Cell surface receptors, ion channels, and transporters are critical components of signaling and excitability in the nervous system. As such, they represent the majority of drug targets currently being explored in the pharmaceutical industry. Basic studies have revealed that these proteins are nonuniformly distributed on neurons and targets and this distribution can be impacted by multiple signal transduction pathways and endogenous regulatory programs. Moreover, many drugs appear to alter the responsiveness, distribution and/or surface abundance of their protein targets following chronic occupancy. Currently, the detection, quantitation and localintion 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, the limited availability of surface domain-selective antibody probes for membrane proteins, the broad emission spectra of available fluorophores and their photochemical degradation. In this proposal we will continue to develop our novel, non-isotopic, labeling strategies with a principal focus on drug-conjugated fluorescent nanocrystals (nanoconjugates) that can permit the imaging and quantitative analysis of cell surface receptor and transporter proteins. Specifically we will: I. Synthesize improved nanocrystal probes. II. Establish dynamic imaging of the serotonin transporter protein. III. Develop pharmacological assays that exploit the unique properties of drug-conjugated nanocrystals. To accomplish these aims we have assembled an interdisciplinary team of chemists, microscopists, pharmacologists and neuroscientists. The experiments proposed here exploit the unique optical properties of fluorescent nanocrystals and cannot be performed with traditional organic fluorophores or fluorescent proteins. In this proposal nanotechnology interfaces with neuroscience in a way that advances both fields.
The completion of this grant will result in a new class of selective, bright, compact drugconjugated nanocrystals for labeling neurotransmitter receptor and transporter proteins. Realtime imaging enabled by these probes may provide a new understanding of neural processes such as depression, addiction, and learning. Assays utilizing these probes may provide an improved platform for discovering better drugs for the treatment of mental illness. Ultimately these probes will enable a new level of inquiry in neuroscience.
|Kovtun, Oleg; Sakrikar, Dhananjay; Tomlinson, Ian D et al. (2015) Single-quantum-dot tracking reveals altered membrane dynamics of an attention-deficit/hyperactivity-disorder-derived dopamine transporter coding variant. ACS Chem Neurosci 6:526-34|
|Gussin, Hélène A; Tomlinson, Ian D; Cao, Dingcai et al. (2013) Quantum dot conjugates of GABA and muscimol: binding to ?1?2?2 and ?1 GABA(A) receptors. ACS Chem Neurosci 4:435-43|
|Chang, Jerry C; Rosenthal, Sandra J (2013) A Bright Light to Reveal Mobility: Single Quantum Dot Tracking Reveals Membrane Dynamics and Cellular Mechanisms. J Phys Chem Lett 4:2858-2866|
|Chang, Jerry C; Tomlinson, Ian D; Warnement, Michael R et al. (2012) Single molecule analysis of serotonin transporter regulation using antagonist-conjugated quantum dots reveals restricted, p38 MAPK-dependent mobilization underlying uptake activation. J Neurosci 32:8919-29|
|Solis Jr, Ernesto; Zdravkovic, Igor; Tomlinson, Ian D et al. (2012) 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter. J Biol Chem 287:8852-63|
|Chang, Jerry C; Kovtun, Oleg; Blakely, Randy D et al. (2012) Labeling of neuronal receptors and transporters with quantum dots. Wiley Interdiscip Rev Nanomed Nanobiotechnol 4:605-19|
|Chang, Jerry C; Rosenthal, Sandra J (2012) Visualization of lipid raft membrane compartmentalization in living RN46A neuronal cells using single quantum dot tracking. ACS Chem Neurosci 3:737-43|
|Tomlinson, Ian D; Iwamoto, Hideki; Blakely, Randy D et al. (2011) Biotin tethered homotryptamine derivatives: high affinity probes of the human serotonin transporter (hSERT). Bioorg Med Chem Lett 21:1678-82|
|Kovtun, Oleg; Tomlinson, Ian D; Sakrikar, Dhananjay S et al. (2011) Visualization of the cocaine-sensitive dopamine transporter with ligand-conjugated quantum dots. ACS Chem Neurosci 2:370-8|
|Rosenthal, Sandra J; Chang, Jerry C; Kovtun, Oleg et al. (2011) Biocompatible quantum dots for biological applications. Chem Biol 18:10-24|
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