Measurements of the Acidification Kinetics of Single SynaptopHluorin Vesicles

AbstractUptake of neurotransmitters into synaptic vesicles is driven by the proton gradient established across the vesicle membrane. The acidification of synaptic vesicles, therefore, is a crucial component of vesicle function. Here we present measurements of acidification rate constants from isolated, single synaptic vesicles. Vesicles were purified from mice expressing a fusion protein termed SynaptopHluorin created by the fusion of VAMP/synaptobrevin to the pH-sensitive super-ecliptic green fluorescent protein. We calibrated SynaptopHluorin fluorescence to determine the relationship between fluorescence intensity and internal vesicle pH, and used these values to measure the rate constant of vesicle acidification. We also measured the effects of ATP, glutamate, and chloride on acidification. We report acidification time constants of 500 ms to 1 s. The rate of acidification increased with increasing extravesicular concentrations of ATP and glutamate. These data provide an upper and a lower bound for vesicle acidification and indicate that vesicle readiness can be regulated by changes in energy and transmitter availability

SVOP Is a Nucleotide Binding Protein

Background: Synaptic Vesicle Protein 2 (SV2) and SV2-related protein (SVOP) are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF) family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1). In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties. Methodology/Principal Findings: We performed in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[c] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9–12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A. Conclusions/Significance: SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately

Loss of the Synaptic Vesicle Protein SV2B Results in Reduced Neurotransmission and Altered Synaptic Vesicle Protein Expression in the Retina

The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content

Levetiracetam Reverses Synaptic Deficits Produced by Overexpression of SV2A

Levetiracetam is an FDA-approved drug used to treat epilepsy and other disorders of the nervous system. Although it is known that levetiracetam binds the synaptic vesicle protein SV2A, how drug binding affects synaptic functioning remains unknown. Here we report that levetiracetam reverses the effects of excess SV2A in autaptic hippocampal neurons. Expression of an SV2A-EGFP fusion protein produced a ∼1.5-fold increase in synaptic levels of SV2, and resulted in reduced synaptic release probability. The overexpression phenotype parallels that seen in neurons from SV2 knockout mice, which experience severe seizures. Overexpression of SV2A also increased synaptic levels of the calcium-sensor protein synaptotagmin, an SV2-binding protein whose stability and trafficking are regulated by SV2. Treatment with levetiracetam rescued normal neurotransmission and restored normal levels of SV2 and synaptotagmin at the synapse. These results indicate that changes in SV2 expression in either direction impact neurotransmission, and suggest that levetiracetam may modulate SV2 protein interactions

Comparison of the nucleotide binding sites in Glut1, SV2A and SVOP.

<p>Comparison of the nucleotide binding sites in Glut1, SV2A and SVOP suggests convergent evolution of nucleotide binding. Predicted 12 transmembrane domains are depicted. Red lines represent the nucleotide binding domains. The amino and carboxy termini of the proteins are indicated with letter N and C.</p

SVOP demonstrates highest affinity for NAD.

<p>SVOP-FLAG was labeled with 100 µM 8-azido-ATP in the absence or presence of ATP (0.25–1.5 mM) or NAD (0.25–1.5 mM). Data were expressed as the percentage of 8-azido-ATP labeling according to control with no ATP or NAD in the reaction. A and B were representative western blot results of SVOP labeling in the presence of different concentration of ATP (A) or NAD (B). C shows the quantification of the western blot results. Error bars represent SEM, n = 3. The half maximum inhibition concentration of NAD and ATP on SVOP ATP binding is about 0.25 mM and 0.75 mM, respectively.</p

SV2AB and CIP data

<p>ATP hydrolysis reactions were conducted at 25°C with purified recombinant SV2-FLAG or commercially available calf intestinal alkaline phosphatase (CIP). An aliquot of CIP was heated at 95 °C for 20 min to inactivate the enzyme.</p> <p>At designated intervals (0 min, 1 min, 2 min, 5 min and 10 min), aliquots were removed from the reaction for analysis by the malachite green assay described as above. Duplicates were assayed for each time point and the average value of the duplicates was used for quantification.</p

Nucleotide specificity of the 8-azido-ATP binding to SVOP.

<p>SVOP-FLAG was labeled with 100 µM 8-azido-ATP in the absence or presence of 1 mM indicated competitive nucleotides. Samples were subjected to SDS-PAGE and western blot followed by quantitative analysis. Panel A shows a representative western blot result. Panel B shows the quantification of the western blot data. The error bars represent SEM, n = 5.</p

The nucleotide binding site in SVOP localizes to the C-terminal half of the protein.

<p>A. 8-azido-ATP photoaffinity labeled SVOP-FLAG was cleaved by hydroxylamine and the samples were subjected to western blot analysis using anti-FLAG, anti-SVOP and ExtrAvidin-HRP. Arrowhead indicated an N-terminal fragment which is recognized by anti-SVOP antibody but not labeled by 8-azido-ATP. The Arrows indicate a C-terminal fragment which is labeled by 8-azido-ATP and anti-FLAG antibody. B. Only the C-terminal half of SVOP shows dominant nucleotide binding. N- and C-terminal halves of SVOP-FLAG were expressed and purified from HEK293 cells. Photoaffinity labeling was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005315#s4" target="_blank"><i>Methods</i></a>. C. Shown are the results of trypsin digestion of 8-azido-ATP labeled SVOP-FLAG. Labeled protein was digested at 37°C in the presence of trypsin. At the time periods indicated, an aliquot was withdrawn and subjected to analysis as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005315#s4" target="_blank"><i>Methods</i></a>. The arrowheads indicate the smallest trypsinized 15 kDa fragment which is labeled by both 8-azido-ATP and anti-FLAG antibody. The proportion of total anti-FLAG and 8-azido-ATP labeling (undigested protein) was similar for the 15 KDa fragment (∼9% in both cases), consistent with it being labeled with the same efficiency as the full-length protein. Therefore the nucleotide-binding site is contained within this 15 KDa fragment. The image of 8-azido-ATP labeling blot was adjusted with contrast to show better results.</p