Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter

Joel W Schwartz,Gaia Novarino,David W Piston,Louis J Defelice

doi:10.1074/jbc.m412923200

Abstract

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 homomultimers, 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 micros. 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.

Highlights

Adrenergic signaling in the central nervous system modulates learning and memory, the fight-or-flight response, and the reception of pain [2]
The human norepinephrine (NE) transporter 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

Summary

Introduction

Adrenergic signaling in the central nervous system modulates learning and memory, the fight-or-flight response, and the reception of pain [2]. Related transporters for serotonin (SERT) and dopamine (DAT) function as multimers [20, 21], suggested by fluorescence resonance energy transfer [22] and cross-linking experiments [23]. Directional cross-linking and Zn2ϩ binding suggest that DAT consists of homodimers (24 –26), and Kocabas et al [19] measured oligomerization by co-immunoprecipitation of tagged NETs. monoamine transporters function as homomultimers [27], but the number of bound substrates per functional unit is unknown. The accepted mechanism for co-transporters is the fixed stoichiometry, alternating access model [29], which predicts NET transports 1NE:1Na:1Cl roughly once per second [30, 31]; NET-. NET Binding Kinetics and Stoichiometry mediated currents are 100 times larger than predicted by this model [32]. Similar currents exist in native cells [33,34,35,36,37,38], and the charge-to-substrate ratio is an important key to mechanism [39]

Methods

Results

Conclusion