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
✍ Scribed by Jason P. Brown; Visaka U.K. Dissanayake; Alex R. Briggs; Mihajlo R. Milic; Nicolas S. Gee
- Publisher
- Elsevier Science
- Year
- 1998
- Tongue
- English
- Weight
- 225 KB
- Volume
- 255
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 delta subunits from porcine brain. This involves sequential chromatography on Q-Sepharose, Cu(2+)-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 Mr of 145,000. Using [3H] 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-[3H]leucine/L-isoleucine. Even in binding assays of crude brain membrane fractions, [3H]leucine proved to be remarkably stable and specific for the alpha 2 delta Ca2+ channel subunit. [3H]Leucine offers several advantages over custom-labeled [3H]gabapentin: it has a higher specific activity, is relatively inexpensive, and is available from commercial sources.