To explore the potential for use of ligand-conjugated nanocrystals to target cell surface receptors, ion channels, and transporters, we explored the ability of serotonin-labeled CdSe nanocrystals (SNACs) to interact with antidepressant-sensitive, human and Drosophila serotonin transporters (hSERT, dSERT) expressed in HeLa and HEK-293 cells. Unlike unconjugated nanocrystals, SNACs were found to dose-dependently inhibit transport of radiolabeled serotonin by hSERT and dSERT, with an estimated half-maximal activity (EC(50)) of 33 (dSERT) and 99 microM (hSERT). When serotonin was conjugated to the nanocrystal through a linker arm (LSNACs), the EC(50) for hSERT was determined to be 115 microM. Electrophysiology measurements indicated that LSNACs did not elicit currents from the serotonin-3 (5HT(3)) receptor but did produce currents when exposed to the transporter, which are similar to those elicited by antagonists. Moreover, fluorescent LSNACs were found to label SERT-transfected cells but did not label either nontransfected cells or transfected cells coincubated with the high-affinity SERT antagonist paroxetine. These findings support further consideration of ligand-conjugated nanocrystals as versatile probes of membrane proteins in living cells.
Norepinephrine (NE) transporters (NETs) terminate noradrenergic synaptic transmission and represent a major therapeutic target for antidepressant medications. NETs and related transporters are under intrinsic regulation by receptor and kinase-linked pathways, and clarification of these pathways may suggest candidates for the development of novel therapeutic approaches. Syntaxin 1A, a presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, interacts with NET and modulates NET intrinsic activity. NETs colocalize with and bind to syntaxin 1A in both native preparations and heterologous systems. Protein kinase C activation disrupts surface NET/syntaxin 1A interactions and downregulates NET activity in a syntaxin-dependent manner. Syntaxin 1A binds the NH(2) terminal domain of NET, and a deletion of this domain both eliminates NET/syntaxin 1A associations and prevents phorbol ester-triggered NET downregulation. Whereas syntaxin 1A supports the surface trafficking of NET proteins, its direct interaction with NET limits transporter catalytic function. These two contradictory roles of syntaxin 1A on NET appear to be linked and reveal a dynamic cycle of interactions that allow for the coordinated control between NE release and reuptake.
Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585-602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine- and dopamine-containing neurons. Intracerebroventricular anti-dopamine beta-hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP-25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states.
The prefrontal cortex (PFC) is a likely site of action for the therapeutic efficacy of antidepressants that inhibit norepinephrine (NE) reuptake. Moreover, drugs that block the NE transporter (NET) increase extracellular levels of both NE and dopamine (DA), an interaction that may contribute to their therapeutic properties. To examine the subcellular localization of NET and to investigate the spatial relationships between presumed NE and DA axons within the rat prelimbic PFC, we combined immunogold-silver localization of NET with immunoperoxidase staining for the catecholamine synthetic enzyme tyrosine hydroxylase (TH). An additional aim was to quantify the proportion of profiles dually labeled for NET and TH to test the common observation that TH immunolabeling is relatively selective for DA axons. NET-immunoreactive (NET-ir) axonal profiles were typically unmyelinated and occasionally were observed to form symmetric axodendritic synapses. The majority of immunogold NET labeling was unexpectedly observed in the cytoplasm rather than on the plasma membrane. Furthermore, in tissue dually labeled for both NET and TH, only 8-10% of profiles contained both markers. Unlike observations for singly labeled profiles, gold-silver particles for NET in dually labeled axons were localized primarily to the plasmalemma. A systematic survey of terminals labeled only for TH revealed that they were typically separated by at least 1.2 mum from NET-ir varicosities, and the two profile types were not seen to contact common targets. These results suggest that, in the rat PFC, NE axons (1) contain predominantly cytoplasmic NET, (2) infrequently contain TH immunolabeling, and (3) may interact with probable DA afferents by means of extrasynaptic mechanisms.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.