Abstracts

Rationale: Levetiracetam (LEV) has been reported to bind to the SV2A vesicular protein (Gillard et al., Eur. J. Pharmacol. 478:1-9, 2003), and to inhibit calcium-induced calcium release in hippocampal neuronal cultures (Nagarkatti et al., Neurosci. Lett. 436:289-293, 2008). Previously, we reported that the divalent cations, Mg²⁺, Ca²⁺, Sr²⁺, and Ba,²⁺, stimulated [³H]LEV binding to guinea pig brain membranes (Bidlack et al., Epilepsia 48(supple. 6): 316-317, 2007). Zinc and copper are endogenous transition metals that can be synaptically released during neuronal activity. Zn²⁺ interacts with many ion channels and functions as an endogenous modulator of neuronal excitability by modulating ligand-gated ion channels such as the NMDA glutamate and the GABA_A receptors. Zn²⁺ and Cu²⁺ concentrations at the synaptic cleft have been estimated to peak at 100 - 300 μM (Horning et al., Brain Res. 852:56-61, 2000). The purpose of this study was to determine if Zn²⁺ and Cu²⁺ altered [³H]LEV binding to neuronal membranes. Methods: [³H]LEV (18 Ci/mmol) was custom labeled by GE Healthcare. Guinea pig brain membranes were incubated at 4^oC with varying concentrations of ZnCl₂ or CuCl₂ and 10 nM [³H]LEV for 4 hrs. Nonspecific binding was measured by the inclusion of 100 μM unlabeled 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 brain membranes was inhibited by Zn²⁺ and Cu²⁺ in a concentration-dependent manner. Zn²⁺ had an IC₅₀ value of 62 ± 4.2 μM for the inhibition of the binding of 10 nM [³H]LEV to neuronal membranes. An I_max value of 74 ± 4.5% was obtained, meaning that approximately 25% of the [³H]LEV binding was not inhibited by Zn²⁺ at concentrations up to 3 mM. CuCl₂ inhibited [³H]LEV binding with an IC₅₀ value of 250 ± 72 μM and an I_max value of 95 ± 5.2%, meaning that Cu²⁺ at physiological concentrations inhibited virtually all of the [³H]LEV binding. Conclusions: [³H]LEV binding is tightly regulated by cations, particularly Zn²⁺ and Cu²⁺. Zn²⁺ and Cu²⁺ are highly concentrated in several brain regions, including the hippocampus. A role for Zn²⁺ in epilepsy was suggested by its modulatory actions on neuronal activity, the prominence of Zn²⁺-containing pathways in seizure-prone brain regions, abnormalities of cerebral Zn²⁺ in epileptic animals, and the effects of Zn²⁺ manipulations on seizures susceptibility (Erickson et al., J. Neurosci. 17:1271-1281, 1997). Collectively, the results from this study suggest that [³H]LEV binds to a protein that is directly modulated by cations, particularly Zn²⁺ and Cu²⁺. By examining [³H]LEV binding to proteins known to bind Zn²⁺ and Cu²⁺, the identification of the LEV binding site involved in its anti-epileptic effects may be determined.