A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity

Abstract

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.

Fig. 1.

Toxin or antisense disruption of syntaxin 1A diminishes NE transport activity. A, Antisense suppression of syntaxin 1A expression reduces NE transport in CADhNET cells. CADhNET cells were transfected with mouse sense or antisense syntaxin 1A oligonucleotides and assayed for syntaxin content or NE transport activity 2 d later. The cells in one well for each transfection were lysed in PBS/1% Triton X-100, assayed for protein, and then immunoblotted for syntaxin 1A content. Cells treated with antisense syntaxin 1A oligonucleotides (8.6 μm) displayed a reduction of syntaxin 1A protein, compared with the cells treated with sense oligonucleotides. In parallel, a dose-dependent effect of syntaxin 1A antisense oligonucleotides on NE transport activity was observed. Results are mean values ± SEM (n = 3); *p < 0.05; Student's t test.B, Treatments with BoNT/affect syntaxin cleavage and reduce NE transport in native rat tissues. Minced rat vas deferens and synaptosomes from rat brain cortex as well as SK-N-SH cells and CADhNET cells were incubated with BoNT/C1 for 1 hr at 37°C before NE transport assay. Aliquots of tissue extracts or cell lysates treated with BONT/C1 (+) or vehicle (−) were immunoblotted for syntaxin 1A content. BONT/C1-treated tissues or cells displayed weak or no immunoreactivity for syntaxin 1A in contrast to vehicle-treated cells. Values reported are mean transport activities ± SEM (n = 3); *p < 0.05; Student'st test.

Fig. 2.

Colocalization of NET and syntaxin 1A in sympathetic axons. Mouse superior cervical ganglion cultures were cultured for 5 d and stained for NET and syntaxin 1A as described in Materials and Methods. In A–C, double staining was performed by using permeabilized, fixed cells to reveal total NET (antibody 43408; A) and syntaxin immunoreactivity (B); a merged image is shown in C. In D, labeling of live, nonpermeabilized cells was achieved with 43408 antibody to detect surface NET protein, followed by permeabilization to detect cytoplasmic labeling of syntaxin 1A (E). Note the overlap in labeling apparent as yellow fluorescence in the merged image (F), particularly evident at varicosities (arrows). Colocalization of NET and syntaxin 1A in rat vas deferens is shown in G–L. Frozen sections of rat vas deferens were double labeled with anti-NET (43411 antibody) and anti-syntaxin 1A as described in Materials and Methods, and the immunofluorescence was detected by confocal microscopy. The merged images (I, L) demonstrate the discontinuous and colocalized expression of NET and syntaxin 1A along sympathetic axonsin vivo. Scale bars: A–C, 15 μm;D–F, 7 μm, G–L, 5 μm.

Fig. 3.

Coimmunoprecipitation of NET and syntaxin 1A.A, Syntaxin 1A coimmunoprecipitates with NET. Solubilized rat vas deferens membranes were immunoprecipitated with NET antisera 43411 or preimmune serum, and complexes were resolved by SDS-PAGE, followed by immunoblotting for syntaxin 1A. An aliquot of the total extracts was blotted in parallel. B, Coimmunoprecipitation of NET and syntaxin 1A is diminished in vas deferens extracts from NET knock-out mice. Extracts were prepared from wild-type C57BL/6 (+/+) and homozygous null (−/−) NET knock-out mice as described for rat preparations and were immunoprecipitated with NET antibody 43411 before syntaxin 1A immunoblot. Total extracts were blotted for syntaxin 1A in parallel and showed no loss of syntaxin as a result of NET deficiency. C, Coimmunoprecipitation of syntaxin 1A and His-NET in cotransfected CHO cells. As noted by others (Bittner et al., 1996; Rowe et al., 1999), reduced concentrations of syntaxin 1A cDNA were required in cotransfection studies to limit the suppression of hNET biosynthetic progression, observed as diminished N-glycosylated cell surface transporters (see below). CHO cells grown in six-well plates were singly or cotransfected with His-tagged hNET (670 ng) and full-length syntaxin 1A (45 ng). Cell lysates were immunoprecipitated with anti-His, resolved on SDS-PAGE, and blotted for syntaxin 1A. Immunoprecipitations also were performed with extracts mixed from separately transfected cells (Mix). Aliquots of total cell lysates show equal expression of syntaxin 1A in each transfection (Total) except for lysates derived from cells transfected with only hNET. CHO cells do not express endogenous syntaxin 1A. D, Coexpression of Munc18 diminishes recovery of syntaxin 1A from NET immunoprecipitates. CHO cells were transfected with syntaxin 1A alone (S, 42 ng), His-hNET (640 ng) and syntaxin 1A (42 ng) (N+S), or His-hNET (640 ng), syntaxin 1A (42 ng), and Munc18 (318 ng) (N+S+M). pcDNA3 was used to adjust transfections to 1 μg of total DNA for N and N+S. Extracts were immunoprecipitated with anti-HIS before SDS-PAGE and syntaxin 1A immunoblots. Blots of total cell lysates reveal equivalent expression of syntaxin 1A. Results presented in A–D are representative of two to six experiments for each condition.

