article published Joel Schwartz author Gaia Novarino author 3E57A680-F248-11E8-B48F-1D18A9856A870000-0002-7673-7178 David Piston author Louis Defelice author 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. American Society for Biochemistry and Molecular Biology2005 eng 0021-9258 1083-351X 1575790410.1074/jbc.M412923200 2801919177 - 19184 yes 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>. J. Schwartz, G. Novarino, D. Piston, L. Defelice, Journal of Biological Chemistry 280 (2005) 19177–19184. 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> 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>. 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> 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. 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. 23072018-12-11T11:56:54Z2026-08-07T09:25:18Z