Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching
Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching
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DOI：
10.1021/bi700429z
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发表时间：
2007-09-18
期刊：
影响因子：
2.9
通讯作者：
Gether, Ulrik
中科院分区：
文献类型：
--
作者：
Adkins, Erika M.;Samuvel, Devadoss J.;Gether, Ulrik
为了研究多巴胺转运蛋白（DAT）的微区相关性，我们采用了FCS（荧光相关光谱）和FRAP（光漂白后的荧光恢复）。在非神经元细胞（HEK 293）中，FCS测量显示YFP-DAT（用黄色荧光蛋白标记的DAT）的扩散系数（D）类似于3.6 x 10（-9）cm（2）/S，与相对自由扩散的蛋白质一致。在神经源性细胞（N2 a）中，由于光漂白，我们无法对质膜相关蛋白进行FCS测量，这表明部分固定。这得到了FRAP测量的支持，FRAP测量揭示了与HEK 293细胞相比，N2 a细胞中YFP-DAT的较低D和移动的部分。与EGFP-EGFR（表皮生长因子受体）和EGFP-beta 2AR（β 2肾上腺素能受体）的比较表明，该观察结果是DAT特异性的。细胞骨架破坏剂细胞松弛素D和胆固醇消耗剂甲基-β-环糊精（m CD）都增加了YFP-DAT的侧向移动性，但没有增加EGFP-EGFR的侧向移动性。DAT与部分膜筏标记物在N2 a细胞和大鼠纹状体突触体评估蔗糖密度梯度离心。通过霍乱毒素B修补进一步证实了N2 a细胞中的筏缔合。此外，观察到胆固醇消耗，从而膜筏破坏，降低了[H-3]多巴胺摄取的V-max和K-M值，而不改变DAT表面表达。总之，我们建议，协会的DAT与脂质微区在质膜和/或细胞骨架用于调节横向流动性的转运蛋白和其运输能力。
To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of similar to 3.6 x 10(-9) cm(2)/S, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta 2AR (beta 2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (m CD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the V-max and K-M values for [H-3]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity.