DRUG-CONJUGATED NANOCRYSTALS AS FLUORESCENT LABELS

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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.