Zachariassen Linda G, Katchan Ljudmila, Jensen Anna G, Pickering Darryl S, Plested Andrew J R, Kristensen Anders S
Department of Drug Design & Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark;
Molecular Neuroscience and Biophysics, Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; Cluster of Excellence NeuroCure, Charité Universitätsmedizin, 10117 Berlin, Germany.
Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):E3950-9. doi: 10.1073/pnas.1601747113. Epub 2016 Jun 16.
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.
α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPARs）是配体门控离子通道，介导中枢神经系统中大部分快速兴奋性神经传递。尽管最近在AMPARs的结构研究方面取得了进展，但关于受体功能背后特定构象变化的信息仍然缺乏。在这里，我们在AMPAR亚基的不同位置进行了GFP变体的单插入和双插入，以便在活细胞中使用荧光共振能量转移（FRET）测量构象变化。我们构建了具有正常活性并显示受体内部FRET的双CFP/YFP标记的GluA2亚基构建体。我们在活的HEK293细胞中使用荧光寿命成像显微镜（FLIM）来确定在不同配体存在下不同的稳态FRET效率，这表明了静息状态的动态情况。对双插入和单插入构建体的膜片钳荧光测定表明，在激活过程中，细胞内C末端结构域（CTD）和M1与M2螺旋之间的环区域都会移动，并且CTD会从膜上脱离。我们的时间分辨测量揭示了这些细胞内结构域内出乎意料的复杂荧光变化，为翻译后修饰与受体功能如何相互作用提供了线索。
