Isolation of the [3H]gabapentin-binding protein/alpha 2 delta Ca2+ channel subunit from porcine brain: development of a radioligand binding assay for alpha 2 delta subunits using [3H]leucine

Analytical Biochemistry
J P BrownN S Gee

Abstract

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.

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Citations

Feb 27, 2008·Proceedings of the National Academy of Sciences of the United States of America·Jan HendrichAnnette C Dolphin
Oct 3, 2009·Circulation Research·John P BannisterJonathan H Jaggar
Sep 15, 2010·Nihon yakurigaku zasshi. Folia pharmacologica Japonica·Yasuo OchiAkio Arakawa
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Jan 16, 2007·Trends in Pharmacological Sciences·David J DooleyDouglas Feltner
Nov 10, 2010·Toxicology and Applied Pharmacology·Elizabeth Martínez-HernándezRicardo Felix
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