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.

Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo.

The primary mechanism for clearance of extracellular dopamine (DA) is uptake mediated by the dopamine transporter (DAT), which is governed, in part, by the number of functional DATs on the cell surface. Previous studies have shown that amphetamine (AMPH) decreases DAT cell surface expression, whereas insulin reverses this effect through the action of phosphatidylinositol 3-kinase (PI3K). Therefore, it is possible that AMPH causes DAT cell surface redistribution by inhibiting basal insulin signaling. Here, we show in a heterologous expression system and in murine striatal synaptosomes that AMPH causes a timedependent decrease in the activity of Akt, a protein kinase immediately downstream of PI3K. This effect was blocked by the DAT inhibitor cocaine, suggesting that AMPH must interact with DAT to inhibit Akt. We also showed that AMPH is able to stimulate Ca 2ϩ /calmodulin-dependent kinase II (CaMKII) activity, both in the heterologous expression system as well as in murine striatal synaptosomes. The ability of AMPH to decrease Akt activity was blocked by the CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93), but not by its inactive analog 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN92). Furthermore, preincubation with KN93 prevented the AMPH-induced decrease in DAT cell surface expression. Thus, AMPH, but not cocaine, decreases Akt activity through a CaMKII-dependent pathway, thereby providing a novel mechanism by which AMPH regulates insulin signaling and DAT trafficking. Dopaminergic neurotransmission is governed in part by the reuptake of extracellular DA via the dopamine transporter (DAT). Because DA uptake capacity is dependent on the DAT turnover rate, the affinity of the transporter for DA, and the number of functional transporters at the cell surface, regulation of DAT cell surface expression is important for tuning DA neurotransmission (Beckman et al., 1998;Robinson, 2001;Kahlig and Galli, 2003). The psychostimulant amphetamine (AMPH) causes DAT to redistribute away from the cell surface, decreasing DA clearance efficiency and providing an additional mechanism by which this DAT substrate increases extracellular DA (Sulzer et al., 2005).A variety of receptor signal transduction pathways have been shown to regulate neurotransmitter transporter trafficking as well as activity (Beckman et al

Norepinephrine (NE) transporters (NETs) are high-affinity transport proteins that mediate the synaptic clearance of NE after vesicular release. NETs represent a major therapeutic target for antidepressants and are targets of multiple psychostimulants including amphetamine (AMPH) and cocaine. Recently, we demonstrated that syntaxin 1A (SYN1A) regulates NET surface expression and, through binding to the transporter's NH 2 terminus, regulates transporter catalytic function. AMPH induces NE efflux and may also regulate transporter trafficking. We monitored NET distribution and function in catecholaminergic cell lines (CAD) stably transfected with either full-length human NET (CAD-hNET) or with an hNET N-terminal deletion (CADhNET⌬ 28 -47 cells). In hNET-CAD cells, AMPH causes a slow and small reduction of surface hNET with a modest increase in hNET/SYN1A associations at the plasma membrane. In contrast, in CAD-hNET⌬ 28 -47 cells, AMPH induces a rapid and substantial reduction in surface hNET⌬ 28 -47 accompanied by a large increase in plasma membrane hNET⌬ 28 -47 /SYN1A complexes. We also found that AMPH in CAD-hNET⌬ 28 -47 cells induces a robust increase in cytosolic Ca 2ϩ and concomitant activation of calcium/calmodulin-dependent protein kinase II (CaMKII). Inhibition of either the increase in intracellular Ca 2ϩ or CaMKII activity blocks AMPH-stimulated hNET⌬ 28 -47 trafficking and the formation of hNET⌬ 28 -47 /SYN1A complexes. Here, we demonstrate that AMPH stimulation of CAMKII stabilizes an hNET/SYN1A complex. This hNET/SYN1A complex rapidly redistributes, upon AMPH treatment, when mechanisms supported by the transporter's NH 2 terminus are eliminated.NET is responsible for the presynaptic elimination of NE after release at noradrenergic synapses (Iversen, 1971;Trendelenburg, 1991). NETs are targets for various psychostimulants, including cocaine and amphetamine (AMPH), and are antagonized by multiple antidepressants (Tatsumi et al., 1997). Topological predictions indicate that NET and its homologs bear 12 transmembrane domains with intracellular NH 2 and COOH termini. The 12 transmembrane domains topology has been supported recently by the high-resolution structure of LeuT, a prokaryotic sodium-dependent leucine transporter with significant homology to NET and related neurotransmitter transporters (Yamashita et al., 2005).The intracellular domains of NET have numerous putative phosphorylation sites for various protein kinases, and multiple protein kinases have been suggested to regulate NET function . For example, muscarinic receptors (e.g., M3) that are able to stimulate phospholipase C and protein kinase C (PKC) can induce the loss of cell surface NETs with a consequent loss of transport activity (Apparsundaram et al., 1998). Consistent with this observation, phorbol esters trigger a loss of NET surface expression in heterologous expression systems, rat vas deferens, and in forebrain synaptosomes (Apparsundaram et al., 1998;Sung et al

No 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.

Basement membrane laminin (laminin-I) is a multidomain glycoprotein that interacts with itself, heparin and cells. The interaction with heparinkeparan sulfate proteglycans is thought to be important for the architectural formation of basement membranes and adhesion to cells. The major heparin binding site has been known to reside in the long arm globular domain (G domain). The G domain is in turn subdivided into five subdomains (G1 -G5). In order to localize the heparin binding regions further, recombinant G domains (rG and rG5) were expressed in Sfp insect cells using baculovirus expression vector. By the limited proteolysis of recombinant G domains followed by either heparin affinity HPLC or overlay with radiolabeled heparin, the relative affinity of each subdomain to heparin was assigned as G l > G 2 = G4>G5>G3, such that G1 bound strongly and G3 not at all. Since the activity in GI-G3 is cryptic in intact laminin long arm [Sung, U., O'Rear, J.

Abstract. The long arm of laminin, which binds heparin and cells, consists of three polypeptides (A, B1, and B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). Previously, we found that recombinant globular domain (rG) supported heparin and myoblast binding (Yurchenco, P. D., U. Sung, M. D. Ward, Y. Yamada, and J. J. O'Rear. 1993. J. Biol. Chem. 268:8356-8365). To further analyze long arm functions, we expressed the distal moiety of the mouse laminin A chain extending from the middle of the rod to the carboxyl terminus (rAiG). This larger glycoprotein, secreted by Sf9 insect ceils infected with recombinant baculovirus, was intercalated in vitro into the corresponding disulfide-linked B chain segments of laminin fragment E8 (distal long arm rod and proximal globule). The hybrid molecule (B-rAiG) possessed a structure similar to laminin long arm as judged by electron microscopy and limited proteolysis. By joining rAiG with E8-B chains, the affinity of G domain for heparin decreased from that observed with rAiG and rG to one similar to native protein. HT1080 cells adhered to E8, rAiG, and B-rAiG, less well to rG, and not to denatured E8/ B-rAiG, the A and B chain moieties of E8, or to a mixture of rG and E8-B chains. Cell adhesion to E8 and B-rAiG, in contrast to rAiG, was inhibited with antibodies specific for or6 and #1 integrin chains. Since intercalation (a) restored a conformationally dependent a6B1 integrin recognition site present in native protein, (b) inactivated a cryptic cell binding activity in the A chain, and (c) inhibited a heparin binding site present in proximal G domain, we conclude that biological activities of laminin are different from that of its isolated subunits.