Novel fluorescence-based approaches for the study of biogenic amine transporter localization, activity, and regulation

Pre-synaptic norepinephrine (NE) and dopamine (DA) transporters (NET and DAT) terminate catecholamine synaptic transmission through reuptake of released neurotransmitter. Recent studies reveal that NET and DAT are tightly regulated by receptor and second messenger-linked signaling pathways. Common a...

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Bibliographic Details
Published in:	Journal of neuroscience methods Vol. 143; no. 1; pp. 3 - 25
Main Authors:	Mason, J.N., Farmer, H., Tomlinson, I.D., Schwartz, J.W., Savchenko, V., DeFelice, L.J., Rosenthal, S.J., Blakely, R.D.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 15-04-2005
Subjects:	Antibodies - chemistry ASP Biological Assay - methods Cadmium Compounds - chemistry Catecholamine Catecholamines - metabolism Cell Line Dopamine Dopamine Plasma Membrane Transport Proteins Enzyme Inhibitors - pharmacology Fluorescent Dyes - chemistry Humans Immunoassay - methods Kinetics Ligands Membrane Glycoproteins - analysis Membrane Glycoproteins - antagonists & inhibitors Membrane Glycoproteins - metabolism Membrane Transport Modulators Membrane Transport Proteins - analysis Membrane Transport Proteins - antagonists & inhibitors Membrane Transport Proteins - metabolism Nerve Tissue Proteins - analysis Nerve Tissue Proteins - antagonists & inhibitors Nerve Tissue Proteins - metabolism Neurochemistry - methods Norepinephrine Norepinephrine Plasma Membrane Transport Proteins Peptides - chemistry Pyridinium Compounds - chemistry Quantum dots Selenium Compounds - chemistry Sulfides - chemistry Symporters - analysis Symporters - antagonists & inhibitors Symporters - metabolism Temperature Zinc Compounds - chemistry Norepinephrine Dopamine Catecholamine Quantum dots ASP
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Summary:	Pre-synaptic norepinephrine (NE) and dopamine (DA) transporters (NET and DAT) terminate catecholamine synaptic transmission through reuptake of released neurotransmitter. Recent studies reveal that NET and DAT are tightly regulated by receptor and second messenger-linked signaling pathways. Common approaches for studying these transporters involve use of radiolabeled substrates or antagonists, methods possessing limited spatial resolution and that bear limited opportunities for repeated monitoring of living preparations. To circumvent these issues, we have explored two novel assay platforms that permit temporally resolved quantitation of transport activity and transporter protein localization. To monitor the binding and transport function of NET and DAT in real-time, we have investigated the uptake of the fluorescent organic compound 4-(4-diethylaminostyryl)- N-methylpyridinium iodide (ASP +). We have extended our previous single cell level application of this substrate to monitor transport activity via high-throughput assay platforms. Compared to radiotracer uptake methods, acquisition of ASP + fluorescence is non-isotopic and allows for continuous, repeated transport measurements on both transfected and native preparations. Secondly, we have extended our application of small-molecule-conjugated fluorescent CdSe/ZnS nanocrystals, or quantum dots (Qdots), to utilize antibody and peptide ligands that can identify surface expressed transporters, receptors and other membrane proteins in living cell systems. Unlike typical organic fluorophores, Qdots are highly resistant to bleaching and can be conjugated to multiple ligands. They can also be illuminated by conventional light sources, yet produce narrow, gaussian emission spectra compatible with multiple target visualization (multiplexing). Together, these approaches offer novel opportunities to investigate changes in transporter function and distribution in real-time with superior spatial and temporal resolution.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0165-0270 1872-678X
DOI:	10.1016/j.jneumeth.2004.09.028