{"intvolume":"       280","publist_id":"4619","publisher":"American Society for Biochemistry and Molecular Biology","abstract":[{"lang":"eng","text":"The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homo-multimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 μs. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP + molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP +/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates."}],"issue":"19","extern":1,"citation":{"chicago":"Schwartz, Joel, Gaia Novarino, David Piston, and Louis Defelice. “Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter.” <i>Journal of Biological Chemistry</i>. American Society for Biochemistry and Molecular Biology, 2005. <a href=\"https://doi.org/10.1074/jbc.M412923200\">https://doi.org/10.1074/jbc.M412923200</a>.","ieee":"J. Schwartz, G. Novarino, D. Piston, and L. Defelice, “Substrate binding stoichiometry and kinetics of the norepinephrine transporter,” <i>Journal of Biological Chemistry</i>, vol. 280, no. 19. American Society for Biochemistry and Molecular Biology, pp. 19177–19184, 2005.","ama":"Schwartz J, Novarino G, Piston D, Defelice L. Substrate binding stoichiometry and kinetics of the norepinephrine transporter. <i>Journal of Biological Chemistry</i>. 2005;280(19):19177-19184. doi:<a href=\"https://doi.org/10.1074/jbc.M412923200\">10.1074/jbc.M412923200</a>","ista":"Schwartz J, Novarino G, Piston D, Defelice L. 2005. Substrate binding stoichiometry and kinetics of the norepinephrine transporter. Journal of Biological Chemistry. 280(19), 19177–19184.","mla":"Schwartz, Joel, et al. “Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter.” <i>Journal of Biological Chemistry</i>, vol. 280, no. 19, American Society for Biochemistry and Molecular Biology, 2005, pp. 19177–84, doi:<a href=\"https://doi.org/10.1074/jbc.M412923200\">10.1074/jbc.M412923200</a>.","apa":"Schwartz, J., Novarino, G., Piston, D., &#38; Defelice, L. (2005). Substrate binding stoichiometry and kinetics of the norepinephrine transporter. <i>Journal of Biological Chemistry</i>. American Society for Biochemistry and Molecular Biology. <a href=\"https://doi.org/10.1074/jbc.M412923200\">https://doi.org/10.1074/jbc.M412923200</a>","short":"J. Schwartz, G. Novarino, D. Piston, L. Defelice, Journal of Biological Chemistry 280 (2005) 19177–19184."},"publication_status":"published","doi":"10.1074/jbc.M412923200","date_published":"2005-05-13T00:00:00Z","author":[{"last_name":"Schwartz","full_name":"Schwartz, Joel W","first_name":"Joel"},{"orcid":"0000-0002-7673-7178","full_name":"Gaia Novarino","last_name":"Novarino","first_name":"Gaia","id":"3E57A680-F248-11E8-B48F-1D18A9856A87"},{"first_name":"David","full_name":"Piston, David W","last_name":"Piston"},{"first_name":"Louis","full_name":"DeFelice, Louis J","last_name":"Defelice"}],"status":"public","year":"2005","type":"journal_article","day":"13","date_created":"2018-12-11T11:56:54Z","publication":"Journal of Biological Chemistry","volume":280,"month":"05","date_updated":"2021-01-12T06:56:40Z","page":"19177 - 19184","_id":"2307","title":"Substrate binding stoichiometry and kinetics of the norepinephrine transporter","quality_controlled":0}