐1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP‐time lapse imaging, we show that GLT‐1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity‐dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT‐1 is more stable than diffuse GLT‐1 and that glutamate increases GLT‐1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT‐1 isoforms expressed in the brain, GLT‐1a and GLT‐1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT‐1b more so. GLT‐1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT‐1 isoforms. Altogether, these data reveal that astrocytic GLT‐1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252–1264