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

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

Role of active metabolites in the use of opioids

The opioid class of drugs, a large group, is mainly used for the treatment of acute and chronic persistent pain. All are eliminated from the body via metabolism involving principally CYP3A4 and the highly polymorphic CYP2D6, which markedly affects the drug's function, and by conjugation reactions mainly by UGT2B7. In many cases, the resultant metabolites have the same pharmacological activity as the parent opioid; however in many cases, plasma metabolite concentrations are too low to make a meaningful contribution to the overall clinical effects of the parent drug. These metabolites are invariably more water soluble and require renal clearance as an important overall elimination pathway. Such metabolites have the potential to accumulate in the elderly and in those with declining renal function with resultant accumulation to a much greater extent than the parent opioid. The best known example is the accumulation of morphine-6-glucuronide from morphine. Some opioids have active metabolites but at different target sites. These are norpethidine, a neurotoxic agent, and nordextropropoxyphene, a cardiotoxic agent. Clinicians need to be aware that many opioids have active metabolites that will become therapeutically important, for example in cases of altered pathology, drug interactions and genetic polymorphisms of drug-metabolizing enzymes. Thus, dose individualisation and the avoidance of adverse effects of opioids due to the accumulation of active metabolites or lack of formation of active metabolites are important considerations when opioids are used.Janet K. Coller, Lona L. Christrup, Andrew A. Somogy