Abstracts

RATIONALE: To identify the protein constituent of the levetiracetam (KEPPRA[reg]) binding site (LBS) in situ, we synthesized the photoaffinity label [³H]ucb 30889, a levetiracetam analogue bearing a substituted aryl group on the 4-position of the pyrrolidone and which binds to the same site. This radioligand was used to study the mapping of the novel binding site within the brain and both its cellular and subcellular distribution.
METHODS: For rat brain autoradiography, 25 [mu]m slices were incubated with 1.3 nM [³H]ucb 30889 for 120 min at 4[degree]C in 50 mM Tris-HCl buffer. Binding assays with rat brain membranes and various neuronal cultures were performed under similar conditions. Non-specific binding was determined by the inclusion of 1 mM levetiracetam in the assay. For photolabeling, membranes were incubated with 40 nM [³H]ucb 30889 for 120 min at 4[degree]C in 50 mM Tris-HCl buffer, followed by irradiation with UV-light for 30 min.
RESULTS: [³H]ucb 30889 binding sites were heterogeneously distributed in the rat brain. While there was no apparent binding in the white matter there was a high level of binding in the dentate gyrus, the superior colliculus, several thalamic nuclei and in the molecular layer of the cerebellum. Binding was less pronounced in the cerebral cortex, the hypothalamus and the striatum. [³H]ucb 30889 binding in whole-cell binding assays (rat and mice brain neuronal primary cultures and PC12 cells) showed high levels of specific binding. After fractionation of rat brain synaptosomes by sucrose gradient and differential centrifugation, the LBS was localized predominantly in synaptic membrane and microsomal fractions, whereas there was no specific binding in the mitochondrial fraction. Upon UV-irradiation, a protein of 97 [plusminus] 10 kDa was specifically photolabeled by [³H]ucb 30889 in rat brain synaptosomes and microsomes. The inclusion of 1 mM levetiracetam in the binding assay prevented this labeling. Biochemical experiments indicated that the photolabeled protein is not N-glycosylated and may likely be a membrane protein.
CONCLUSIONS: The localization of [³H]ucb 30889 binding sites in rat brain does not match the distribution of classical receptors and ion channels such as the glutamate receptors, the GABA[sub]A[/sub] receptor as well as the Na⁺ and Ca²⁺ channels. This novel binding site is present in various neuronal cell types and brain areas.
[Supported by: UCB S.A., Pharma Sector]; (Disclosure: Salary - UCB S.A., Grant - UCB S.A.)