Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A

Abstract

The norepinephrine (NE) transporter (NET) terminates noradrenergic signaling by clearing released NE at synapses. The activity of NET can be rapidly regulated by depolarization and receptor activation via Ca 2+ and kinase/phosphatase linked pathways. The SNARE protein syntaxin 1A (SYN1A) interacts with NET and influences transporter surface trafficking and catalytic activity. In this study, we establish a link between changes in intracellular Ca 2+ and SYN1A/NET interactions. SYN1A influenced NE transport only in the presence of Ca 2+ in brain cortical synaptosomes. Although NET/SYN1A associations were sensitive to manipulations of Ca 2+ in CHO cells, in vitro binding experiments using purified NET and SYN1A fusion proteins demonstrated a lack of direct Ca 2+ sensitivity. Disruption of NET/SYN1A interaction abolished inhibition of NE transport by phorbol ester (PMA) to activate protein kinase C (PKC), but had no effect on transport inhibition by the Ca 2+ calmodulin kinase (CaMK) inhibitor KN93. Furthermore, PMA enhanced Ca 2+ dependent modulation of NE transport in synaptosomes. Our data reveal roles for SYN1A in the Ca 2+ -dependent regulation of NET, likely reliant on regulation by PKC signaling, but independent of CaMK.

Ganglion-Specific Impairment of the Norepinephrine Transporter in the Hypertensive Rat

“…37 Similarly Mas receptor stimulation can upregulate NET via Akt and Erk1/2-dependent pathways in the SHR as a potential compensation pathway to maintain appropriate NE levels. 32 Regulation of transporter function by interaction of components of the vesicle release SNARE complex (predominantly syntaxin-1A) and NET have also been observed, 38,39 revealing a Ca 2+ -dependent NET regulation by a protein kinase C-dependent, calmodulin-activated kinase independent mechanism. This results in both an increase in surface trafficking of NET, but also a direct interaction that acts to limit NET catalytic function.…”

“…This results in both an increase in surface trafficking of NET, but also a direct interaction that acts to limit NET catalytic function. 38,39 When these studies are viewed together they highlight the complexity of the NET reuptake pathways, because NET dysregulation in the SHR could be residing at several intracellular sites.…”

Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization

“…4). Some of these NET interacting proteins modulate PKC-mediated NET regulation (5)(6)(7)(8). Signals linked to G protein-coupled receptor (GPCR) activation are also known to regulate NET (9,10).…”

“…Data were derived from three separate experiments, each in triplicate are given as mean Ϯ S.E. * indicates significant change (p Ͻ 0.01) in NE transport (one-way analysis of variance; Dunnett's test: F (4,8) ϭ 10.99; p Ͻ 0.01). B and C, surface biotinylation: synaptosomes treated with drugs as above were biotinylated and biotinylated NET or NK1R were isolated and analyzed as described under "Experimental Procedures."…”

LeuT: A prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure

“…Indeed, the SNARE protein syntaxin 1A complexes with the N-terminus to regulate ion conductance (refer to "Ion Dependence" section below) in the glycine transporter (GlyT1/GlyT2), 71 GAT1, 57 DAT, 58 NET 72 and SERT, 56 presumably by recognizing negatively-charged aspartate residues, 56 perhaps via a Ca 2+ -dependent process. 73 From a clinical perspective, the missing sections of EL2 as well as the N-and C-termini are significant because several disease-associated polymorphisms that affect transporter trafficking and regulation reside within these areas. 74 …”