The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam

Abstract

Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of approximately 90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.

Figures

Fig. 1.

Photolabeling of the LEV-binding site in synaptic vesicles from rat brain. After photoaffinity labeling with [3H]ucb 30889, SDS-solubilized proteins were run on an SDS/PAGE gel, and then the gel was cut into thin slices and counted for 3H content. Photoaffinity labeling of crude synaptosomal (P2) (•) and synaptic vesicle (LP2) (□) fractions by using [3H]ucb 30889 is shown. Photoaffinity labeling identifies a protein of ≈90 kDa enriched in the synaptic vesicle fraction.

Fig. 2.

Binding of [3H]ucb 30889 to WT and KO brain membranes and synaptic vesicles. (A) Western blot of brain membranes from WT and homozygous KO mice probed with an anti-SV2 monoclonal antibody (cross-reactive to all isoforms) or with an anti-SV2A-specific polyclonal antibody. Lanes 1, WT; lanes 2, SV2A-/- KO; lanes 3, SV2B-/- KO; lanes 4, SV2A-/-/B-/- double KO. (B) Binding of [3H]ucb 30889 to brain membranes from WT, SV2A-/-, SV2B-/-, and SV2A-/-/SV2B-/- KO mice. Binding is observed only to membranes from animals expressing SV2A. □, [3H]ucb 30889 alone; ▪, [3H]ucb 30889 plus 1 mM LEV. Error bars are the SD of experiments performed with five WT brains and four KO brains, with three replicates within each experiment. (C) Purification of synaptic vesicles enriches for the synaptic vesicle proteins and LEV binding. Shown are blots of mouse brain homogenate (H), crude synaptosomes (P2), plasma and heavy membranes (LP1), and synaptic vesicles (LP2) (2 μg of each fraction) that were probed for the synaptic vesicle proteins SV2A (Upper) and synaptophysin (Lower). The synaptic vesicle fraction from WT animals displays enrichment of both synaptic vesicle proteins and LEV-binding proteins, whereas material from SV2A KOs shows enrichment of synaptophysin only. (D) Binding to the different fractions using [3H]ucb 30889 shows significant binding only to the WT LP2 fraction, containing SV2A-rich synaptic vesicles. Shown are [3H]ucb 30889 alone (open bars) and [3H]ucb 30889 plus 1 mM LEV (filled bars). Shown are representative examples of two experiments. Error bars are the SD of two replicates.

Fig. 3.

Binding of [3H]ucb 30889 to heterologously expressed SV2 isoforms. (A) Western blot with cross-reactive anti-SV2 monoclonal showing roughly equivalent amounts of hSV2A (lane A), hSV2B (lane B), and hSV2C (lane C). There is no detectable SV2 immunoreactivity in the β-gal-transfected cells (β-gal, fourth lane). In the case of hSV2C, three times as much total protein was loaded as used for the other samples. (B) Binding of [3H]ucb 30889 to hSV2A, hSV2B, or hSV2C (three times the number of cells as other samples) transiently expressed in COS-7 cells. Significant binding is observed only to hSV2A, not to hSV2B or hSV2C. Shown are [3H]ucb 30889 alone (open bars) and [3H]ucb 30889 plus 1 mM LEV (filled bars).

Fig. 4.

Binding of [3H]ucb 30889 to immunoprecipitated SV2A. (A) Western blot of immunoprecipitated SV2A. Immunoprecipitation was performed with a goat antibody against SV2A, or normal goat IgG. Western blotting with the SV2A antibody revealed the presence of the protein in the former condition only. Mb, membrane; IP, immunoprecipitate. (B) Binding of [3H]ucb 30889 to SV2A purified by IP from a detergent-soluble fraction of rat cortical membranes. Binding occurs only to the DM-solubilized membrane or anti-SV2A immunoprecipitate, not to the control IgG immunoprecipitate. Shown are [3H]ucb 30889 alone (open bars) and [3H]ucb 30889 plus 1 mM LEV (filled bars). Shown is a representative example of three experiments.

Fig. 5.

Binding affinities of selected LEV derivatives for human SV2A. (A)IC50 curves of LEV, ucb L060, and ucb 30889 against hSV2A transiently expressed in COS-7, using [3H]ucb 30889. Shown are LEV (▪), ucb 30889 (▪), and ucb L060 (•). Error bars are SEM, n = 3. (B) Correlation of binding of a series of LEV compounds to mouse brain and to hSV2A. Shown are pIC50s measured by using [3H]ucb 30889. There is a high degree of correlation between binding affinity of these compounds to hSV2A and to mouse brain membranes. pIC50 values are the mean of two independent experiments, where each determination lies within 0.2 log units of the mean.

Fig. 6.

Correlation between binding affinity and antiseizure potency of LEV derivatives. Correlation of binding of a series of LEV-related compounds to hSV2A assayed in transiently transfected COS-7 cells (pIC50s measured by using [3H]ucb 30889), and of antiseizure potencies shown as the -log ED50s (pED50s) in the mouse audiogenic seizure model. There is a good correlation between antiseizure potency in audiogenic mice and affinity to hSV2A.