Fig. 4.

Syntaxin 1A binds NET directly via sequences in the NH2 terminus of the transporter. A, GST-SynΔTM pull-down of NET protein. COS-7 cells were transfected with HA-tagged hNET, and detergent lysates were incubated with glutathione beads precoated with either GST (GST) or GST-SynΔTM (Syn). Proteins bound to the beads were eluted and subjected to SDS-PAGE, followed by immunoblotting with anti-HA. Unlike GST beads, GST-SynΔTM beads retrieved NET proteins both in the immature and mature forms. B, Direct binding of syntaxin 1A cytoplasmic domain to the hNET NH₂ terminus. Amylose resins, precoated with equimolar MBP, MBP-hNET NH₂ terminal protein (MBP-N), or MBP-hNET COOH terminal protein (MBP-C), were incubated with GST-SynΔTM as described in Materials and Methods, followed by elution of bound material, SDS-PAGE, and immunoblotting for syntaxin 1A. Only MBP-N retained GST-SynΔTM. Membranes subsequently were stained with Coomassie brilliant blue to reveal equivalent amounts of MBP fusion proteins used in the experiments. C, An hNET NH₂ terminal deletion disrupts NET/syntaxin 1A coimmunoprecipitation. The top panel shows an immunoblot of hNET along with the NH₂ terminal deletion mutants that were used. CHO cells were transfected with either His-hNET (wtNET) or His-hNET mutants NΔ2–42 or NΔ43–64. Aliquots of extracts were analyzed in SDS-PAGE and probed with polyclonal anti-His antibody to reveal equivalent expression. The bottom panel shows results of cotransfection of N or NET mutants (670 ng) with syntaxin 1A (45 ng)/coimmunoprecipitation experiments, immunoprecipitating with anti-His and probing for syntaxin 1A. The NΔ2–42 mutant significantly diminished syntaxin 1A recovery relative to wt hNET or NΔ43–64. Syntaxin 1A expression was equivalent in all samples as assessed with syntaxin 1A immunoblots of total cell extracts. Results presented in A–C are representative of three to six experiments for each condition.

Fig. 5.

Phorbol ester and okadaic acid modulate NE transport and levels of NET/syntaxin 1A complexes in rat vas deferens.A, NE transport activity of rat vas deferens. Minced vas deferens was pretreated with DMSO (Veh), 1 μm β-PMA (PMA), or 1 μmokadaic acid (OK) for 30 min at 37°C and subjected to evaluation of NE transport activity. MeanK_m and V_maxvalues were obtained from three separate experiments; forV_max, control = 6.9 ± 0.7 pmol/mg per min, PMA = 4.8 ± 0.47 pmol/mg per min, and OK = 4.1 ± 0.55 pmol/mg per min.V_max values of PMA and OK are different from the control value in the analysis of one-way ANOVA, followed by Tukey's test;p < 0.05. The mean K_mvalues were control = 416 ± 32 nm, PMA = 391 ± 22 nm, and OK = 364 ± 41 nm. The differences of K_m values are not statistically significant. B,Top, Evaluation of NET/syntaxin coimmunoprecipitation after phorbol ester or okadaic acid treatment. Minced rat vas deferens was pretreated with DMSO (Veh), 1 μm PMA (PMA), or 1 μm okadaic acid (OK), as described in A, before extraction, immunoprecipitation with anti-NET sera 43411, SDS-PAGE, and immunoblotting for syntaxin 1A. Total extracts from all treatments were blotted in parallel for syntaxin 1A and revealed equivalent levels.Bottom, Average syntaxin band density ± SEM from three different immunoprecipitation experiments conducted as intop panel were quantitated by densitometric scanning; the values obtained after phorbol ester or okadaic acid treatments are expressed as a percentage of syntaxin 1A levels found in the vehicle-treated sample. *Significant loss of syntaxin 1A from NET immunoprecipitates as assessed by one-way ANOVA, followed by Tukey's comparisons of group means; p < 0.05.C, Phorbol ester-induced downregulation of NET activity in rat synaptosomes is lost after BONT/C1 pretreatment. Rat cortical synaptosomes, prepared as described in Materials and Methods, were pretreated with BONT/C1 (100 nm) 1 hr before treatment with either vehicle or 1 μm PMA for 30 min, followed by assay of [³H]NE transport as described in Materials and Methods. Results reflect the mean of three experiments ± SEM; *p < 0.05; Student's t test.

Fig. 6.

