Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter

Schwartz, Janet; Novarino, Gaia; Piston, David W.; DeFelice, Louis J.

doi:10.1074/jbc.m412923200

Cited by 57 publications

(67 citation statements)

References 63 publications

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“…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.…”

Section: ■ Discussionmentioning

confidence: 66%

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

Karpowicz

Dunn

Sulzer

et al. 2013

ACS Chem. Neurosci.

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ABSTRACT:We have previously introduced fluorescent false neurotransmitters (FFNs) as optical reporters that enable visualization of individual dopaminergic presynaptic terminals and their activity in the brain. In this context, we examined the fluorescent pyridinium dye 4-(4-dimethylamino)phenyl-1-methylpyridinium (APP+), a fluorescent analogue of the dopaminergic neurotoxin MPP+, in acute mouse brain tissue. APP+ is a substrate for the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT), and as such represented a candidate for the development of new FFN probes. Here we report that APP+ labels cell bodies of catecholaminergic neurons in the midbrain in a DAT-and NET-dependent manner, as well as fine dopaminergic axonal processes in the dorsal striatum. APP+ destaining from presynaptic terminals in the dorsal striatum was also examined under the conditions inducing depolarization and exocytotic neurotransmitter release. Application of KCl led to a small but significant degree of destaining (approximately 15% compared to control), which stands in contrast to a nearly complete destaining of the new generation FFN agent, FFN102. Electrical stimulation of brain slices at 10 Hz afforded no significant change in the APP+ signal. These results indicate that the majority of the APP+ signal in axonal processes originates from labeled organelles including mitochondria, whereas only a minor component of the APP+ signal represents the releasable synaptic vesicular pool. These results also show that APP+ may serve as a useful probe for identifying catecholaminergic innervations in the brain, although it is a poor candidate for the development of FFNs.

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Section: ■ Discussionmentioning

confidence: 66%

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

Karpowicz

Dunn

Sulzer

et al. 2013

ACS Chem. Neurosci.

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“…Multimeric transporters bind one substrate per gene product, and thousands of binding and unbinding events appear to occur before transport ensues. On the basis of such data, Schwartz and colleagues (50) suggest that ASP + acts as a ligand that gates a substrate-permeant channel and evokes a substantial depolarizing current but is itself inefficiently transported. They propose that the rate-limiting step for ASP + translocation is not a large conformational change of the protein occurring continually throughout the transport cycle but rather reflects the low probability of transport following binding and may be more consonant with a gated-pore model of permeation.…”

Section: Substrate Binding and Transport: Watching Transporters At Workmentioning

confidence: 99%

“…Nonspecific surface labeling is expected due to the relatively low spatial resolution of confocal microscopy and cellar movement during the 2-min incubation. To improve resolution, total internal reflectance microscopy was implemented, identifying membrane subdomains enriched for ASP + labeling (50). These studies are the first to resolve binding and transport of a biogenic amine transporter substrate at the single-cell level and reveal nonuniformities in sites of expression whose presence may relate to membrane subdomains that support transporter regulation (25,35).…”

Section: Substrate Binding and Transport: Watching Transporters At Workmentioning

confidence: 99%

“…To gather insights into stoichiometric relationships between substrate and protein targets, ASP + labeling in combination with visualization of green fluorescent protein-tagged hNETs was used to measure substrate-transporter kinetics and stoichiometry (50). Calibrated confocal microscopy and fluorescence correlation spectroscopy revealed that hNETs, which exist as homomultimers, bind one substrate molecule per transporter subunit with an average dwell time (average substrate residence time on the transporter) of 526 Ϯ 25 s (FIGURE 1 ) emits a strong, easily detected red signal only when it is bound.…”

Section: Substrate Binding and Transport: Watching Transporters At Workmentioning

confidence: 99%

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Biogenic Amine Neurotransmitter Transporters: Just When You Thought You Knew Them

2005

Self Cite

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Beginning in the early 1990s, a new era dawned in studies of neurotransmitter transporter structure, function, and regulation, illuminated by the cloning of transporter cDNAs and genes, the development of transporter-specific gene and protein probes, and the characterization of heterologous expression systems suitable for advanced biophysical analyses. Among the advances in this field over the next decade were the elucidation of critical domains and residues supporting substrate and antagonist recognition, the discovery that transporters exist as multimeric protein complexes, the definition of N-glycosylation and phosphorylation as important posttranslational modifications, the recognition that transporters exhibited ion-channel states, and the elucidation of the first human disorders associated with transporter mutations. Many reviews are available that highlight these and other areas (4-6, 8, 15, 20, 31, 51). Together, they illustrate a field reaching maturity, yet poised for renaissance, in which long-held ideas are challenged by new techniques and data. In this short review, we describe several new approaches and findings from our labs and from colleagues in the field that have challenged us to reconsider aspects of biogenic amine transporter structure, function, and regulation. Substrate Binding and Transport: Watching Transporters at WorkThe measurements of substrate flux through neurotransmitter transporters has relied extensively on radiolabeled substrates. Although such molecules are sensitive probes for transporter activity from populations of transporter proteins, radiolabeled substrates have limited utility for the real-time monitoring of either binding or transport; they cannot illuminate a substrate's local environment or how it changes during substrate flux, and they cannot be used to identify cellular subdomains or relate population properties to individual molecules. Amperometric approaches offer some solutions to these limitations, as we will note below, but they are limited to paradigms such as in vitro (26, 45) or in vivo clearance measurements (10,14), in which the presence or absence of oxidizeable amine changes significantly as a function of time. Binding and transfer events that occur before changes in extracellular levels of diffusible neurotransmitter are achieved cannot be resolved. To move beyond these approaches, Schwartz et al. 1548-9213/05 8.00

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“…Therefore, examining DAT activity in its native environment or in heterologous expression system when D2R is blocked or removed via genetic manipulations [60] may confound the outcome since D2R is shown to interact with DAT and regulate the baseline and substrateinduced activity of the transporter [61]. and fine-tuned the methodology for utilization of ASP + to monitor DA uptake through DAT [62][63][64].This approach was later adopted by other groups [61,65,66]. [87][88][89].…”

mentioning

confidence: 99%

α-synuclein regulation of dopamine transporter

Butler

Saha

Khoshbouei

2012

Translational Neuroscience

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The development of effective therapeutic interventions for neurodegeneration requires a better understanding of the early events that precede neuronal loss. Recent work in various disease models has begun to emphasize the significance of presynaptic dysfunction as an early event that occurs before manifestation of neurological disorders. Dysregulation of dopamine (DA) homeostasis is implicated in neurodegenerative diseases, drug addiction, and neuropsychiatric disorders. The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. α-synuclein is a 140-amino acid protein that forms a stable complex with DAT and is linked to the pathogenesis of neurodegenerative disease. In this review we will examine the prevailing hypotheses for α-synuclein-regulation of DAT biology.

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Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter

Cited by 57 publications

References 63 publications

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

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

Biogenic Amine Neurotransmitter Transporters: Just When You Thought You Knew Them

α-synuclein regulation of dopamine transporter

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