Abstract
Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin) is a novel anticonvulsant drug, with a mechanism of action apparently dissimilar to that of other antiepileptic agents. We report here the isolation and characterization of a [3H]gabapentin-binding protein from pig cerebral cortex membranes. The detergent-solubilized binding protein was purified 1022-fold, in a six-step column-chromatographic procedure, with a yield of 3.9%. The purified protein had an apparent subunit Mr of 130,000, and was heavily glycosylated. The partial N-terminal amino acid sequence of the Mr 130,000 polypeptide, EPFPSAVTIK, was identical to that reported for the alpha2delta subunit of the L-type Ca2+ channel from rabbit skeletal muscle (Hamilton, S. L., Hawkes, M. J., Brush, K., Cook, R., Chang, R. J., and Smilowitz, H. M. (1989) Biochemistry 28, 7820-7828). High levels of [3H]gabapentin binding sites were found in membranes prepared from rat brain, heart and skeletal muscle. Binding of [3H]gabapentin to COS-7 cells transfected with alpha2delta cDNA was elevated >10-fold over controls, consistent with the expression of alpha2 delta protein, as measured by Western blotting. Finally, purified L-type Ca2+ channel complexes were fractionated, under dissociating conditions, on an ion-exchange column; [3H]gabapentin binding activity closely followed the elution of the alpha2 delta subunit. [3H]Gabapentin is the first pharmacological agent described that interacts with an alpha2delta subunit of a voltage-dependent Ca2+ channel.
Highlights
Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin) is a novel anticonvulsant drug, with a mechanism of action apparently dissimilar to that of other antiepileptic agents
Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin) is a novel antiepileptic drug that is orally active in various animal models of epilepsy, including maximal electroshock in rats and pentylenetetrazol- or audiogenically induced seizures in mice [1,2,3]
Binding of [3H]gabapentin to membranes prepared from various pig brain regions was examined
Summary
Materials—Pig brains were obtained fresh from the local abattoir and transported to the laboratory on ice. The sample was loaded at 1 ml/min on to a Sephacryl S-400 column (2.6 cm (internal diameter) ϫ 92 cm) equilibrated in 0.08% Tween 20, 150 mM NaCl, 10 mM Hepes/KOH, pH 7.4. After washing with 20 ml of 1 mM NaCl containing 0.08% Tween 20, the column was eluted at 5 ml/h with a linear gradient of 0 – 0.2 M Na2HPO4/H3PO4, pH 7.4, in a total volume of 100 ml of equilibration buffer. Dishes were incubated for 18 h at 37 °C in an atmosphere of 5% CO2 in air and transfected with 5 g of plasmid DNA by Lipofectamine-mediated transfection After another 48 h, cell membranes were prepared and assayed for [3H]gabapentin binding activity as described above. Protein Determinations—Protein concentration was determined by the method of Bradford [18] using bovine serum albumin as a standard
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