Isolation of the [3H]gabapentin-binding protein α2δ Ca2+ channel subunit from porcine brain:: Development of a radioligand binding assay for α2δ subunits using [3H]leucine

The novel antiepileptic agent gabapentin (Neurontin) binds with high affinity to the alpha(2) delta subunit of a voltage-dependent Ca2+ channel. We report here a simple purification scheme for detergent-solubilized alpha(2) beta subunits from porcine brain. This involves sequential chromatography on Q-Sepharose, Cu2+-charged iminodiacetic acid-Sepharose, wheat germ lectin-agarose, and Mono Q. The purified protein was essentially homogeneous by SDS-polyacrylamide gel electrophoresis with a subunit M-r of 145,000. Using [H-3]gabapentin as the radiolabeled tracer and (S)-3-isobutyl gamma-aminobutyric acid to define nonspecific binding, the overall purification factor was 2760-fold and the apparent 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 for the alpha(2) beta Ca2+ channel subunit. [H-3]Leucine offers several advantages over custom-labeled [H-3]gabapentin: it has a higher specific activity, is relatively inexpensive, and is available from commercial sources. (C) 1998 Academic Press.