Center for Structural and Functional Neuroscience, University of Montana, Missoula, MT 59812, USA.
Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14980-5. doi: 10.1073/pnas.1108785108. Epub 2011 Aug 22.
A prominent aqueous cavity is formed by the junction of three identical subunits in the excitatory amino acid transporter (EAAT) family. To investigate the effect of this structure on the interaction of ligands with the transporter, we recorded currents in voltage-clamped Xenopus oocytes expressing EAATs and used concentration jumps to measure binding and unbinding rates of a high-affinity aspartate analog that competitively blocks transport (β-2-fluorenyl-aspartylamide; 2-FAA). The binding rates of the blocker were approximately one order of magnitude slower than l-Glu and were not significantly different for EAAT1, EAAT2, or EAAT3, but 2-FAA exhibited higher affinity for the neuronal transporter EAAT3 as a result of a slower dissociation rate. Unexpectedly, the rate of recovery from block was increased by l-Glu in a saturable and concentration-dependent manner, ruling out a first-order mechanism and suggesting that following unbinding, there is a significant probability of ligand rebinding to the same or neighboring subunits within a trimer. Consistent with such a mechanism, coexpression of wild-type subunits with mutant (R447C) subunits that do not bind glutamate or 2-FAA also increased the unblocking rate. The data suggest that electrostatic and steric factors result in an effective dissociation rate that is approximately sevenfold slower than the microscopic subunit unbinding rate. The quaternary structure, which has been conserved through evolution, is expected to increase the transporters' capture efficiency by increasing the probability that following unbinding, a ligand will rebind as opposed to being lost to diffusion.
在兴奋性氨基酸转运体（EAAT）家族中，三个相同亚基的连接形成了一个显著的水性腔。为了研究该结构对配体与转运体相互作用的影响，我们在表达 EAAT 的电压钳制非洲爪蟾卵母细胞中记录电流，并使用浓度跃变来测量高亲和力天冬氨酸类似物（竞争性阻断转运的β-2-氟苯丙氨酸酰胺；2-FAA）的结合和解离速率。阻断剂的结合速率比 l-Glu 慢约一个数量级，并且对于 EAAT1、EAAT2 或 EAAT3 没有显著差异，但由于解离速率较慢，2-FAA 对神经元转运体 EAAT3 表现出更高的亲和力。出乎意料的是，l-Glu 以饱和和浓度依赖的方式增加了从阻断状态的恢复速率，排除了一级机制，并表明在结合配体后，配体重新结合到同一或相邻亚基的可能性很大。与这种机制一致，野生型亚基与突变体（R447C）共表达，突变体亚基不结合谷氨酸或 2-FAA，也增加了去阻断速率。数据表明，静电和空间位阻因素导致有效解离速率比微观亚基解离速率慢约 7 倍。这种四级结构在进化中得到了保守，预计会通过增加结合配体重新结合的可能性，而不是被扩散丢失，从而提高转运体的捕获效率。
