Abstract
Neurotransmitter gamma-aminobutyric acid (GABA) release to the synaptic clefts is mediated by the formation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which includes two target SNAREs syntaxin 1A and SNAP-25 and one vesicle SNARE VAMP-2. The target SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the putative intermediate of the SNARE complex. Neurotransmitter GABA clearance from synaptic clefts is carried out by the reuptake function of its transporters to terminate the postsynaptic signaling. Syntaxin 1A directly binds to the neuronal GABA transporter GAT-1 and inhibits its reuptake function. However, whether other SNARE proteins or SNARE complex regulates GABA reuptake remains unknown. Here we demonstrate that SNAP-25 efficiently inhibits GAT-1 reuptake function in the presence of syntaxin 1A. This inhibition depends on SNAP-25/syntaxin 1A complex formation. The H3 domain of syntaxin 1A is identified as the binding sites for both SNAP-25 and GAT-1. SNAP-25 binding to syntaxin 1A greatly potentiates the physical interaction of syntaxin 1A with GAT-1 and significantly enhances the syntaxin 1A-mediated inhibition of GAT-1 reuptake function. Furthermore, nitric oxide, which promotes SNAP-25 binding to syntaxin 1A to form the SNARE complex, also potentiates the interaction of syntaxin 1A with GAT-1 and suppresses GABA reuptake by GAT-1. Thus our findings delineate a further molecular mechanism for the regulation of GABA reuptake by a target SNARE complex and suggest a direct coordination between GABA release and reuptake.
Highlights
GABA is cleared away rapidly from synaptic clefts to terminate synaptic transmission through the reuptake function of its specific, high affinity, sodium- and chloride-dependent transporters [6], which are located on presynaptic terminals and surrounding glial cells [7]
synaptosomal associated protein of 25 kDa (SNAP-25)/Syntaxin 1A Are Intrinsically Involved in the Inhibition of GAT-1 Uptake Function—It has been shown that syntaxin 1A inhibits the uptake function of GABA transporter GAT-1 in hippocampal neurons [20, 22]
Because the inhibition of GAT-1 uptake function is mediated by syntaxin 1A, to further identify whether the inhibition by syntaxin 1A is special for the uptake function of GABA transporter GAT-1 subtype (599 amino acids), we examined the effects of syntaxin 1A on the uptake function of GAT-2 (602 amino acids) or GAT-3 subtype (627 amino acids)
Summary
Plasmids—The full lengths of SNAP-25 and VAMP-2 were cloned into modified pcDNA3 vector in-frame with HA at the N terminus. [3H]GABA Uptake Assay—GABA uptake in cultured neurons and transfected PC12 or HEK293 cells was performed as described previously [27]. The PC12 cells cotransfected FLAG-syntaxin 1A and/or HA-SNAP-25 with GAT-1-GFP were incubated with mouse anti-FLAG (1:500, Sigma) and/or rabbit anti-HA antibody (1:500, Sigma) overnight at 4 °C, followed by second antibodies conjugated with indocarbocyanine (Cy3) and indodicarbocyanine (Cy5) (1:100; Jackson ImmunoResearch). NOC-18 was added into the culture medium without serum to stimulate PC12 cells transfected with GAT-1-GFP and were washed away before immunohistochemistry. The supernatants were treated with protein G-agarose beads (Amersham Biosciences) followed by incubation with the immunoprecipitated antibodies (mouse anti-syntaxin 1A, mouse anti-SNAP25, rabbit anti-GAT-1, and mouse anti-HA) at 4 °C for 2– 4 h. Statistics differences were determined by Student’s t test for two group comparisons
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