Regulated association of NET and syntaxin 1A in cotransfected CHO cells. A, Phorbol ester or okadaic acid treatments diminish recovery of syntaxin 1A from NET immunoprecipitations. CHO cells, cotransfected with His-hNET and syntaxin 1A, were preincubated with DMSO (Veh), 1 μm β-PMA (PMA), or 1 μmokadaic acid (OK) for 30 min at 37°C before immunoprecipitation with anti-His, SDS-PAGE, and immunoblotting for syntaxin 1A. Total cell extracts for each condition were blotted for syntaxin 1A in parallel. Both PMA and okadaic acid diminished recovery of syntaxin 1A relative to vehicle-treated samples. B, Muscarinic receptor activation diminishes recovery of syntaxin 1A from NET immunoprecipitates. Stable M3 muscarinic receptor-transfected CHO cells that had been cotransfected transiently with His-hNET and syntaxin 1A were treated with the indicated concentrations (in μm) of the muscarinic agonists methacholine or carbachol (30 min) before extraction and immunoprecipitation of complexes with anti-His, SDS-PAGE, and blotting for syntaxin 1A. In parallel, syntaxin 1A was blotted from total cell extracts and is evident at equivalent levels in all conditions. C, Phorbol ester regulation of the interaction between NET and syntaxin 1A occurs with plasma membrane-localized complexes. CHO cells, cotransfected with His-hNET and syntaxin 1A, were treated with DMSO (Veh) or 1 μm β-PMA for 30 min at 37°C. Surface proteins were labeled with NHS-sulfo-biotin at 4°C before cell lysis and recovery of surface complexes (Surface) on avidin beads. Bound proteins were eluted with 2 mm biotin, immunoprecipitated with anti-His, resolved on SDS-PAGE, and immunoblotted for syntaxin 1A. Nonbound (Intra) extracts were immunoprecipitated and blotted in parallel. Phorbol ester-induced reduction in syntaxin 1A in NET immunoprecipitates is evident in surface fractions, but not in intracellular complexes. Blots of total cell extracts (Total) and nonbiotinylated, intracellular samples (Intra Total) show no impact of phorbol ester on syntaxin 1A content. PMA increased syntaxin 1A contents in total biotinylated pools (Surface Total). The bar graph on the right is a quantitation of syntaxin 1A recovery in the immunoprecipitates. D, hNET NH₂ terminal deletion that disrupts NET/syntaxin 1A interactions diminishes phorbol ester-mediated NET downregulation. CHO cells were transfected with His-hNET (wt) or hNET NΔ2–42 as described in Figure 4, followed by treatment of cells with 1 μm β-PMA or vehicle for 30 min. Cells receiving hNET NΔ2–42 were significantly less sensitive to phorbol ester treatment with respect to NE transport activity (n = 3; *p < 0.05; Student's ttest).

Fig. 7.

Syntaxin reversibly inhibits NET-mediated single channel currents. A, Four representative current traces of single channel activity measured from an individual inside-out membrane excised patch held at –80 mV with sequential application of GST (3 μm), GST-SynΔTM (3 μm), and cocaine (20 μm). The patch pipette was filled with 30 μm NE (inset) to open maximally the NET-dependent channels. B, Amplitude histograms for each trace were analyzed to calculate the cumulative open probabilityNP_o for the NET-mediated channel-like events. Statistical analysis of the NP_o determined from four independent experiments is shown. TheNP_o of the channel decreased by 83% after perfusion of the membrane patch with GST-syntaxin 1AΔTM, compared with GST alone; NP_o = 10.2 ± 2.8 to 1.7 ± 0.3%, respectively. The channel-like activity was recovered by perfusion with GST alone (NP_o = 9.2 ± 1.9%). Cocaine sensitivity, tested after completion of the reversal experiments, revealed the hNET origin of the channel-like activity, reducingNP_o to 1.4 ± 0.4%. *p < 0.05 by one-way ANOVA, followed by Tukey's test.

Fig. 8.

Fusion-incompetent form of syntaxin (SynΔTM) increases NET surface expression in CAD cells without an increase in NE transport activity. A, HA-hNET-transfected (100 ng) CAD cells (N) were transfected additionally with 100 ng SynΔTM (N+SΔ) or 150 ng SynΔTM (N+1.5SΔ). Surface proteins of transfected cells were labeled with NHS-sulfo-S-S-biotin, extracted, and isolated on streptavidin beads. Bound proteins were eluted, resolved by SDS-PAGE, and immunoblotted for NET by using anti-HA. SynΔTM increases recovery of surface biotinylated NET (top). Probing for NET in the whole extracts reveals no impact of SynΔTM on total NET levels (bottom). B, Impact of SynΔTM on NET activity in cells monitored for surface NET expression in parallel. Transfection, biotinylation, and NE transport assays were performed as described above, with one set of samples used for surface biotinylation/immunoblot with anti-HA and the other used for NE transport determinations. SynΔTM increases surface NET but does not increase NE transport activity. C, Analysis of the impact of SynΔTM on NET surface expression and NE transport activity in CAD cells. For cotransfection of HA-NET with SynΔTM (line with filled circles), CAD cells were transfected with 100–200 ng of HA-hNET and variable amounts of SynΔTM (50–300 ng). Surface NET proteins in immunoblots were quantitated and normalized to the amount of surface NET obtained in the absence of SynΔTM (set at 100%; open circle withX). Similarly, NE transport activity in these assays was compared with the activity of NET in the absence of SynΔTM cotransfection. The dotted line represents a comparison of surface NET density and transport activity when surface density was varied by varying the concentration of HA-hNET cDNA. Note that, whereas a comparable range of surface abundance is achieved via either of these two methods, transfection with SynΔTM blunts the ability of increasing hNET surface protein to result in an increase in NE transport activity.