Membrane Mobility and Microdomain Association of the Dopamine Transporter Studied with Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching

详细信息    查看全文

• 作者：Erika M. Adkins ; Devadoss J. Samuvel ; Jacob U. Fog ; Jacob Eriksen ; Lankupalle D. Jayanthi ; Christian Bjerggaard Vaegter ; Sammanda Ramamoorthy ; Ulrik Gether
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：September 18, 2007
• 年：2007
• 卷：46
• 期：37
• 页码：10484 - 10497
• 全文大小：851K
• 年卷期：v.46,no.37(September 18, 2007)
• ISSN：1520-4995

文摘

To investigate microdomain association of the dopamine transporter (DAT), we employedFCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). Innon-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellowfluorescent protein) a diffusion coefficient (D) of ~3.6 × 10^-9 cm²/s, consistent with a relatively freelydiffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements onplasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This wassupported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2acells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor)and the EGFP-2AR (2 adrenergic receptor) demonstrated that this observation was DAT specific. Boththe cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl--cyclodextrin(mCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DATassociated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes asassessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2acells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and therebymembrane raft disruption, decreased both the V_max and K_M values for [³H]dopamine uptake without alteringDAT surface expression. In summary, we propose that association of the DAT with lipid microdomainsin the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporterand its transport capacity.