Combs-Bachmann Rosamund E, Johnson Jeffreys Nate, Vytla Devaiah, Hussey Amanda M, Kilfoil Maria L, Chambers James J
Neuroscience and Behavior Program, University of Massachusetts, Amherst, Massachusetts, USA.
J Neurochem. 2015 May;133(3):320-9. doi: 10.1111/jnc.13051. Epub 2015 Mar 13.
Subcellular trafficking of neuronal receptors is known to play a key role in synaptic development, homeostasis, and plasticity. We have developed a ligand-targeted and photo-cleavable probe for delivering a synthetic fluorophore to AMPA receptors natively expressed in neurons. After a receptor is bound to the ligand portion of the probe molecule, a proteinaceous nucleophile reacts with an electrophile on the probe, covalently bonding the two species. The ligand may then be removed by photolysis, returning the receptor to its non-liganded state while leaving intact the new covalent bond between the receptor and the fluorophore. This strategy was used to label polyamine-sensitive receptors, including calcium-permeable AMPA receptors, in live hippocampal neurons from rats. Here, we describe experiments where we examined specificity, competition, and concentration on labeling efficacy as well as quantified receptor trafficking. Pharmacological competition during the labeling step with either a competitive or non-competitive glutamate receptor antagonist prevented the majority of labeling observed without a blocker. In other experiments, labeled receptors were observed to alter their locations and we were able to track and quantify their movements. We used a small molecule, ligand-directed probe to deliver synthetic fluorophores to endogenously expressed glutamate receptors for the purpose of tracking these receptors on live, hippocampal neurons. We found that clusters of receptors appear to move at similar rates to previous studies. We also found that the polyamine toxin pharmacophore likely binds to receptors in addition to calcium-permeable AMPA receptors.
已知神经元受体的亚细胞运输在突触发育、稳态和可塑性中起关键作用。我们开发了一种配体靶向且可光裂解的探针，用于将合成荧光团递送至神经元中天然表达的AMPA受体。受体与探针分子的配体部分结合后，一种蛋白质亲核试剂与探针上的亲电试剂发生反应，使这两种物质共价结合。然后可通过光解去除配体，使受体恢复到未结合配体的状态，同时受体与荧光团之间的新共价键保持完整。该策略用于标记来自大鼠的活海马神经元中的多胺敏感受体，包括钙通透AMPA受体。在此，我们描述了一些实验，在这些实验中我们研究了特异性、竞争性和浓度对标记效率的影响，并对受体运输进行了定量。在标记步骤中，用竞争性或非竞争性谷氨酸受体拮抗剂进行药理学竞争，可阻止在没有阻滞剂的情况下观察到的大部分标记。在其他实验中，观察到标记的受体改变了它们的位置，并且我们能够追踪和量化它们的移动。我们使用一种小分子、配体导向的探针将合成荧光团递送至内源性表达的谷氨酸受体，以便在活的海马神经元上追踪这些受体。我们发现受体簇的移动速度似乎与先前的研究相似。我们还发现，除了钙通透AMPA受体外，多胺毒素药效基团可能还与其他受体结合。
