ISOLATION OF THE [H-3]GABAPENTIN-BINDING PROTEIN ALPHA(2)DELTA CA2- DEVELOPMENT OF A RADIOLIGAND BINDING ASSAY FOR ALPHA(2)DELTA SUBUNITS USING [H-3]LEUCINE( CHANNEL SUBUNIT FROM PORCINE BRAIN )

The novel antiepileptic agent gabapentin (Neurontin) binds with high a ffinity to the alpha(2) delta subunit of a voltage-dependent Ca2+ chan nel. We report here a simple purification scheme for detergent-solubil ized alpha(2) beta subunits from porcine brain. This involves sequenti al chromatography on Q-Sepharose, Cu2+-charged iminodiacetic acid-Seph arose, wheat germ lectin-agarose, and Mono Q. The purified protein was essentially homogeneous by SDS-polyacrylamide gel electrophoresis wit h a subunit M-r of 145,000. Using [H-3]gabapentin as the radiolabeled tracer and (S)-3-isobutyl gamma-aminobutyric acid to define nonspecifi c binding, the overall purification factor was 2760-fold and the appar ent yield 26.6%. We also developed and validated a novel binding assay for alpha(2) delta Ca2+ channel subunits using the ligand pair L-[H-3 ]leucine/L-isoleucine. Even in binding assays of crude brain membrane fractions, [H-3]leucine proved to be remarkably stable and specific fo r the alpha(2) beta Ca2+ channel subunit. [H-3]Leucine offers several advantages over custom-labeled [H-3]gabapentin: it has a higher specif ic activity, is relatively inexpensive, and is available from commerci al sources. (C) 1998 Academic Press.