Abstracts

Rationale: Levetiracetam (LEV) is the S-enantiomer of α-ethyl-2-oxo-l-pyrrolidine acetamide. LEV has been reported to bind to the SV2A vesicular protein, which is present on all presynaptic vesicles. The function of the SV2A protein is not known. However, it is difficult to hypothesize a mechanism whereby a protein that is expressed on all synaptic vesicles can regulate either excitatory or inhibitory activity. To further address the LEV binding site that is responsible for LEV's anticonvulsant effects, the binding of [³H]LEV to neuronal membranes was performed and compared to previously reported results.Methods: Guinea pig brain membranes were prepared by homogenizing guinea pig brains in 50 mM Tris-HCl, pH 7.5, followed by three centrifugal washes and incubation at 37^oC for 30 min to remove any endogenous ligands associated with the binding site. Nonspecific binding was measured by the inclusion of 100 μM unlabelled LEV. To terminate binding, membranes were filtered through glass fiber filters, soaked in 0.25% polyethyleneimine. The filters were washed with cold 50 mM Tris-HCl, pH 7.5, and were counted in a scintillation counter. Results: [³H]LEV binding to guinea pig brain membranes was dependent on the protein concentration, time and temperature. At 25^oC, [³H]LEV binding reached equilibrium within 30 min. At 4^oC, binding continued to gradually increase until equilibrium was reached at 4 hr. Inclusion of MgCl₂ stimulated [³H]LEV binding up to 80% at Mg²⁺ concentrations of 8 mM and higher. Neither NaCl nor GTP had any effect on [³H]LEV binding. Scatchard plots of [³H]LEV saturation binding data were biphasic, suggesting more than one [³H]LEV binding site.Conclusions: [³H]LEV binding to guinea pig brain membranes was favored at 4^oC in comparison to 25^oC, suggesting that binding was enthalpy-driven, which has been observed for agonist binding to many neuronal receptors (Lohse et al., Mol. Pharmacol., 1984). The fact that Mg²⁺ stimulated [³H]LEV binding, while Na⁺ and GTP did not have any effect on binding strongly suggests that [³H]LEV does not bind to a G Protein-coupled receptor (GPCR). Agonist binding to GPCR is inhibited by mono- and di- valent cations and the guanine nucleotide GTP. A biphasic Scatchard plot of [³H]LEV saturation binding data suggests that [³H]LEV is binding to more than one site. Previously, biphasic Scatchard plots and biphasic kinetic data were obtained when the binding of an analogue of LEV, [³H]ucb 30889, was measured in human brain membranes (Gillard et al., Eur. J. Pharmacol., 2006). Collectively, this current study and previously published studies suggest that LEV binds to more than one site in brain.