Binding and Transport in Norepinephrine Transporters

Abstract

Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP ؉ (4-(4-(dimethylamino)-styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP ؉ accumulation is Na ؉ -, Cl ؊ -, cocaine-, and desipraminesensitive and temperature-dependent, and that ASP ؉ competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na ؉ and Cl ؊ , binding is independent of Na ؉ and actually increases in low Cl؊ . We further demonstrate that ASP ؉ interacts with transporters not only in transfected cells but in cultured neurons. ASP ؉ is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery.

Evidence for Distinct Sodium-, Dopamine-, and Cocaine-dependent Conformational Changes in Transmembrane Segments 7 and 8 of the Dopamine Transporter

“…9). Because dopamine and other substrates have been shown to bind to the hDAT and related transporters with unchanged affinity even in the absence of Na ϩ (23)(24)(25), this observation strongly supports the idea that the protection of M371C observed only in the presence of Na ϩ is the consequence of a transport-associated conformational change. Data from our previous studies also argue against position 371 being a direct part of the substrate binding crevice.…”

“…8), we were able to assess whether dopamine protection of this construct was Na ϩ -dependent. Transport is strictly Na ϩ -dependent, but increasing evidence suggests that dopamine and other substrates can bind to hDAT and related transporters with unchanged affinity even in the absence of Na ϩ (23)(24)(25). Accordingly, assessing the Na ϩ dependence of dopamine protection should enable us to distinguish between direct steric protection and a dopamineinduced conformational change that decreases the accessibility of M371C to MTSET.…”

APP+, a Fluorescent Analogue of the Neurotoxin MPP+, Is a Marker of Catecholamine Neurons in Brain Tissue, but Not a Fluorescent False Neurotransmitter

“…23 Further, the fluorescent analogues of MPP+, ASP+ and APP+, were shown to act as substrates for DAT and other monoamine transporters. [24][25][26]32,33 These observations therefore suggested that a fluorescent analogue of MPP+ might function as an FFN and provided the rationale for the present study.…”

“…29 This property was ascribed to light emission quenching via a twisted intramolecular charge transfer (TICT) mechanism, enabled by the perpendicular conformation of the two arene rings. These photophysical properties are shared by the other members of the pyridinium class of dyes, including ASP+, 24 2-(4-dimethylaminostyryl)-N-methylpyridinium (DASPMI), 30 and the FM dyes. 31 Recently, APP+ was also shown to be a substrate for DAT, NET, and SERT, providing a reporter substrate for this important group of plasma membrane transporters and enabling cell-based assays for examining their function and inhibition.…”

A Nonradioactive High-Throughput / High-Content Assay for Measurement of the Human Serotonin Reuptake Transporter Function In Vitro

“…1) has been used to stain nerve termini 9 and is a known substrate for the organic cation transporter (OCT). 10 Schwartz et al 11 recently reported that ASP may be used to measure the biophysical properties of monoamine transporters as its accumulation in norepinephrine reuptake transporter (hNET)−expressing cells (measured by fluorescence microscopy) was Na + , Cl − , and temperature dependent. In this article, we describe the use of ASP to develop a microplate-based high-throughput screen for hSERT function, using a microplate fluorimeter.…”