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 Ϸ90 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.

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The proto‐oncogene c‐kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white‐spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 are severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. Wv and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co‐culture assay. We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c‐kit coding sequence (W37 E‐‐‐‐K at position 582; Wv T‐‐‐‐M position 660 and W41 V‐‐‐‐M position 831), which affect the c‐kit associated tyrosine kinase to varying degrees. The c‐kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c‐kitW37 protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c‐kit protein. A 125 kd c‐kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The proto-oncogene c-k/t, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-k/t is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-k/t associated tyrosine-specific protein kinase is functionally impaired in W/W v mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.