Abstracts

Rationale: The synaptic vesicle protein 2A (SV2A) is the brain binding site of Levetiracetam (LEV), a new generation AED. There are two additional isoforms of SV2: SV2B and SV2C. Using the difference in binding of two LEV derivatives, ucb 30889 and ucb 101275, to the three SV2 isoforms, we tested a series of chimeric constructs between SV2A and the other isoforms for their binding properties. After successfully identifying regions of SV2 responsible for binding differences between the three isoforms, we made additional mutations in the most critical region and identified specific residues important for binding of LEV and derivatives.Methods: Two labelled LEV derivatives, [3H]ucb-30889 and [3H]ucb-101275, were utilized as probes of binding to rat SV2A and SV2C, chimeras between the two, and point mutants of rSV2A. Binding was studied in proteins expressed heterologously from a pDEST40 vector (Invitrogen) under CMV promoter control. Chimeras and mutants were constructed using a QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene).Results: The LEV derivative ucb 30889 binds selectively to the SV2A isoform. Here, we report that an alternate ligand, ucb 101275, binds with similar affinities to both the rSV2A and rSV2C isoforms. We used a strategy to identify binding site residues in the SV2 proteins by analyzing chimeras between isoforms for their binding to the above LEV derivatives. Chimeras between rSV2A and rSV2B/C isoforms identified a region of rSV2A comprising residues 594-742 (TM8-12) as most important for binding differences between the three isoforms. Fourteen residues in TM8-12 that differ among all three isoforms were mutated to Ala in rSV2A. Two of the mutated positions near the center of TM10 were shown to have altered affinity to the ligands. Additional Ala substitutions in flanking residues in TM10 identified a third residue, which also appears to be involved in ligand binding.Conclusions: Here we report that a LEV derivative, ucb 101275, has similar affinities for the SV2A and SV2C isoforms, while showing no measurable affinity for SV2B. The new ligand was used to demonstrate that LEV has a weaker affinity to the SV2C isoform than to SV2A. The different relative binding affinities of the two ligands ucb 30889 and ucb 101275 towards SV2A and SV2C was utilized to identify regions of SV2A involved in binding to LEV and related compounds. Following an iterative process, a region of SV2 that includes TM helices 8-12 was identified as containing residues responsible for the difference in affinities of the two ligands. Ala substitution of selected non-conserved residues in this region resulted in the identification of specific residues in transmembrane helix 10 (TM10) that appear to play a role in binding levetiracetam and related racetams.