Imoukhuede P I, Moss Fraser J, Michael Darren J, Chow Robert H, Lester Henry A
Bioengineering Division, California Institute of Technology Pasadena, California 91125, USA.
Biophys J. 2009 Apr 8;96(7):2949-60. doi: 10.1016/j.bpj.2008.11.070.
A high density of neurotransmitter transporters on axons and presynaptic boutons is required for the efficient clearance of neurotransmitters from the synapse. Therefore, regulators of transporter trafficking (insertion, retrieval, and confinement) can play an important role in maintaining the transporter density necessary for effective function. We determined the interactions that confine GAT1 at the membrane by investigating the lateral mobility of GAT1-yellow fluorescent protein-8 (YFP8) expressed in neuroblastoma 2a cells. Through fluorescence recovery after photobleaching, we found that a significant fraction ( approximately 50%) of membrane-localized GAT1 is immobile on the time scale investigated ( approximately 150 s). The mobility of the transporter can be increased by depolymerizing actin or by interrupting the GAT1 postsynaptic density 95/Discs large/zona occludens 1 (PDZ)-interacting domain. Microtubule depolymerization, in contrast, does not affect GAT1 membrane mobility. We also identified ezrin as a major GAT1 adaptor to actin. Förster resonance energy transfer suggests that GAT1-YFP8 and cyan fluorescent (CFP) tagged ezrin (ezrin-CFP) exist within a complex that has a Förster resonance energy transfer efficiency of 19% +/- 2%. This interaction can be diminished by disrupting the actin cytoskeleton. In addition, the disruption of actin results in a >3-fold increase in gamma-aminobutyric acid uptake, apparently via a mechanism distinct from the PDZ-interacting protein. Our data reveal that actin confines GAT1 to the plasma membrane via ezrin, and this interaction is mediated through the PDZ-interacting domain of GAT1.
轴突和突触前终扣上高密度的神经递质转运体对于从突触有效清除神经递质是必需的。因此，转运体运输（插入、回收和限制）的调节因子在维持有效功能所需的转运体密度方面可发挥重要作用。我们通过研究在神经母细胞瘤2a细胞中表达的GAT1-黄色荧光蛋白8（YFP8）的横向流动性，确定了将GAT1限制在膜上的相互作用。通过光漂白后的荧光恢复，我们发现在所研究的时间尺度（约150秒）上，相当一部分（约50%）膜定位的GAT1是不移动的。通过使肌动蛋白解聚或中断GAT1突触后密度95/盘状大蛋白/紧密连接蛋白1（PDZ）相互作用结构域，可以增加转运体的流动性。相比之下，微管解聚不影响GAT1的膜流动性。我们还确定埃兹蛋白是GAT1与肌动蛋白的主要衔接蛋白。荧光共振能量转移表明，GAT1-YFP8和青色荧光（CFP）标记的埃兹蛋白（埃兹蛋白-CFP）存在于一个复合物中，其荧光共振能量转移效率为19%±2%。这种相互作用可通过破坏肌动蛋白细胞骨架而减弱。此外，肌动蛋白的破坏导致γ-氨基丁酸摄取增加>3倍，显然是通过一种不同于PDZ相互作用蛋白的机制。我们的数据表明，肌动蛋白通过埃兹蛋白将GAT1限制在质膜上，这种相互作用是通过GAT1的PDZ相互作用结构域介导的。
