DRUG-CONJUGATED NANOCRYSTALS AS FLUORESCENT LABELS

细胞表面受体、离子通道和转运蛋白是神经系统和靶位点的信号传导和兴奋性的关键组成部分。 因此，它们代表了制药行业目前探索的大多数药物靶点。 目前，膜蛋白的检测、定量和定位主要是使用放射性标记的配体或间接使用抗体技术来实现的。 这些方法是有限的，由于放射性示踪剂研究的空间分辨率差和膜蛋白的表面结构域选择性抗体探针的有限可用性。在拟议的工作中，一种新的荧光标记策略，使用药物共轭纳米晶体将开发。 该方法将使得能够使用荧光显微镜在真实的时间内监测蛋白质运输。 将使用多巴胺、去甲肾上腺素和5-羟色胺转运蛋白以及5-羟色胺（5 HT 2）受体对该策略进行测试。 将合成苯托烷缀合的纳米晶体，并通过对转运蛋白转染的细胞进行氚化多巴胺、去甲肾上腺素和5-羟色胺通量测定来确定其对转运蛋白的亲和力。 将合成苯基异丙胺-N-羟乙基氨基甲烷缀合物，并在受体转染的细胞中使用竞争结合和磷酸肌醇水解测定来确定其标记5 HT 2受体的能力。 将使用落射荧光和共聚焦成像来确定纳米缀合物是否提供用于检测非透化细胞中的转运蛋白和受体的合适信号。 为了用荧光显微镜跟踪转运蛋白或受体的运输，将纳米标记的细胞暴露于已知引起细胞表面转运蛋白和受体的内化和再分布的外源性调节剂。

Cell surface receptors, ion channels and transporters are critical components of signaling and excitability in the nervous system and at target sites. As such they represent the majority of drug targets currently explored in the pharmaceutical industry. Currently the detection, quantitation and localization of membrane proteins is achieved largely using radiolabeled ligands or indirectly with antibody techniques. These approaches are limited due to the poor spatial resolution of radiotracer studies and the limited availability of surface domain-selective antibody probes for membrane proteins. In the proposed work a novel fluorescence labeling strategy using drug-conjugated nanocrystals will be developed. This method will enable the monitoring of protein trafficking in real time using fluorescence microscopy. This strategy will be tested using dopamine, norepinephrine and serotonin transporters as well as serotonin (5HT2) receptors. Phenyltropane-conjugated nanocrystals will be synthesized and their affinity for the transporters will be determined by tritiated dopamine, norepinephrine, and serotonin flux assays on transporter transfected cells. Phenylisopropylamine-nanocrystal conjugates will be synthesized and their ability to label 5HT2 receptors will be determined using competition binding and phosphoinositide hydrolysis assays in receptor transfected cells. Epifluorescence and confocal imaging will be used to determine whether the nanoconjugates provide suitable signal for detection of the transporters and receptors in nonpermeabilized cells. To follow transporter or receptor trafficking with fluorescence microscopy nanocrystal-labeled cells will be exposed to exogenus modulators known to cause internalizatioin and redistribution of cell surface transporters and receptors.